U.S. patent application number 14/511778 was filed with the patent office on 2015-02-05 for methods and devices for micro-isolation, extraction, and/or analysis of microscale components.
The applicant listed for this patent is CALIFORNIA INSTITUTE OF TECHNOLOGY, UNIVERSITY OF SOUTHERN CALIFORNIA. Invention is credited to Emil P. KARTALOV, Darryl SHIBATA, Clive TAYLOR, Lawrence A. WADE.
Application Number | 20150038379 14/511778 |
Document ID | / |
Family ID | 44277846 |
Filed Date | 2015-02-05 |
United States Patent
Application |
20150038379 |
Kind Code |
A1 |
KARTALOV; Emil P. ; et
al. |
February 5, 2015 |
METHODS AND DEVICES FOR MICRO-ISOLATION, EXTRACTION, AND/OR
ANALYSIS OF MICROSCALE COMPONENTS
Abstract
Provided herein are devices and methods for the micro-isolation
of biological cellular material. A micro-isolation apparatus
described can comprise a photomask that protects regions of
interest against DNA-destroying illumination. The micro-isolation
apparatus can further comprise photosensitive material defining
access wells following illumination and subsequent developing of
the photosensitive material. The micro-isolation apparatus can
further comprise a chambered microfluidic device comprising
channels providing access to wells defined in photosensitive
material. The micro-isolation apparatus can comprise a chambered
microfluidic device without access wells defined in photosensitive
material where valves control the flow of gases or liquids through
the channels of the microfluidic device. Also included are methods
for selectively isolating cellular material using the apparatuses
described herein, as are methods for biochemical analysis of
individual regions of interest of cellular material using the
devices described herein. Further included are methods of making
masking arrays useful for the methods described herein.
Inventors: |
KARTALOV; Emil P.; (LOS
ANGELES, CA) ; SHIBATA; Darryl; (ROLLING HILLS
ESTATES, CA) ; TAYLOR; Clive; (MALIBU, CA) ;
WADE; Lawrence A.; (LA CANADA-FLINTRIDGE, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CALIFORNIA INSTITUTE OF TECHNOLOGY
UNIVERSITY OF SOUTHERN CALIFORNIA |
Pasadena
Los Angeles |
CA
CA |
US
US |
|
|
Family ID: |
44277846 |
Appl. No.: |
14/511778 |
Filed: |
October 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13010761 |
Jan 20, 2011 |
8889416 |
|
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14511778 |
|
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61297207 |
Jan 21, 2010 |
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61309292 |
Mar 1, 2010 |
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Current U.S.
Class: |
506/40 ;
435/289.1 |
Current CPC
Class: |
C12M 23/12 20130101;
C12M 25/04 20130101; C12M 35/02 20130101; C12M 25/14 20130101; C12M
23/16 20130101; C12M 37/04 20130101; G01N 33/54366 20130101; C12N
13/00 20130101; C12M 23/22 20130101; C12M 47/04 20130101 |
Class at
Publication: |
506/40 ;
435/289.1 |
International
Class: |
C12M 1/42 20060101
C12M001/42; C12M 1/32 20060101 C12M001/32; C12M 3/06 20060101
C12M003/06; C12M 1/12 20060101 C12M001/12 |
Claims
1-7. (canceled)
8. A micro-isolation apparatus comprising a support, a cellular
material mounted upon the support, and a photosensitive material
deposited on the cellular material, wherein the photosensitive
material comprises an access well positioned to correspond to a
region of interest of the cellular material.
9. The micro-isolation apparatus of claim 8, further comprising a
chambered microfluidic device comprising at least one channel that
provides access to the access well and is positioned adjacent to a
photosensitive material located above the cellular material.
10. The micro-isolation apparatus of claim 9, wherein the
photosensitive material comprises a plurality of access wells
corresponding to a plurality of respective areas of interest of the
cellular material.
11. The micro-isolation apparatus of claim 10, wherein the
plurality of access wells are interconnected through a series of
channels in the microfluidic device.
12. The micro-isolation apparatus of claim 11, wherein the series
of channels are connected to one or more inputs and one or more
outputs.
13. A micro-isolation apparatus comprising a first photosensitive
material deposited on cellular material, adapted to be contained in
the apparatus, wherein the first photosensitive material comprises
a first access well positioned to correspond to a region of
interest of the cellular material, a first chambered microfluidic
device comprising at least one channel that provides access to the
first access well and is positioned adjacent to the first
photosensitive material, a second photosensitive material adapted
to be deposited on the cellular material opposite to the first
photosensitive material, wherein the second photosensitive material
comprises a second access well positioned to correspond to an
opposing side of the region of interest of the cellular material,
and a second chambered microfluidic device comprising at least one
channel that provides access to the second access well and is
positioned adjacent to the second photosensitive material.
14. The micro-isolation apparatus of claim 13, wherein the channel
or channels of the first chambered microfluidic device provide an
input to the region of interest of the tissue and/or cells and the
channel or channels of the second chambered microfluidic device
provide an output from the region of interest of the tissue and/or
cells.
15. The micro-isolation apparatus of claim 13, wherein the
photosensitive material comprises a plurality of access wells
corresponding to a plurality of respective areas of interest.
16. A maskless micro-isolation apparatus comprising cellular
material, a first chambered microfluidic device comprising multiple
channels, wherein each channel provides access to respective
regions of interest of the cellular material, and a second
chambered microfluidic device comprising multiple channels
positioned to correspond to the regions of interest of the cellular
material that are opposite to the regions of interest corresponding
to the first chambered microfluidic device.
17. The maskless micro-isolation apparatus of claim 16, wherein the
channels comprises valves that control flow of gases or liquids
through the channels of the first and second chambered microfluidic
devices.
18. The maskless micro-isolation apparatus of claim 16, wherein the
first and second chambered microfluidic device comprise a dense
pore matrix.
19-43. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. patent
application Ser. No. 13/010,761 filed on Jan. 20, 2011, and
incorporated herein by reference in its entirety, which, in turn,
claims priority to U.S. Provisional Application No. 61/297,207,
filed on Jan. 21, 2010, and U.S. Provisional Application No.
61/309,292, filed on Mar. 1, 2010, each of which is incorporated
herein by reference in its entirety.
STATEMENT OF GOVERNMENT GRANT
[0002] The invention described herein was made in the performance
of work under a NASA contract, and is subject to the provisions of
Public Law 96-517 (35 USC 202) in which the Contractor has elected
to retain title.
FIELD
[0003] The present disclosure relates to micro-isolation of
microscale components such as tissue and/or cell samples. More
specifically, it relates to methods and devices for such
micro-isolation.
BACKGROUND
[0004] The isolation of certain microscale components is an
important factor in several applications where the ability to
differentially analyze properties exhibited by varying types of
components (e.g. cell types) is desired.
[0005] For example, the ability to recognize properties typical of
a component included in a matrix with other similar components, can
be of importance in various fields, including in particular
biological fields. In particular, the ability to identify
properties which cause a cell to behave in a certain way is
expected to promote an understanding of how cells behave both
normally and abnormally. For example, the ability to selectively
analyze cancerous cells is expected to provide insight into the
particular biochemical activities of those cells relative to normal
cells.
[0006] However, separating different cell types and in particular
cancerous cells from non-cancerous cells can be a difficult
endeavor.
SUMMARY
[0007] Provided herein are apparatuses and methods for the
micro-isolation of micro-scale components such as cellular
material, which in several embodiments provide for the selective
biochemical analysis of desired components.
[0008] According to a first aspect, a micro-isolation apparatus is
provided. The micro-isolation apparatus comprises a support, a
cellular material mounted upon the support, a photomask comprising
a transparent region and a non-transparent blocking region, the
non-transparent blocking region covering at least a portion of the
transparent region, wherein the photomask is placed over the
cellular material such that the blocking region is positioned to
correspond to a region of interest of the cellular material to
minimize damage to the cellular material in the region of interest
by illumination.
[0009] According to a second aspect, a micro-isolation apparatus is
provided. The micro-isolation apparatus comprises a support, a
cellular material mounted upon the support, and a photosensitive
material deposited on the cellular material wherein the
photosensitive material comprises an access well positioned to
correspond to a region of interest of the cellular material. Some
embodiments can further include a chambered microfluidic device
comprising at least one channel that provides access to the access
well and is positioned adjacent to a photosensitive material
located above the cellular material.
[0010] According to a third aspect, a micro-isolation apparatus is
provided. The micro-isolation apparatus comprises a first
photosensitive material deposited on cellular material adapted to
be contained in the apparatus, wherein the first photosensitive
material comprises a first access well positioned to correspond to
a region of interest of the cellular material, a first chambered
microfluidic device comprising at least one channel that provides
access to the first access well and is positioned adjacent to the
first photosensitive material, a second photosensitive material
adapted to be deposited on the cellular material opposite to the
first photosensitive material wherein the second photosensitive
material comprises a second access well positioned to correspond to
an opposing side of the region of interest of the cellular
material, and a second chambered microfluidic device comprising at
least one channel that provides access to the second access well
and is positioned adjacent to the second photosensitive material.
In some embodiments, the first chambered microfluidic device
provides an input to the region of interest of the tissue and/or
cells and the channel or channels of the second chambered
microfluidic device provide an output from the region of interest
of the tissue and/or cells.
[0011] According to a fourth aspect, a maskless micro-isolation
apparatus is provided. The apparatus comprises cellular material, a
first chambered microfluidic device comprising multiple channels,
wherein each channel provides access to respective regions of
interest of the cellular material, and a second chambered
microfluidic device comprising multiple channels positioned to
correspond to the regions of interest of the cellular material that
are opposite to the regions of interest corresponding to the first
chambered microfluidic device.
[0012] According to a fifth aspect, a method for selectively
isolating cellular material is provided. The method comprises
positioning cellular material on a support, placing a photomask
comprising a blocking region covering at least part of a blocking
support over the cellular material such that the blocking region
corresponds to a region of interest of the cellular material, and
exposing the cellular material to photons wherein the photons
penetrate the blocking support without penetrating the blocking
region so that the cellular material in the region of interest is
preserved and the cellular material that is not in the region of
interest is damaged.
[0013] According to a sixth aspect, a method for selectively
isolating cellular material is provided. The method comprises
positioning cellular material on a support, depositing a
photosensitive material on the cellular material, applying a
photomask comprising a blocking region onto the photosensitive
material, exposing the photosensitive material through a light
accessible region of the photomask to photons in order to generate
a lithographic pattern on the photosensitive material, removing the
photomask, and applying a developer to the photosensitive material
in order to define an access well corresponding to a region of
interest of the cellular material.
[0014] According to a seventh aspect, a method for selectively
isolating cellular material is provided. The method comprises
positioning cellular material on a support, depositing a
photosensitive material on the cellular material, exposing the
photosensitive material to photons in order to generate a
lithographic pattern on the photosensitive material, removing the
photomask, and applying a developer to the photosensitive material
in order to define an access well corresponding to a region of
interest of the cellular material.
[0015] According to an eighth aspect, a method for selectively
isolating cellular material is provided. The method comprises
positioning cellular material on a support, and exposing an
unwanted region of the cellular material to photons to selectively
damage DNA in the unwanted region of the cellular material while
not exposing a wanted region of the cellular material to minimize
damage to the DNA in the wanted region.
[0016] According to a ninth aspect, a method to analyze a
biological sample is provided. The method comprises forming
microfluidic access wells in a substrate, filling the microfluidic
access wells with a reaction mixture comprising digestion agents
and components necessary for a desired reaction, evaporating the
mixture to uniformly decrease the reaction mixture level, aligning
a support comprising cellular material facing downward on top of
the microfluidic access wells such that the cellular material is
exposed to the reaction mixture, vertically turning over the access
wells comprising the support and cellular material, allowing the
reaction mixture to flow by gravity to cover the cellular material,
allowing the digestion agents to break down the cellular material,
releasing contents from the cellular material into the microfluidic
access wells, and performing the reaction simultaneously but
separately in each of the microfluidic access wells.
[0017] According to a tenth aspect, a method to analyze a
biological sample is provided. The method comprises positioning
cellular material on a first support, applying a maskless
microisolation apparatus comprising one or more randomly placed
first access wells, filling the one or more first access wells with
a reaction mixture comprising digestion agents and components
necessary for a desired reaction, allowing the digestion agents to
digest the cellular material thereby releasing cellular contents
into the reaction mixture, inactivating the digestion agents,
filling one or more second access wells in a second support
corresponding to the one or more first access wells with an
analytical reaction mixture, evaporating a fraction of the mixture
to uniformly decrease the reaction mixture level, aligning the
first support on top of the second support such that the one or
more first access wells face the one or more second access wells,
securing the first and second support, inverting the secured
support to allow the analytical reaction mixture to contact the
cellular contents, and performing a reaction simultaneously but
separately in each well of the array.
[0018] According to an eleventh aspect, a method to isolate a
region of interest of cellular material is provided. The method
comprises positioning cellular material on a support, depositing a
negative photosensitive material on the cellular material,
capturing an image of the cellular material through the negative
photosensitive material as reflected on a processing mirror or
mirror array, positioning the processing mirror or programming the
mirror array such that photons are directed to the negative
photosensitive material over an unwanted region of the cellular
material to laminate the negative photosensitive material over the
unwanted region of the cellular material while leaving a region of
interest of the cellular material non-laminated, and removing the
negative photosensitive material that has not been laminated so
that the region of interest of the cellular material is exposed
while the unwanted region of the cellular material is sealed.
[0019] According to a twelfth aspect, a method to isolate a region
of interest of cellular material is provided. The method comprises
positioning cellular material on a support, capturing an image of
the cellular material as reflected on a processing mirror or mirror
array, positioning the processing mirror, or programming a mirror
array, such that photons are directed to an unwanted region of the
cellular material to damage DNA in the unwanted region of the
cellular material while leaving DNA in a cellular region of
interest intact.
[0020] According to a thirteenth aspect, a method for making a
micro-isolation apparatus is provided. The method comprises
positioning cellular material on a support, identifying an unwanted
region of interest of the cellular material, converting a selection
into a digital image, transferring the digital image to a plate
comprising a layer of photosensitive material over a metal, using a
laser to trace a digital mask on the photosensitive material,
developing the photosensitive material to remove exposed
photosensitive material, chemically etching the metal in the area
where the photosensitive material has been removed, and removing
remaining photosensitive material to produce a plate comprising a
layer of metal in a pattern where the metal is absent corresponding
to the unwanted region of interest of the cellular material and the
metal remaining corresponds to a region of interest of the cellular
material.
[0021] According to a fourteenth aspect, a method of making an
active masking array is provided. The method comprising positioning
cellular material on a support, identifying a region of interest of
the cellular material, polarizing an illumination light along a
first axis, directing the illumination light through (e.g. liquid
crystal) polarizer elements that are aligned or programmed in such
a way that the illumination light is absorbed in the polarizer
elements over regions of interest of the cellular material while
allowing the illumination light to damage DNA or cellular material
in an unwanted region of the cellular material to preserve the DNA
or cellular material in the region of interest of the cellular
material.
[0022] According to a fifteenth aspect, a method of making an
active array is provided. The method comprising positioning
cellular material on a support, identifying a region of interest of
the cellular material, and generating an active array of
illuminators that target unwanted regions of the cellular material
while allowing the region of interest of the cellular material to
be non-illuminated.
[0023] According to a sixteenth aspect, a method of analyzing a
biological sample is provided. The method comprising positioning
cellular material on a first support, depositing a negative
photosensitive material on the cellular material, applying a
photomask comprising a blocking region onto the negative
photosensitive material, exposing the negative photosensitive
material to photons in order to generate a lithographic pattern on
the negative photosensitive material, removing the photomask,
applying a developer to the negative photosensitive material in
order to define one or more first access wells filling the one or
more access wells with a reaction mixture comprising digestion
agents and components necessary for a desired reaction, allowing
the digestion agents to digest the cellular material thereby
releasing the cellular contents into the reaction mixture,
inactivating the digestion agents, filling one or more second
access wells in a second support corresponding to the one or more
first access wells with an analytical reaction mixture, evaporating
a fraction of the mixture to uniformly decrease the reaction
mixture level, aligning the first support on top of the second
support such that the one or more first access wells face the one
or more second access wells securing the first and second support,
inverting the secured support to allow the analytical reaction
mixture to contact the cellular contents, and performing a reaction
simultaneously but separately in each well of the array.
[0024] According to a seventeenth aspect, a micro-isolation
apparatus is provided. The apparatus comprising a support, cellular
material mounted upon the support, a photosensitive material
deposited on the cellular material, wherein the photosensitive
material comprises an access well positioned to correspond to a
region of interest of the cellular material, and a microfluidic
device that provides access to the access well and is positioned
adjacent to the photosensitive material.
[0025] According to an eighteenth aspect, a method for selectively
isolating cellular material is provided. The method comprising
positioning cellular material on a support, depositing a
photosensitive material on the cellular material, applying a
photomask comprising a blocking region onto the photosensitive
material, exposing the photosensitive material through a light
accessible region of the photomask to photons in order to generate
a lithographic pattern on the photosensitive material, removing the
photomask, applying a developer to the photosensitive material in
order to define an access well corresponding to a region of
interest of the cellular material, positioning a microfluidic
device that provides access to the access well and is positioned
adjacent to a photosensitive material located above the cellular
material, and analyzing cellular material from the regions of
interest.
[0026] According to a nineteenth aspect, a method for selectively
isolating cellular material is provided. The method comprising
positioning cellular material on a support, depositing a
photosensitive material on the cellular material, exposing the
photosensitive material to photons in order to generate a
lithographic pattern on the photosensitive material, removing the
photomask applying a developer to the photosensitive material in
order to define an access well corresponding to a region of
interest of the cellular material, positioning a microfluidic
device that provides access to the access well and is positioned
adjacent to a photosensitive material located above the cellular
material, and analyzing cellular material from the region of
interest.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The accompanying drawings, which are incorporated into and
constitute a part of this specification, illustrate one or more
embodiments of the present disclosure and, together with the
detailed description and the examples, serve to explain the
principles and implementations of the disclosure.
[0028] FIG. 1 shows a cross-sectional view of a series of steps in
which tissue masking selectively destroys DNA.
[0029] FIG. 2 shows a cross-sectional view of a series of steps
where tissue isolation or masking is performed through lamination
with a photosensitive material.
[0030] FIG. 3 shows a top view of an embodiment in which tissue
isolation targets multiple areas of interest occurring by
lamination with a photosensitive material.
[0031] FIG. 4 shows a cross sectional view where a tissue is
integrated with microfluidic elements.
[0032] FIG. 5A is related to FIG. 3, and illustrates a top view of
a customized chambered microfluidic device. FIG. 5B is related to
FIG. 3, and illustrates a top view of a standardized chambered
microfluidic device.
[0033] FIG. 6 shows a cross-sectional view of an embodiment where
tissue encapsulation is performed.
[0034] FIG. 7 illustrates an embodiment in which a maskless
chambered microfluidic device encapsulates the tissue encapsulates
a tissue without customized photomasking while specificity of
micro-isolation is achieved through active control of arrays of
valves allowing specificity of microisolation to be achieved
through active control of arrays of valves.
[0035] FIG. 8 illustrates a top view of a matrix of microfluidic
wells.
[0036] FIG. 9 shows a series of steps for the parallel processing
of isolated tissue subsections.
[0037] FIG. 10 shows a series of steps for the parallel processing
of isolated tissue subsections that allows step-wise administration
of biochemical agents.
[0038] FIG. 11 illustrates an optical setup for dynamic optical
array masking.
[0039] FIG. 12 provides optical microscope optical microscope
images as an Example of the techniques described herein.
DETAILED DESCRIPTION
[0040] Methods and systems are provided herein that allow in
several embodiments the integration of microfluidic techniques with
micro-isolation of light sensitive microscale components, such as
cells and/or tissue.
[0041] The term "microfluidic" refers to a system or device for
handling, processing, ejecting and/or analyzing a fluid sample
including at least one channel having microscale dimensions.
Microfluidic tissue isolation can be customized morphologically,
functionally, and a combination of the two.
[0042] The term "micro-isolation" refers to the isolation of
micro-scale components (components having a size or measure in the
order of micrometers) and in particular of light sensitive
microscale components, which includes, but is not necessarily
limited to, one or more biological components. A biological
component refers to any organized substance forming part of a
living matter, e.g. a cell, cellular material, membranes,
organelles, proteins, nucleic acids and/or living organisms of any
dimensions or a part thereof (e.g. tissues or various cell
extracts). Micro-isolation as used herein can refer to the
isolation of a single nucleus, a single cell or a biological
component thereof, a group of individual cells, or a cluster of
cells, or a group of clusters of cells, or a specific region of a
tissue or a portion thereof, or even cellular organelles (e.g. cell
nuclei). In particular, in some embodiments, methods and system
herein described allow one to simultaneously address a distributed
group of regions of interest across a tissue slide while each
region can be a single nucleus, a single cell, a cluster of cells
or a biological component thereof.
[0043] In some embodiments, the proposed integration of
microfluidic techniques and micro-isolation of cellular material
molds the microfluidic architectures in accordance with the
particular structure of each specific biological component to be
isolated. In particular, in some of those embodiments, the approach
described herein is mainly built around the cellular material,
following the tissue structure such that the devices and methods
described herein are adapted to the specific geometry of particular
tissues or other biological components. By contrast, in some of the
traditional approaches microfluidic devices are structured taking
only in accordance with engineering considerations (e.g. path
minimization, fluidic efficiency), while the biological components
in applied devices are forced to comply with these engineering
presets and are not taken into consideration.
[0044] In some embodiments, the subject matter described represents
a paradigm shift in microfluidic technology because 1) microfluidic
devices are directly integrated with, onto, or around tissue
samples, in contrast to certain conventional method of off-chip
sample extraction followed by sample insertion in microfluidic
devices, 2) architectural and operational principles of
microfluidic devices are mainly subordinated to suit specific
tissue structure and needs, in contrast to certain conventional
method of building devices according to fluidic function alone and
without regard to tissue structure, and/or 3) sample acquisition
from tissue is to be performed on-chip and is to be integrated with
the diagnostic measurement within the same device, in contrast to
the conventional method of off-chip sample prep and subsequent
insertion into a diagnostic device.
[0045] In an embodiment, a particular sample is hardwired using
photolithographically defined masks.
[0046] FIG. 1 illustrates exemplary hardwired masks. Cells (110)
inside a tissue slice (120) on a tissue support (e.g. glass slide)
(130) are exposed to ultraviolet (UV) light (140) through a
photomask (150) comprising blocking regions (e.g. chrome regions)
(160) patterned on a transparent blocking support (e.g. glass
slide) (170). The blocking regions (160) are patterned in
correspondence to cells of interest (180). The illuminating UV
light (140) passes through a region of the transparent blocking
support that is not blocked (175) and is prevented from exposing an
area (195) protected by the blocking region (160). DNA in exposed
cells is destroyed (185) but protected DNA inside the cells of
interest is preserved (190).
[0047] The term "illumination" refers to the exposure of light.
Light can be visible or non-visible light, and can be one or more
of UV, two-photon, or multi-photon light or additional examples of
light of various wavelength suitable to be used in connection with
biological components which are identifiable by a skilled person
upon reading of the present disclosure
[0048] The term "hardwired" refers to devices, methods and systems
herein described or portions thereof, that are tailored for a
specific biological component of interest. For example, a
particular device is hardwired if it is configured to be suitable
for a specific component, e.g. a specific tissue to be investigated
using methods and systems herein described. In some embodiments,
hardwired devices, methods and systems are herein described that
are tailored not only for a specific biological component of
interest, but also for a specific investigative approach of
interest. For example, in some embodiments, hardwired masks are
described that allow UV light to be directed only to areas of no
interest so that the DNA, protein, or other biological material in
those areas is damaged to various extents and even destroyed by
photodamage. In some of those embodiments, damage in areas of
interest not exposed to UV is minimized to various extents and in
some cases even remain intact. The term "destroy" as used in the
present disclosure with reference to an item indicates a damage
level able to impact at least one biological activity associated to
the item. The term "intact" as used in the present disclosure with
reference to an item indicates a molecule that preserve all the
biological activities associated to the item.
[0049] In an embodiment, cells (or another biological components or
microscale component) of interest can be identified, for example,
using a microscopic computerized image of the slide and appropriate
custom software, which can convert the selection into a digital
image. The digital mask can be fed into a direct laser writer, e.g.
Heidelberg DWL66, which transfers a digital mask onto the
photosensitive material by direct writing with a resolution of 2
microns or higher (see Example 1). One skilled in the art would
recognize that other laser writers or means for transferring
digital masks with even higher resolution can be used with the
claimed subject matter described herein.
[0050] The term "micro-isolation apparatus" refers to a device that
aids in the micro-isolation of a microscale component (e.g. a
biological component, which relates to biology, life and/or living
processes, such as a cellular material).
[0051] The term "cellular material" refers to biological material
pertaining to a biological cell. As used herein, it can refer to
sub-components of a biological cell, a single intact biological
cell, a group of biological cells, or a tissue.
[0052] The term "region of interest" as used herein pertains to a
targeted area within cellular material. Definition of a targeted
area can be of any dimensions and include one or more cellular
material depending on the experimental design of choice. For
example, the region of interest can be an area that is sought to be
preserved, or an area that is sought to be damaged or even
destroyed. In a further example, the region of interest can be as
small as a DNA molecule, or as large as an entire tissue sample, a
group of topologically non-contiguous targeted areas in the tissue
sample, which are all to be isolated and/or extracted at a same or
a different time.
[0053] The term "support" as used herein refers to any type of
support in which cellular material can be mounted. One type of
support is a glass slide, although one skilled in the art would
recognize that many materials can provide support for cellular
material.
[0054] The term "photomask" as used herein refers to the blocking
support comprising a blocking region and a light accessible region.
The term "blocking" refers to the ability of an item to hinder the
passage of light through the item. The term "light accessible" as
used herein refers to the ability of an item to allow passage of
light through the item. In some embodiments, the photomask can be
any type of transparent support (light accessible region) having a
non-transparent region (blocking region). In some embodiments, the
transparent support can further be at least in part,
semi-transparent, or translucent and/or include different blocking
portions with different blocking and light accessible capabilities
(e.g. limited to one or more selected wavelengths for one or more
areas of the photomask). In some embodiments, the photomask can be
a physical object (e.g. a glass slide partially covered with
chrome, or a transparency partially covered with ink or other
blocking material). In some embodiments, the photomask can be
purely or partially digital. For example, in some embodiments, the
photomask can comprise a series of instructions to a micro-mirror
array, which operates so that some mirror elements are activated
while others are not. In some of those embodiments, they activate
mirror elements to form a photomask pattern on a sample with
respect to an illumination light reflected onto the sample by the
micro-mirror array. Additional embodiments are encompassed by the
present disclosure wherein a photomask is dynamic photomask, as the
instructions are dynamically defined in addition or in the
alternative to photomask wherein physical blocking material (e.g.
chrome coating) blocks light on a suitable support (e.g. glass
slide).
[0055] The term "blocking region" refers to a region of a blocking
support that functions to block photons (e.g. non-transparent
region blocking photons by absorption). In an embodiment, a blocked
region can be a region of chrome covering the blocking support. One
skilled in the art would appreciate that many metals or other
materials could be used, for example, to block photon exposure. For
example, titanium can be deposited on platinum, covered with a
photosensitive material, and then similarly oxidized to produce a
chemically and mechanically resistant oxide that can serve as a
photon shield for agents damaging to micro-components such as
cellular material, such as UV exposure. In some embodiments, a
material forming a blocking region can also further be a polarizer
as will be understood by a skilled person.
[0056] The term "blocking support" as used herein refers to a
support that can be used to support a blocking region. For example,
a blocking support can be transparent, or in general configured for
allowing passage of a desired lighting in one or more areas where
there is no blocking region.
[0057] Embodiments of the present disclosure can use illumination
light other than UV. For example, a two-photon or other
multi-photon approach would use illumination of larger wavelengths
where the resulting excitation would have an effective wavelength
that is half or a smaller fraction of the illumination light. Such
illumination wavelengths provide increased resolution because the
light intensity is a strong function of the distance from the focal
point, which allows more precise focusing of the illumination
laser.
[0058] Hardwired masking can also be accomplished by other methods,
for example, methods using polarizers and/or polarizer arrays. A
polarizer surface can in principle be chemically or optically
modified to produce contrast between treated and untreated regions
to be used as a mask for the embodiments described herein. Some
polarizers can function on the principle of aligned molecular
structure of polymers, while others are based on metal wire arrays.
In either case, disrupting the structural order in chosen regions,
for example by heating or melting, would make such regions lose
their polarizer property, which would produce the desired contrast
between the regions of interest. Any hardwired method used produces
a sample where the only remaining DNA and/or other biological
material of interest provides the sample of interest. Thus, the
destroyed and/or other biological material of interest does not
contribute noise to the signal.
[0059] In an embodiment, cellular material can be micro-isolated by
lamination of unwanted areas.
[0060] In an embodiment, micro-isolation by lamination provides
mutual isolation of non-contiguous regions of interest, while
preventing the material from contiguous or non-contiguous unwanted
regions from interfering with subsequent reactions. In some of
these embodiments, unwanted regions (e.g. DNA or proteins of
interest) can be destroyed in subsequent processing of the
biological component of interest. In other embodiments, unwanted
regions do not necessarily need to be damaged. In particular, in
one embodiment DNA or other regions of interest (damaged or not)
can be locked inside the laminate thus minimizing the possibility
that contamination of the region of interest can occur. In an
embodiment, a lamination approach further allows that each area of
interest can be contacted with additional microfluidics for
individual addressability and individual extraction. In an
embodiment, the nature of the photomasking (hardwired or dynamic)
is orthogonal to the choice of isolation method (e.g. DNA damaging
or tissue lamination). In some of those embodiments, both
photomasking methods are compatible with both isolation
methods.
[0061] In an embodiment, after identifying desired cells, a
suitable lithography mask can be generated to protect the contents
of desired cells. In some of those embodiments, biological material
such as DNA or protein from the protected cells can be used in a
number of downstream applications including, but not limited to,
DNA sequencing, protein analysis, etc. The purity of such specimens
will greatly enhance the value and information of downstream
applications. In some of these embodiments, the lamination method
does not ensure destruction of unwanted biological material, but
still maximizes survival of the wanted biological material, because
the latter is protected by the blocked regions of the photomask,
due to the requirements of the negative photoresist (see e.g. FIG.
2).
[0062] In an embodiment, the lamination-based method of
micro-isolation relies on the fact that DNA and many other
biological materials in tissue slice samples are neither damaged
nor removed by organic solvents (e.g. ethylene, acetone, xylene).
Thus, it is possible to produce a layer of photocurable material
directly onto a tissue slice containing cellular material, cure in
situ by photoexposure with a desirable mask pattern, and then
remove the uncured sections with organic solvents without damaging
the biological materials of interest. In those embodiments,
photolithographic masking can thus be viewed as a way to use a
photosensitive material as a microfluidic element, which can be
dynamically defined by optical methods and made to match the
morphology and analytical needs of the particular tissue
sample.
[0063] FIG. 2 is an example of tissue isolation by lamination. (A)
A tissue (280) containing a cell of interest (205) is fixed on a
tissue support (220). (B) A photosensitive material (210) is then
deposited onto the tissue (280). The photosensitive material can be
deposited on the tissue, e.g. by simple application, by spinning
the substance down on a spincoater, by kinetic mounting, or by
using spacers (e.g. microspheres of fixed dimensions) and
mechanical contact with a flat surface. (C) A photomask (240)
comprising a blocking region (245) is then applied onto the
photosensitive material (210). (D) The tissue (280) is then exposed
to UV light (230) through the photomask (240) and the
photosensitive material (210). (E) Photoexposure through the
photomask (240) produces a lithographic pattern (250) inside the
photosensitive material (210). (F) The photomask (240) is removed.
(G) A developer is applied (not shown) to remove non-cured sections
of the photosensitive material, which leaves the areas of interest
(260) open to interaction with the outside world. (H). The cell of
interest (205) is unprotected and subjected to removal (270) for
subsequent biochemical analysis (e.g. extraction or in-situ
measurements) whereas unwanted cells (215) are left
inaccessible.
[0064] Those skilled in the art would see that the described
process utilizes "negative" photosensitive materials
(photosensitive material in which the protected areas are the areas
that get removed), but the same technique can be applied with
"positive" materials as well, by inversion of the mask.
[0065] The term "developer" as used herein refers to a chemical
that reacts with a chemical (e.g. a photosensitive material) that
has been exposed to light.
[0066] The term "lamination" or "to laminate" as used herein refers
to the placement or layering of a material, e.g. a photosensitive
material, over a sample, including but not limited to, a tissue or
cell sample, and the (thermal, photolithographic, or otherwise)
thickening or hardening of the laminating material into a
"laminate," so that the biological components under the laminate
are locked by it and cannot contaminate the biological components
in the non-laminated areas.
[0067] The term "photosensitive material" as used herein refers to
any organic soluble or water soluble material that experiences a
change in solubility in a developer solution when exposed to light,
such as UV light.
[0068] In an embodiment, one type of photosensitive material that
can be used is photoresist. One skilled in the art would recognize
that many different types of photoresist materials can be used such
as negative (SU8) and positive (SPR and AZ) photoresists and
additional photoresist identifiable by a skilled person. One
skilled in the art would further understand that other
photosensitive materials, or other photocurable polymers, can be
used with the embodiments described herein. Any photo-curable
material, which can include, but is not limited to, many types of
polymer, elastomer, or epoxy, can be used as a negative
photosensitive material. Any substance that becomes soluble after
UV exposure can act as a positive photosensitive material, for
example, urethane or Polymethylmethacrilate (PMMA) and additional
materials identifiable by a skilled person.
[0069] The use of a negative photosensitive material is shown in
FIG. 2. The "dark" areas of the mask are designed or programmed to
allow access to the protected areas once the developer is applied
because negative photoresists use organic solvent developers, which
neither damage nor extract DNA and RNA during the development
process, because the DNA and RNA are charged and hydrophilic.
Subsequently, the DNA and RNA can be extracted or analyzed in situ
in aqueous solutions.
[0070] The use of positive photosensitive materials causes the
UV-exposed photosensitive material to be soluble in developer, such
as an aqueous alkaline developer, so that areas not protected by
the mask would be open to interaction (not shown). This allows
hydrophobic molecules (e.g. hydrophobic lipid-soluble proteins) to
be extracted by organic solvents that do not damage the positive
photoresist coating of the unwanted areas.
[0071] One skilled in the art would recognize that DNA and other
biological material can be damaged by UV light. Nonetheless, one
skilled in the art would also recognize that DNA most strongly
absorbs light with a wavelength below about 320 nm, while many
photosensitive materials are activated at higher wavelengths, for
example, at 400 nm. Although some light processing systems use a
wide wavelength range, it can be appreciated that cut-off filters
or other means that are commonly known can be used to limit the
excitation light to wavelengths that are too long to damage DNA but
are short enough to expose the photoresist, which can include, but
is not limited to, wavelengths between about 350 nm and about 400
nm as will be understood by a skilled person.
[0072] Photoexposure can occur by any means known in the art, which
includes, but is not limited to, UV exposure, light emitting
diodes, or photonic crystal devices, or alternatively or in
addition, reflected onto the sample by micro-mirror arrays.
[0073] FIG. 3 shows from a top view the same process of tissue
isolation by lamination as FIG. 2. (A) Clusters (310) of potential
cancer cells within a tissue sample (320) are selected. The
clusters provide a plurality of cells, each of which are targeted
in a manner described in FIG. 2. (B) The selection is reflected in
a photomask (either hardwired or dynamically defined) of black
spots (330). (C) A photosensitive material (340) is deposited onto
the tissue sample (320) and a tissue support (not shown); the
photomask (330) is aligned on top, and UV light (not shown) is
directed through the photomask (330) to expose the photosensitive
material (340) over unprotected unwanted regions. (350) (D) The
tissue slide (not shown) is treated (e.g. with a developer), which
removes unexposed photosensitive material above the areas of
interest. Defined access wells (370) in the photosensitive material
(340) ensure that only the wanted areas can be extracted with
suitable methods (e.g. chemically) for further analysis.
[0074] The term "well" or "wells" or "access wells" or "defined
access wells" as used herein describes an area around a tissue
region of interest that provides access to that particular region
of interest.
[0075] In an embodiment, the tissue lamination approach is coupled
with microfluidic devices placed on top of the photosensitive
material in order to individually analyze regions of interest.
[0076] A photolithographically masked tissue containing cellular
material can be integrated with a microfluidic chip (e.g.
consisting of a series of chambers matching the mask) which can be
fabricated in situ or separately. The chip can be used to extract
the subsamples of the selected area. The chip can also be used to
supply reagents for in-situ analysis (e.g. immunoassay, PCR,
RT-PCR), which allows unequaled flexibility and parallelism in the
microfluidic dynamic selection of tissue areas of interest for each
individual slide.
[0077] As shown in FIG. 4, access wells (410) defined in a
photosensitive material (440) over cells of interest (430) placed
on a tissue support (450) can be accessed microfluidically by
producing and aligning a chambered microfluidic device (e.g. a
microfluidic chip) (420) comprising channels (470) having an input
(480) and an output (490).
[0078] As described herein, the term "microfluidic chip" or "chip"
as used herein refers to at least one substrate having microfluidic
structures contained therein or thereon. For example in an
embodiment, the chips can be one-layer, e.g. made of silicone,
where horizontal channels are confined in a single 2-D plane, with
vertical channels only for input/output operations. The chips can
also be multi-layer devices [ref. 2], where each layer of the
material contains its own network of channels. Such networks can be
connected with vertical connecting channels called "vias" [ref. 3].
The chip can contain valves, or a plurality of valves and arrays of
valves. One skilled in the art would appreciate that any chip that
can be integrated with microfluidic wells defined in photosensitive
material to allow for highly specific extraction of desired cells
can be used.
[0079] The term "channel" or "channels" or "chamber" or "chambers"
as used herein refers to a pathway formed in or through a medium
that allows for the movement of fluids, such as liquids and gases.
The term "input" or "inputs" as defined herein is intended to refer
to areas of the microfluidic device where materials (e.g.
proteolysis agents, gases, other liquids) can be introduced through
the chambers to the regions of interest. The inputs further allow
introduction of materials to multiple areas of interest through
channels connected to the chambers.
[0080] In various embodiments, a chip herein described can provide
a multitude of analytical functions. The term "analyze" and
"analytical" refer to activities related to process of the
microscale component at issue for the purpose of detecting
information related to the component. For example, in embodiments
where the microscale component is formed by biological material,
analytical functions comprise activities directed to process the
biological material to identify information concerning and/or
originating from the material which are identifiable by a skilled
person. For example, in an embodiment, the integrated chip can
uptake micro-isolated cells, lyse such cells, capture DNA, RNA,
and/or other biological materials by specific or general
hybridization assays to magnetic nanoparticles, which can then be
extracted from the chip for traditional PCR analysis off-chip. In
an embodiment, the chip can also uptake the micro-isolated cells,
lyse such cells, and perform PCR on-chip. In some of those
embodiments, on-chip PCR allows subsequent detection of specific
genes or mutations by molecular beacons or on-chip specific
hybridization arrays. In an embodiment, the chip can also uptake a
biological components such as micro-isolated cells, lyse such
cells, and then perform immunoassays on intracellular proteins,
e.g. for proteomics-level expression analysis. In an embodiment,
the chip can also be used to extract the cell regions
micro-isolated by the lamination techniques described herein,
separate the clusters into single cells, then feed each cell into a
single-cell analysis device, e.g. for DNA sequencing, mutation
detection, or gene expression analysis. Such devices as the
enumerated examples can be used in fundamental research, and can
also be adapted as biomedical diagnostic tools, e.g. in oncology
and pathology. Additional variations of these devices, wherein same
or further functionalities are added or combined are encompassed by
the present disclosure as would be understood by a skilled
person.
[0081] The term `output" or "outputs" as defined herein is intended
to refer to areas where the cellular material from the regions of
interest can be extracted for further analysis. Input/output
operations can be conducted by microfluidics, thus preventing
contamination, waiting for diffusion, and excessive dilution of the
sample. Also, the chip itself can have analytical function, e.g.
microfluidic immunoassays or microfluidic PCR and RT-PCR.
[0082] In some embodiments, the described integration of tissue
lamination micro-isolation with microfluidic devices allows the
leveraging of the advantages of microfluidic handling (e.g. micro
scale, fast diffusion, parallelism, integrated mechanical and/or
chemical functionalities) with the advantages of the described
micro-isolation technique (e.g. high specificity, parallelism, low
cost, flexibility, and/or dynamic masking). In some of those
embodiments, this architecture provides flexible sample-specific
interface between the unique patterns of the particular sample
morphology and the hardwired architectures of conventional
microfluidic devices.
[0083] FIG. 5A shows a top view demonstrating a hardwired
multi-chambered approach wherein regions of interest (from FIG. 3)
are connected via channels. A laminated tissue sample (550) is
integrated with a chambered microfluidic device (as shown in FIG.
4), whose channels (510) connect to chambers (as described in FIG.
4) over the laminated tissue's cellular areas of interest (540). An
input (520) allows introduction of components necessary to collect
extracts into a single output (530). FIG. 5A shows a single input
and output, although multiple inputs and outputs are possible. In
this particular approach, the entire chip is customized to the
extraction needs of the particular sample, although as described
herein, multiple approaches are possible.
[0084] In an embodiment, the lamination is customized to the
particular sample, but the rest of the microfluidic device can be
standardized and thus used universally with any and all tissue
samples. As used herein, a universal microfluidic device is a
device that can be standardized and used with multiple tissue
samples that have been selectively or non-selectively isolated. For
example FIG. 5B shows a top view demonstrating a standardized
multi-chambered approach. In this approach, a laminated tissue
(550) is integrated with a chambered microfluidic device (as shown
in FIG. 4), whose standardized matrix of channels (560) connect to
chambers (as described in FIG. 4) over the laminated tissue's
cellular areas of interest (540). An input (520) allows
introduction of components necessary to collect extracts into a
single output (530). FIG. 5B shows a single input and output,
although multiple inputs and outputs are possible. Those skilled in
the art would appreciated that a standardized multi-chambered chip
can be configured in one of many possible ways in order to allow
microfluidic channels to cover a tissue surface adequately in order
to extract desired cells from randomly distributed locations. For
example, the grid can be rectangular as shown in FIG. 5B, or a
binary tree of parallel channels. In addition, a universal
extractor device can be combined with a sample-specific
micro-isolation technique. In embodiments, wherein the spacing in a
grid or mesh of channels is not bigger than the expected size of
wanted clusters, a fluidic connection between one or more wanted
cluster with at least one of the extraction channels can be
performed. Thus in some of those embodiments a universal extractor
device can be combined with a sample-specific micro-isolation
technique (e.g. the described lamination method). In some
embodiments, in particular the described combination can have the
benefits of both techniques in the same system--low cost and
universal applicability offered by the standardized chip with the
high specificity and sample-specific customization of the
micro-isolation technique.
[0085] In an embodiment, a tissue is completely encapsulated inside
a microfluidic device, to allow for full surface access.
[0086] Tissue encapsulation captures a tissue of interest between
two separate microfluidic devices, which allow simultaneous access
to two surfaces. If the slice is sufficiently thin, fluidic
communication is ensured through the slice. Such communication
allows more efficient and reliable extraction of desired samples,
as a resuspension liquid can be used to push desired material out
of the tissue matrix. This approach allows extraction of desired
material by microfluidic/hydraulic means without the need for more
aggressive chemical treatments.
[0087] FIG. 6 illustrates an embodiment where complete
encapsulation is shown. A tissue slice (630) covered in a
photosensitive material both above (620) and below (625) the tissue
slice and defined by access wells both above (660) and below (665)
the tissue slice that can be integrated with microfluidic devices
(e.g. microfluidic chips) positioned above (610) and below (615)
the cells of interest and having chambers from both above (670) and
below (680) the tissue slice. This architecture allows a more
efficient input (640) and output (650), while an area of contact
(690) can be doubled for better access to the cells of interest
(695).
[0088] In an embodiment, tissue encapsulation allows the use of 3D
polymerization for in-situ chip construction around the 3D tissue
sample. This can be done, e.g. by using direct laser writing and 3D
rastering to build the desired architectures such that the monomer
material for the chip can be spread thick over the tissue sample.
The laser can then polymerize the chip material in the desired
shape over the tissue sample. The tissue is completely submerged in
a monomer, while a 3D chip is built around it, thus allowing
microfluidic access to the sample from all directions. In some
embodiments, performed according to this approach, the photocurable
polymer of the chip material itself can be used as a photosensitive
material, wherein a tissue slice can be placed inside the polymer
prior to 3-D photopatterning (e.g. before a 3-D photopatterning
commences).
[0089] In an embodiment, multiple areas of interest are addressed
with individual channels without masking.
[0090] Individually and collectively controlled arrays of
microvalves, which allow the same architecture to address a
customizable subset of chambers of access loci within the matrix,
can provide the ability to match particular regions of interest on
the particular tissue sample. A chip with a dense pore matrix
allows the differential opening of particular pores for a short
time such that only material confluent with the pore would flow
from the sample into the chip for analysis without masking.
[0091] Maskless microfluidic encapsulation is shown in FIG. 7. A
tissue slice (710) is encapsulated in a dense pore matrix
microfluidic device both above (745) and below (750) the tissue.
Individually addressable valves that are closed are shown marked
with an "X" (770) as opposed to valves that are open (760) to allow
flow through the open channel (755). The open valve (760) forces a
pressure drop that ensures input flow (720) and forces material
from a cell (740) through a pore (730) into an output channel (780)
and through an output (790) for analysis.
[0092] An embodiment provides tissue micro-isolation by
microfluidic matrices for parallel analysis of subsamples with
preserved morphological context.
[0093] According to an embodiment, microfluidic matrices with
highly parallel single-cell analysis is based on the combination of
a nanofabricated microfluidic matrix and a tissue section. This
allows a matrix of millions of microfluidic wells to be filled with
biochemical reagents and contacted to a tissue section deposited on
a support. For example, each well can contain Proteinase K and
sequencing reagents. The Proteinase K digests the tissue and
releases the contents of each cell into its adjoining well, where
they mix with the PCR reagents by diffusion. The entire system is
contacted to a thermally controlled aluminum plate, to perform
standard or isothermal PCR. Mutant genes can be amplified by
appropriate selection of primers and reported by fluorescent
probes. Signal detection is done by scanning the entire slide on a
fluorescence scanner or by fluorescence microscopy performed one
sector at a time followed by digital assembly. The result is a
highly parallelized single-cell (genetic) analysis of the entire
tissue.
[0094] An exemplary matrix of microfluidic wells according to an
embodiment herein described is illustrated in FIG. 8, which shows a
top view of access wells (810) inside a matrix (820) (built in e.g.
glass silicon, or silicon-on-insulator). Access wells (810) can be
defined, for example, by spreading a photoreactive material on a
substrate, exposing the photoreactive material to UV light through
a photomask, developing the photoreactive material, and etching the
exposed areas. Following removal of the photoreactive material, the
result is defined access wells in the substrate with the same
geometry as the photomask. The term "dense-pore matrix" as used
herein refers to a matrix having dense pores.
[0095] FIG. 9 shows an exemplary illustration of how a matrix of
microfluidic wells can provide access to individual cell nuclei for
independent reactions. Microfluidic access wells (910) are defined
in a substrate (920) (e.g. glass or silicon). Next, the access
wells (910) are filled with a fluid mixture (930) containing
digestion and reaction agents (e.g. Polymerase Chain Reaction (PCR)
reagents, and fluorescent probes). Next, evaporation is performed
to uniformly decrease the fluid mixture level (940), leaving space
at the top (945) of the microfluidic access wells. Next, a tissue
support (950) is aligned on top with a tissue slice (960) facing
downward. Next, an assembled construct (955) is clamped together
and vertically turned over, allowing the fluid mixture to flow by
gravity and cover (975) the tissue. Next, the digestion agents
contained in the fluid mixture break down the tissue (965),
releasing the cellular contents into the microfluidic access wells
(910). The reaction reagents within the fluid mixture complete the
reactions and fluorescent probes (980) reveal results.
[0096] In an embodiment, releasing the cellular content of the
tissue can be performed by digestion of the tissue performed using
Proteinase K. or other techniques of chemical digestion such as the
ones described in [ref. 4,5] which allow multiple analysis of
different molecules in the cellular component. For example, in an
embodiment techniques can be used that allow immunoassay analysis
of the extracts as well as DNA and protein analysis at various
scales as it will be understandable by a skilled person.
[0097] In several embodiments, the nanomatrix technique herein
described can be modified in view of the specific reagents,
biological component, desired result and experimental design as
will be understood by a skilled person. For example, in an
embodiment, a matrix of microfluidic wells can provide access to
individual cell nuclei where a two-step process allows separate
biochemical reactions to occur. By way of example, FIG. 10 shows a
process in which cells can be digested with Proteinase K prior to a
PCR reaction. In the illustration of FIG. 10 tissue (1030) is
placed atop of a support (1010) and a photosensitive material (e.g.
negative photoresist) (1020) covers the tissue (Panel A). Next, a
photomask (1050) having--blocking regions (1055) is placed over the
photosensitive material and exposed to UV light (1040) (Panel B).
Next, the photomask is removed and an organic solvent developer
removes the photosensitive material from the unprotected areas,
leaving defined access wells (1060) (Panel C). The defined access
wells are filled with solution containing Proteinase K (1065)
(Panel D), which digests exposed tissue (1070) and releases the DNA
into the solution in each well (Panel E). Heating deactivates the
Proteinase K and lyophilizes DNA in place in each respective well
(1075) (Panel F). Next, a corresponding matrix of wells is etched
in a well support (e.g. silicon, glass, or silicon-on-insulator)
(1080), which is filled a solution containing PCR reagents (1085)
(Panel G). The assembly is then mechanically secured together (e.g.
clamped) providing water-tightness between compartments, and then
turned over, allowing the PCR solution to resuspend the lyophilate
within each well (1090) (Panel H). PCR can proceed simultaneously
yet separately, in which fluorescent probes (1095) reveal results
of the reaction (see starbursts Panel I). Data acquisition can be
performed e.g. on a fluorescence scanner or by an optical
fluorescence microscope, where the wells are optically accessed by
the side of the glass slide in panel I.
[0098] In the embodiment exemplified in FIG. 10, when the surface
density and size of the wells are correctly chosen, most wells will
adjoin one and only one cell. In some embodiments, wherein the
biological component is formed by cells, an expected optimal well
size is about the same as the size of a mammalian cell (.about.20
.mu.m). However, in those embodiments, one skilled in the art would
recognize that the size and spacing of the wells can be optimized
to ensure that the overwhelming majority of wells contain just one
cell. This would maximize purity of the sample in each well, and
thus maximize specificity and reduce noise in an analytical
determination.
[0099] In an embodiment, a maskless microisolation apparatus
methods and systems are described that are configured according to
the microscale component of interest and experimental design and do
not require (although they could include) photomasks and/or
photosensitive material. For example, in some of those embodiments,
a maskless microisolation apparatus can have randomly placed access
wells that are configured relative to the biological material of
interest and the experimental design. In particular, in embodiments
where the biological material is comprises cells and the
experimental design is directed to isolate and/or analyze
individual cells, a dense porous "honeycomb" arrangement of
predetermined access wells can be used in a device, methods or
systems herein described such that the pre-existing access wells
can be placed accordingly over cellular regions according to the
specific analysis of choice (e.g. to perform protein and/or DNA
analysis of regions of interest selected).
[0100] In some embodiments, the configuration of a matrix of access
wells herein described is not limited to desirable areas alone. In
some of those embodiments, some or all wells can be analyzed
simultaneously but separately, so that no predetermined regions are
necessary. Accordingly, in an embodiment, of devices methods and
systems herein described a photomask can be designed to single out
only the areas of interest and/or to include a repeating regular or
irregular geometric pattern of choice (e.g. circles, squares, or
hexagons in rectangular, checkered, or honeycomb formation) of
appropriately chosen size and spacing, e.g. to contain only one
microscale component or portion thereof (e.g. one cell per
well).
[0101] In several embodiments, wells of a masked or maskless matrix
of access can include one or more reaction mixtures. A reaction
mixture can be any mixture containing components necessary for a
biochemical reaction to occur. Reaction mixtures can include, but
are not limited to, components necessary for PCR, real-time PCR,
RT-PCR, flow cytometry, fluorescent labeling, FRET, DNA sequencing,
protein-protein interaction assays, immunoassays, protein-nucleic
acid assays, and any other biological reaction known in the
art.
[0102] A matrix of microfluidic wells allows incomparable
parallelism in extracting the sequencing information while
preserving the morphological and contextual information from the
tissue sample. It enables large-scale mapping of two-dimensional
spatial distribution of mutations across a tumor section. If such
maps are made of consecutive sections of the same tumor, a
three-dimensional distribution of mutations within a tumor can be
digitally assembled. Furthermore, such 3D maps can be generated for
analogous tumors in multiple test animals at different temporal
points of tumor evolution. Thus, the temporal evolution of a 3D
distribution of mutations can be assembled.
[0103] In many embodiments, the optimal well size is the same size
as a mammalian cell (approximately 20 .mu.m), although one skilled
in the art will recognize that different well sizes can be used for
different applications.
[0104] In an embodiment, an active array of masking material
replaces a physical mask with micro-mirror arrays. As illustrated
in FIG. 11, which is a dynamic process allows the targeted
positioning of tissue selection. First, cells and/or tissue (1160)
are shown to an operator and a camera (1110) takes an image of
cells and/or tissue through an adjustable mirror (1120), a Digital
Light Processing (DLP) mirror (1130) and a photosensitive material
(1140). The image shows the cells and/or tissue (1160) placed on a
tissue support (1150). Upon observation of the image, an area of
interest is selected by programmable patterning the DLP mirror
(1130). Second, the adjustable mirror (1120) is adapted to be
positioned accordingly so that UV light from a lamp (1170) can be
directed through a long-pass filter (1180) and through the
programmed DLP mirror (1130) onto the photosensitive material
(1140) and directed to destroy the DNA of the cells in the region
of interest (1160).
[0105] A long-pass filter indicates a device that operates to allow
all light coming from the UV light having a wavelength above a
certain value, e.g. about 350 nm. Long-pass filters are standard
optical elements known to people skilled in the art. The particular
long-pass filter suitable for the embodiment exemplified in FIG. 11
is configured to ensure that virtually all light coming from the
illumination source has a wavelength above the cut-off value of
about 350 nm. The usual structure of long pass filters is a Bragg
stack of layers of dielectric materials with carefully controlled
thicknesses. The thickness and refractive index of each layer sets
up destructive interference for a narrow band of wavelengths that
are meant to be stopped. Making a stack of such layers ensures that
a wider cumulative range of wavelengths is stopped by the filter.
In this particular case, the cut-off value is 350 nm, because
wavelengths above it are too long to damage DNA when DNA is chosen
as microscale component of interest, but short enough to expose the
photoresist correctly. In the exemplary system of FIG. 11 the long
pass filter is for the tissue lamination method, which necessitates
the exposure of the photoresist which becomes the laminate. People
skilled in the art (e.g. optics and engineering) understand all the
possible variations of long-pass filters in devices, methods and
filters herein described.
[0106] The term "digital image" refers to an image generated by a
computer or other suitable electronic device. In an embodiment, a
digital image can be provided, for example, by a set of
instructions in software on a computer controlling the optical
hardware. In an embodiment, the image can be a 2-D image. In an
embodiment, the digital image can also be 3-D, e.g. in embodiments,
when a device is provided for tissue encapsulation by 3-D rastering
of the photocuring illumination, as described herein. A "digital
mask" refers to the masking of a region of interest of cellular
material based on a digital image as opposed to a physical mask,
e.g. a chrome mask.
[0107] In an embodiment, tissues can be micro-isolated without the
need for a physical mask for UV shielding. Instead, a UV laser,
e.g. Heidelberg DWL66.RTM., can be focused directly onto the
necessary spots in the photoresist on top of the tissue for
lamination or in the tissue itself for destruction of biological
material in the tissue such as DNA. The resolution can be 2 microns
or better, and the desired cells can be skipped in the rastering
process. Different laser heads can be used for the different
regions of the slide. For example, appropriate software can guide
the laser with a 2-micron head around the immediate vicinity of the
cells of interest, while the rest of the slide area is exposed by
broader strokes, e.g. with a 30-micron head.
[0108] In an embodiment, active masking arrays utilize LCDs (liquid
crystal displays). An illumination light would be polarized along
one axis, while the LCD elements would be polarized along one axis
to disallow and another axis to allow the passage of the UV light.
The cells of interest are protected by having the corresponding
elements in the array be perpendicularly aligned, while the
unwanted cells would have their elements aligned in parallel with
incident UV illumination.
[0109] In an embodiment, dynamic masking using fiber optics can be
produced by arrays of LEDs (light emitting diodes). This approach
allows the utilization of increasing smaller wavelengths as current
technology builds LEDs at smaller wavelengths. Individually
addressable elements can be built at the microscale, producing
macro-sized arrays of thousands or millions of individually
addressable LED elements. Such individually addressable LED
elements allow respective areas on the photosensitive material to
be individually photopolymerized to provide the tissue lamination
methods described herein.
[0110] In an embodiment, fiber optics is used in a way similar to
intensified CCD cameras. Bundles of fiber optic cables are arranged
to produce an active array of illuminators. This bundle can be
coupled to an LCD array at the input of illumination light, while
the output is coupled to the tissue slide. Then the output size of
each fiber can be made smaller than the input size, producing both
light transduction and size reduction.
[0111] In an embodiment, active masking array uses photonic
circuitry to define dynamic optical arrays. A photonic circuit can
in principle be built to generate an array of individually
addressable optical outputs. When positioned over a tissue slide,
the individual addressability of optical outputs provides the
capability for individual UV exposure of tissue areas that are
chosen to be discarded.
[0112] In an embodiment, active masking array uses photonic
circuitry to define dynamic optical arrays. A photonic circuit can
in principle be built to generate an array of individually
addressable optical outputs. When positioned over a tissue slide,
the individual addressability of optical outputs provides the
capability for individual UV exposure of tissue areas that are
chosen to be discarded.
[0113] In an embodiment, active masking array uses micro- and
nano-lasers for dynamic arrays. These lasers can be fabricated in
arrays, where each laser is still individually addressable.
Software and electrical outputs control which laser is active, e.g.
by electrical pumping or electrical control of polarization
shielding against pumping illumination. Microfluidic devices can
further follow a combination of morphological and functional
customization. For example, in the particular technique of
multi-layer elastomer microfluidics, the elastomeric layer that
contacts the sample can have a photolithographically defined
morphology that matches the regions of interest in the tissue
sample, while other layers can follow a matrix or array structure
built for functional programmability As an example, see FIG. 5A,
where a uniform matrix of channels overlays the wells of the
regions of interest, ensuring extraction. Thus the extraction
matrix can be standardized and thus produced inexpensively, while
the laminating layer is kept specific to the particular tissue
sample. One skilled in the art would appreciate that such a
combination is clearly not limited to extraction alone, because a
device of any processing or analytical function can be integrated
with a sample-specific micro-isolation stage. In some embodiments,
the specific functionality or purpose of the device can be combined
with the high specificity and sample-specific customization offered
by the described micro-isolation techniques. In some of those
embodiments, this approach provides a low cost of standardization
with a high specificity of sample-specific extraction
[0114] In some embodiments, the methods and devices described
herein overcome various problems e.g. by providing a general
microfluidic bottoms-up sample-specific customization method. Such
a method naturally leads to rapid, parallelized, and highly
specific micro-isolation of the desired cell subpopulation (e.g.
cancer cells from a tumor) directly from tissue samples. In
particular, in some embodiments, devices, methods and systems
herein described preserve the structural integrity of most of the
tissue, thus preserving its inherent morphological information. In
some embodiments, a PCR nanomatrix technique is described that
allows parallelized large-scale high-throughput genomic mappings
across the tissue sample.
[0115] In several embodiments, methods herein described allow
convenient application in a number of methods to separate normal
from cancer cells (See Ref. 1). Methods herein described are not
necessarily dependent on use of fresh tissues, and are applicable
to most human cancer specimens, which are usually fixed in formalin
and paraffin-embedded. In some embodiments, devices methods and
systems herein described allow to process wanted cells (e.g. cancer
cells) minimizing the background noise of unwanted cells (e.g. non
cancerous cells). In some of those embodiments, devices, methods
and systems herein described allow a less expensive and less
labor-intensive of certain methods of the art where a trained
operator must manually identify and then individually address each
cell to be analyzed using an expensive and complex laser microscopy
system.
[0116] In some embodiments, devices, methods and systems herein
described can be performed in microfluidics. In particular, in
certain embodiments, microfluidics is the micro-manipulation of
fluids, and can be integrated with biochemical applications for
microscale analyses of cells with further implementation
identifiable by a skilled person.
[0117] In several embodiments, devices methods and systems herein
described are configured to combine sample specific microisolation
with standardized processing and/or analysis chip. Microisolation
can be used to control and in particular increases up to maximize
specificity of selection. Use of a standardized processing and/or
analysis chip can provide an economic and industry friendly way to
materialize the technique in practice.
[0118] According to several embodiments, microisolation devices
methods and systems are described wherein: cellular material or
other microscale component is positioned on a substrate; a sample
specific micro isolation method herein described is applied to the
cellular material or other component to obtain a substrate
presenting the cellular material, a microfluidic universal device
is connected with or contacted to the substrate presenting the
cellular material, the universal device being analysis specific and
sample non specific; and a microisolation methods is used to
extract the material from the desired regions on the particular
sample and the universal device is used to collect and/or analyze
the extracted sample
[0119] Although any methods and materials similar or equivalent to
those described herein can be used in the practice for testing of
the products, methods and system of the present disclosure,
exemplary appropriate materials and methods are described herein as
examples.
EXAMPLES
[0120] The devices, methods and systems herein described are
further illustrated in the following examples, which are provided
by way of illustration and are not intended to be limiting.
[0121] In particular, the following examples illustrate exemplary
hardwired and laminated devices and related methods and systems. A
person skilled in the art will appreciate the applicability and the
necessary modifications to adapt the features described in detail
in the present section, to additional solutions, devices,
arrangements, methods and systems according to embodiments of the
present disclosure.
Example 1
Hardwired Masking Using Photolithographically Defined Chrome
Masks
[0122] Cells of interest are identified using a microscopic
computerized image of the tissue slide and appropriate custom
software, which converts the selection into a digital image. The
digital mask is fed into a direct laser writer, the Heidelberg
DWL66.RTM., which transfers a digital mask onto the "positive"
photosensitive material deposited on top of a chrome-covered plate,
by direct writing with a resolution of 2 microns. The plate is then
developed to remove the exposed photoresist, which leaves the
exposed areas susceptible to chemical etching. The etching removes
the unprotected chrome, and the rest of the photosensitive material
is removed, e.g. by overdevelopment or exposure to a strongly
alkaline solution. The remaining chrome pattern is quickly oxidized
by atmospheric exposure, typically within 30 sec, which produces a
chrome mask specific to the particular tissue sample. An exemplary
hardwired masking using photolithographically defined chrome masks
is illustrated in FIG. 1.
Example 2
Application of Tissue Lamination Approach to Adrenal Gland Tissue
Slides
[0123] The tissue lamination technique was applied to adrenal gland
tissue slides prepared by routine clinical methods. Photoresist
SU8-2005 was deposited onto a tissue by spinning the slide on a
WS-400B-6NNP/LITE spincoater. The slide was pre-baked at 65.degree.
C. Next, the slide was exposed to UV filtered with a 368-nm
high-pass filter at an MA-6 mask aligner, through a chrome-on-glass
mask bearing the pattern of a USAF 1951 resolution chart. The chart
was chosen as a mask to provide an easily identifiable reference in
terms of size of the defined features in photoresist on top of the
tissue.
[0124] The slide was then post-exposure baked at 95.degree. C. and
developed in SU8 developer, which contains organic solvents.
Finally, each slide was characterized on a profilometer (Alpha-Step
500) to measure the height of the fabricated features. Tissue slice
thickness was measured up to 5 .mu.m tissue, while the photoresist
layer was .about.7 .mu.m high. FIG. 12 shows the "windows" defined
in the photoresist. The dimension defined on the tissue was
.about.12 .mu.m width, which is smaller than a typical mammalian
cell (20 .mu.m).
[0125] The tissue section is essentially unchanged after
photolithography (see FIG. 12A), except for the discoloration of
unmasked areas due to the leeching of the hematoxylin and eosin
staining by the organic solvent of the photoresist developer. Some
of this discoloration extends under the mask (see FIG. 12A, 12D),
likely because the organic solvent is a very small molecule that
can penetrate through the tissue to reach the masked areas. An
alternative explanation is that the dye can diffuse out into the
wells during the digestion and extraction process, leaving the
areas of immediate proximity to the wells. It is noted however that
the nuclei remain in the unwanted areas but are extracted from the
wanted areas--therefore, the unwanted DNA cannot diffuse out the
way the dye can.
[0126] The laminated areas of the tissue appear far brighter than
the exposed tissue (see FIG. 12A) because the refractive index of
the photoresist matches the refractive index of the tissue better
than air, while the photoresist also mechanically smoothens the
surface roughness of the tissue. Thus surface light scattering and
refractive divergence are significantly reduced, and the intensity
of the detected light is increased over the laminated areas, in
comparison to non-laminated tissue.
[0127] To extract the exposed tissue, a drop of extractions
solution (10 mM Tris-HCl, 2 mM EDTA, pH 8.0, with 10 mg/ml
Proteinase K) is placed on top of the masked slide and incubated at
56.degree. C. in a humidity chamber. The Proteinase K digests the
tissue, releasing the DNA into solution, which is then suitable for
amplification by PCR. The slide after digestion (FIGS. 12B, 12C,
12D) shows the removal, with sharp boundaries defined by the mask,
because Proteinase K is a large protein and thus unable to diffuse
through the tissue. As seen in FIG. 12B, the digestion is less
efficient with smaller features, because the photoresist is
hydrophobic and so surface tension works as counter pressure
against the entry of the extraction solution into the smaller
holes.
[0128] The embodiments and examples set forth above are provided to
give those of ordinary skill in the art a complete disclosure and
description of how to make and use the embodiments of the devices,
systems and methods of the disclosure, and are not intended to
limit the scope of what the inventors regard as their
disclosure.
[0129] The examples set forth above are provided to give those of
ordinary skill in the art a complete disclosure and description of
how to make and use the embodiments of the arrangements, devices,
compositions, systems and methods of the disclosure, and are not
intended to limit the scope of what the inventors regard as their
disclosure. All patents and publications mentioned in the
specification are indicative of the levels of skill of those
skilled in the art to which the disclosure pertains.
[0130] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background,
Summary, Detailed Description, and Examples is hereby incorporated
herein by reference. All references cited in this disclosure are
incorporated by reference to the same extent as if each reference
had been incorporated by reference in its entirety individually.
However, if any inconsistency arises between a cited reference and
the present disclosure, the present disclosure takes
precedence.
[0131] The terms and expressions which have been employed herein
are used as terms of description and not of limitation, and there
is no intention in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the disclosure claimed Thus, it
should be understood that although the disclosure has been
specifically disclosed by preferred embodiments, exemplary
embodiments and optional features, modification and variation of
the concepts herein disclosed can be resorted to by those skilled
in the art, and that such modifications and variations are
considered to be within the scope of this disclosure as defined by
the appended claims.
[0132] It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting. 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. The term "plurality" includes two or more referents
unless the content clearly dictates otherwise. Unless defined
otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which the disclosure pertains.
[0133] When a Markush group or other grouping is used herein, all
individual members of the group and all combinations and possible
subcombinations of the group are intended to be individually
included in the disclosure. Every combination of components or
materials described or exemplified herein can be used to practice
the disclosure, unless otherwise stated. One of ordinary skill in
the art will appreciate that methods, device elements, and
materials other than those specifically exemplified can be employed
in the practice of the disclosure without resort to undue
experimentation. All art-known functional equivalents, of any such
methods, device elements, and materials are intended to be included
in this disclosure. Whenever a range is given in the specification,
for example, a temperature range, a frequency range, a time range,
or a composition range, all intermediate ranges and all subranges,
as well as, all individual values included in the ranges given are
intended to be included in the disclosure. Any one or more
individual members of a range or group disclosed herein can be
excluded from a claim of this disclosure. The disclosure
illustratively described herein suitably can be practiced in the
absence of any element or elements, limitation or limitations which
is not specifically disclosed herein.
[0134] A number of embodiments of the disclosure have been
described. The specific embodiments provided herein are examples of
useful embodiments of the disclosure and it will be apparent to one
skilled in the art that the disclosure can be carried out using a
large number of variations of the devices, device components,
methods steps set forth in the present description. As will be
obvious to one of skill in the art, methods and devices useful for
the present methods can include a large number of optional
composition and processing elements and steps.
[0135] In particular, it will be understood that various
modifications may be made without departing from the spirit and
scope of the present disclosure. Accordingly, other embodiments are
within the scope of the following claims.
REFERENCES
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