U.S. patent application number 14/285297 was filed with the patent office on 2015-01-15 for methods for providing and delivering an environmentally sealed chip.
This patent application is currently assigned to Pacific Biosciences of California, Inc.. The applicant listed for this patent is Pacific Biosciences of California, Inc.. Invention is credited to William R. Hanson, Paul Newby.
Application Number | 20150018252 14/285297 |
Document ID | / |
Family ID | 44309250 |
Filed Date | 2015-01-15 |
United States Patent
Application |
20150018252 |
Kind Code |
A1 |
Newby; Paul ; et
al. |
January 15, 2015 |
METHODS FOR PROVIDING AND DELIVERING AN ENVIRONMENTALLY SEALED
CHIP
Abstract
The invention provides methods relating methods for
environmentally sealing, protecting, and providing analysis chips
for processing and analysis. The analysis chips are bonded directly
or indirectly to chip carriers which are held within the chambers
of an environmental packaging strip. The chambers are sealed with a
sealing film such that the chip carriers are extracted using a
piercing tool and an extraction tool.
Inventors: |
Newby; Paul; (Palo Alto,
CA) ; Hanson; William R.; (Mountain View,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pacific Biosciences of California, Inc. |
Menlo Park |
CA |
US |
|
|
Assignee: |
Pacific Biosciences of California,
Inc.
Menlo Park
CA
|
Family ID: |
44309250 |
Appl. No.: |
14/285297 |
Filed: |
May 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12939900 |
Nov 4, 2010 |
8772016 |
|
|
14285297 |
|
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|
|
61261212 |
Nov 13, 2009 |
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Current U.S.
Class: |
506/23 |
Current CPC
Class: |
H01L 21/67356 20130101;
Y10T 428/24331 20150115; B01L 2300/0672 20130101; B01L 3/50855
20130101 |
Class at
Publication: |
506/23 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Claims
1. A method of providing an analysis chip comprising: a) providing
a strip having a plurality of sealed chambers, each chamber having
a bottom, a top, and sidewalls, the top of each chamber sealed with
a sealing film; wherein a chip carrier comprising an analysis chip
is disposed within each chamber; b) piercing the sealing layer of
at least one of the chambers with a piercing tool; and c)
extracting the chip carrier from the chamber with an extraction
tool.
2. The method of claim 1 further comprising transporting the
extracted chip carrier to a holder which holds the chip as reagents
are added to the chip.
3. The method of claim 1 wherein the piercing tool comprises one or
more ridges, each ridge configured to make a cut in the sealing
film.
4. The method of claim 1 wherein the tool has four ridges.
5. The method of claim 1 wherein the piercing tool extends through
the sealing film and pushes down the film to create an opening
through which the chip carrier is extracted.
6. The method of claim 5 wherein the piercing tool has four ridges
such that when the piercing tool extends through the film, four
cuts are made to produce four flaps which are pushed down into the
chamber.
7. The method of claim 1 wherein the top of each chamber is sealed
with a sealing film, and the chip carriers are disposed in the
bottom portion of each chamber whereby the extraction tool extends
through the top of the chamber.
8. The method of claim 7 wherein the chip carriers are disposed
such that the top of each chip carrier is below the level of the
sealing film by a distance that is at least one half of a
cross-sectional dimension of the chamber.
9. The method of claim 7 wherein the chambers have a square or
rectangular cross-sectional profile, and the chip carriers are
disposed such that the top of each chip carrier is below the level
of the sealing film by a distance that is at least one half of the
length of a side of the square or rectangular profile.
10. The method of claim 1 wherein the piercing tool and the
extraction tool are incorporated into a single piercing/extraction
tool.
11. The method of claim 10 wherein the piercing portion of the
piercing tool is inside the single piercing/extraction tool, and
wherein the piercing tool extends to pierce the film, and
subsequently retracts within the single tool to allow the
extraction tool to engage with the chip carrier.
12. The method of claim 7 wherein the chip carrier is attached to
the strip; and the extraction tool grips the chip carrier and
displaces the chip carrier to detach the chip carrier from the
strip.
13. The method of claim 12 wherein the chip carrier is attached to
the chip through break-away tabs.
14. The method of claim 13 wherein the break-away tabs are molded
tabs.
15. The method of claim 12 wherein the chip carrier is displaced
down by the extraction tool to detach the chip carrier.
16. The method of claim 1 wherein the strip is a linear strip
having from 2 to 12 chambers.
17. The method of claim 1 wherein the sealing film comprises a
foil, a polymer, or a combination thereof.
18. The method of claim 2 wherein the process is carried out on an
automated robot.
19. The method of claim 1 wherein the analysis chip is part of a
chip assembly which is bonded to the to the chip carrier.
20. The method of claim 19 wherein the chip carrier has a top and a
base and the chip assembly is bonded to the base of the chip
carrier whereby the analysis chip extends below the chip
carrier.
21. The method of claim 20 wherein after the chip carrier is
extracted, it is placed in a processing station where reagents are
added, and is subsequently moved to an analysis station wherein an
edges of the analysis chip is used to orient the analysis chip in
the analysis station.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/939,900 filed Nov. 4, 2010 which claims the
benefit of Provisional U.S. Patent Application No. 61/261,212,
filed Nov. 13, 2009; the full disclosure of which is hereby
incorporated by reference in its entireties for all purposes
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Not Applicable.
BACKGROUND OF THE INVENTION
[0003] The measurement of large numbers of chemical species at one
time in a high throughput manner is important in a number of areas
including drug discovery and genomics. Advances in the
understanding of molecular biology and genetics and the promise of
biotechnology have created a need for improved tools. For example,
new information provided by projects such as the Human Genome
Project has created even more demand for faster, higher throughput
methods for sequencing DNA. The tremendous efforts put into
sequencing in the last decade have helped researchers begin to
understand fundamental cell function. These efforts have
accelerated the pace of research and discoveries and have created a
growing need for improved tools for analyzing a large variety of
molecules in addition to DNA. The benefits to mankind in medicine,
agriculture and for the environment, as well as the economic
potential that these fields promise, are driving researchers to
decipher the function of individual genes, molecules and the cells
that contain them. By sequencing an organisms' DNA and analyzing
the molecules that make up its cells, researchers are able to
develop an understanding of the systems and structure that make it
function.
[0004] Optical methods can provide the sensitivity and reliability
required for high throughput analysis. In order to optically
interrogate samples, one must have a means for introducing a sample
into an instrument which is capable of optically engaging the
samples. For example, as described by Eid, et al. Science, 323,
133-138, 2009 an array of optical confinements can be used to carry
out thousands of sequencing reactions simultaneously using a chip
having thousands of optical confinements. One way to introduce such
a sample into an optical instrument is to provide a consumable
element such as a chip which is precisely manufactured for high
throughput optical analysis of a sample introduced onto the chip.
Such a consumable should be environmentally protected from the
point of manufacture to the point where it is brought into contact
with a sample and analyzed in the instrument in order to avoid
contamination. There is a need for packaging systems and for
methods of providing a high-throughput analysis chip that is sealed
for shipping, handling, and storing, yet can be quickly and
reliably removed from the sealed environment for the addition of
samples and reagents for analysis. These are particularly important
for analyses which are carried out by automated, robotic
equipment.
BRIEF SUMMARY OF THE INVENTION
[0005] In one aspect the invention comprises an environmentally
sealed strip having a plurality of chambers, each chamber having a
top, a bottom, and side walls, wherein at least one of the top,
bottom, or side walls of each chamber is sealed with a sealing
film, wherein each chamber has sealed therein a chip carrier,
wherein the chip carrier comprises an analysis chip bonded
thereto.
[0006] In some embodiments the strip is a linear strip having from
2 to 12 chambers. In some embodiments each chip carrier is
reversibly held in place within each chamber. In some embodiments
each chip carrier is held in place through one or more break-away
features that are broken away to extract the chip carrier from the
chamber. In some embodiments the chip carrier is molded into a
strip housing, and the break-away features comprise molded
break-away tabs. In some embodiments each chamber is sealed with a
sealing film, and the chip carriers are disposed in the bottom
portion of each chamber.
[0007] In some embodiments the chip carriers are disposed such that
the top of each chip carrier is below the level of the sealing film
by a distance that is at least one half of a cross-sectional
dimension of the chamber. In some embodiments the chambers have a
square or rectangular cross-sectional profile, and the chip
carriers are disposed such that the top of each chip carrier is
below the level of the sealing film by a distance that is at least
one half of the length of a side of the square or rectangular
profile. In some embodiments each analysis chip is bonded to the
bottom of each chip carrier such that a portion of the chip extends
below the chip carrier to provide for physical access to sides of
the chip. In some embodiments the analysis chip is part of a chip
assembly, and the analysis chip is bonded to the chip carrier via
the chip assembly. In some embodiments the top of each chip carrier
comprises a collar which is capable of being gripped by an
extraction tool.
[0008] In some embodiments the strip further comprises a strip
capping piece on top of the sealing film, such strip capping piece
having an opening corresponding to each chamber. In some
embodiments the strip further comprises a base piece attached to
the bottom of the strip.
[0009] In some embodiments the bottom of the chambers is sealed
with a sealing film, and the base piece is attached below the
sealing film. In some embodiments the sealing film comprises a
foil, a polymer, or a combination thereof. In some embodiments the
one or more side walls of the chambers are transparent or have
transparent portions. In some embodiments the analysis chip is part
of an analysis chip assembly comprising a substantially planar
analysis chip bonded to a well-forming piece, whereby a well is
formed in the assembly allowing for the addition and containment of
liquid reagent in contact with the analysis chip. In some
embodiments the analysis chip assembly is bonded to the chip
carrier with adhesive. In some embodiments the chip comprises an
array of optical confinements. In some embodiments the optical
confinements comprise ZMWs.
[0010] In one aspect the invention comprises a molded part having a
plurality of conduits extending therethrough, each conduit having
an open top, and open bottom, and side walls, wherein within each
conduit a chip carrier having a top and a base is held in the
conduit by break-away tabs, the chip carrier configured to allow
for the bonding of an analysis chip assembly to the base of the
chip carrier, and the tops and bottoms of the conduits configured
to be environmentally sealed.
[0011] In one aspect the invention comprises a chip assembly
comprising two pieces bonded together, the first piece comprising a
square or rectangular fused silica sheet having a cladding layer
with a plurality of optical confinements on its top surface; and
the second piece comprising a fused silica chip having a hole
extending therethrough, whereby the assembly comprises a well into
which a liquid can be added to contact the optical confinements
wherein the well acts to contain the liquid, and wherein the
exposed portion of the chip comprise the bottom of the well.
[0012] In some embodiments the plurality of optical confinements
comprises more than 10,000 optical confinements. In some
embodiments the plurality of optical confinements comprises between
than 10,000 and 10 million optical confinements. In some
embodiments the optical confinements comprise ZMWs.
[0013] In some embodiments the chip comprises a trench which
extends around the perimeter of the bottom of the bottom of the
well to arrest the flow of adhesive used to bond the first piece
and the second piece.
[0014] In one aspect the invention comprises a method of providing
an analysis chip comprising: (a) providing a strip having a
plurality of sealed chambers, each chamber having a bottom, a top,
and sidewalls, the top of each chamber sealed with a sealing film;
wherein a chip carrier comprising an analysis chip is disposed
within each chamber; (b) piercing the sealing layer of at least one
of the chambers with a piercing tool; and (c) extracting the chip
carrier from the chamber with an extraction tool.
[0015] In some embodiments the method further comprises
transporting the extracted chip carrier to a holder which holds the
chip as reagents are added to the chip. In some embodiments the
piercing tool comprises one or more ridges, each ridge configured
to make a cut in the sealing film. In some embodiments the tool has
four ridges.
[0016] In some embodiments the piercing tool extends through the
sealing film and pushes down the film to create an opening through
which the chip carrier is extracted. In some embodiments the
piercing tool has four ridges such that when the piercing tool
extends through the film, four cuts are made to produce four flaps
which are pushed down into the chamber. In some embodiments the top
of each chamber is sealed with a sealing film, and the chip
carriers are disposed in the bottom portion of each chamber whereby
the extraction tool extends through the top of the chamber.
[0017] In some embodiments the chip carriers are disposed such that
the top of each chip carrier is below the level of the sealing film
by a distance that is at least one half of a cross-sectional
dimension of the chamber. In some embodiments the chambers have a
square or rectangular cross-sectional profile, and the chip
carriers are disposed such that the top of each chip carrier is
below the level of the sealing film by a distance that is at least
one half of the length of a side of the square or rectangular
profile.
[0018] In some embodiments the piercing tool and the extraction
tool are incorporated into a single piercing/extraction tool. In
some embodiments the piercing portion of the piercing tool is
inside the single piercing/extraction tool, and wherein the
piercing tool extends to pierce the film, and subsequently retracts
within the single tool to allow the extraction tool to engage with
the chip carrier. In some embodiments the chip carrier is attached
to the strip; and the extraction tool grips the chip carrier and
displaces the chip carrier to detach the chip carrier from the
strip.
[0019] In some embodiments the chip carrier is attached to the chip
through break-away tabs. In some embodiments the break-away tabs
are molded tabs. In some embodiments the chip carrier is displaced
down by the extraction tool to detach the chip carrier. In some
embodiments the strip is a linear strip having from 2 toll
chambers. In some embodiments the sealing film comprises a foil, a
polymer, or a combination thereof.
[0020] In some embodiments the method is carried out on an
automated robot.
[0021] In some embodiments the analysis chip is part of a chip
assembly which is bonded to the to the chip carrier. In some
embodiments wherein the chip carrier has a top and a base and the
chip assembly is bonded to the base of the chip carrier whereby the
analysis chip extends below the chip carrier. In some embodiments,
after the chip carrier is extracted, it is placed in a processing
station where reagents are added, and is subsequently moved to an
analysis station wherein an edges of the analysis chip is used to
orient the analysis chip in the analysis station.
[0022] In one aspect the invention comprises a method for producing
a sealed chip carrying strip comprising; providing a molded part
having a plurality of conduits extending therethrough, each conduit
having an open top, and open bottom, and side walls, wherein within
each conduit is a chip carrier having a top and a base that is held
in the conduit by break-away tabs; attaching a chip assembly
comprising an analysis chip to the base of the chip carrier;
sealing the open bottom and the open top to environmentally seal
the analysis chips within the conduits; wherein at least one of the
open top and open bottom is sealed with a sealing film.
[0023] In some embodiments the open top is sealed with a sealing
film further comprising attaching a strip capping piece, wherein
the strip capping piece comprises openings corresponding to teach
of the conduits. In some embodiments both the open top and open
bottom are sealed with a sealing film, further comprising attaching
a bottom piece which protects the sealing film on the open bottoms
during handling.
[0024] In one aspect the invention comprises an analysis instrument
comprising: (a) a station for receiving a sealed environmental
strip having a plurality of sealed chambers, each chamber having a
bottom, a top, and sidewalls, the top of each chamber sealed with a
sealing film; wherein a chip carrier comprising an analysis chip is
disposed within each chamber; (b) an automated chip extractor
comprising (i) a piercing tool that pierces the sealing layer of at
least one of the chambers; and (ii) an extraction tool that
extracts the chip carrier from the chamber and transports the
analysis chip to an automated processing station; (c) an automated
processing station that prepares the chips for analysis by adding
at least one reagent, and at least one sample to the chip; (d) a
tool for transporting the analysis chip from the processing station
to an analysis station; and (e) an analysis station which holds the
chip in place during analysis.
[0025] In some embodiments the analysis comprises nucleic acid
sequencing.
[0026] In some embodiments the piercing tool comprises one or more
ridges, each ridge configured to make a cut in the sealing film. In
some embodiments the tool has four ridges.
[0027] In some embodiments the piercing tool extends through the
sealing film and pushes down the film to create an opening through
which the chip carrier is extracted. In some embodiments the
piercing tool has four ridges such that when the piercing tool
extends through the film, four cuts are made to produce four flaps
which are pushed down into the chamber. In some embodiments the top
of each chamber is sealed with a sealing film, and the chip
carriers are disposed in the bottom portion of each chamber whereby
the extraction tool extends through the top of the chamber.
[0028] In some embodiments n the piercing tool and the extraction
tool are incorporated into a single piercing/extraction tool. In
some embodiments the piercing portion of the piercing tool is
inside the single piercing/extraction tool, and wherein the
piercing tool extends to pierce the film, and subsequently retracts
within the single tool to allow the extraction tool to engage with
the chip carrier. In some embodiments the chip carrier is attached
to the strip; and the extraction tool grips the chip carrier and
displaces the chip carrier to detach the chip carrier from the
strip. In some embodiments the chip carrier is attached to the chip
through break-away tabs. In some embodiments the break-away tabs
are molded tabs. In some embodiments the chip carrier is displaced
down by the extraction tool to detach the chip carrier. In some
embodiments the strip is a linear strip having from 2 to 12
chambers. In some embodiments the sealing film comprises a foil, a
polymer, or a combination thereof. In some embodiments the analysis
chip is bonded to the chip carrier whereby the analysis chip
extends below the chip carrier. In some embodiments the analysis
chip is placed in the analysis station, one or more edges of the
chip are used to physically position the analysis chip.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a cross-sectional drawing illustrating an
environmentally sealed strip package of the invention.
[0030] FIG. 2 shows an exploded view of parts to be assembled to
produce an environmentally sealed strip.
[0031] FIG. 3 shows an assembled environmentally sealed strip.
[0032] FIG. 4 is a cross-sectional drawing of a molded
strip-housing.
[0033] FIG. 5 shows a cross section of a chip assembly wherein the
chip comprises a trench to arrest the flow of adhesive.
[0034] FIGS. 6(A) to 6(D) illustrate a method of the invention for
extracting a chip from a sealed strip.
[0035] FIGS. 7(A) and 7(B) provide a cross-sectional drawing of a
piercing/extraction tool within a chamber of a strip package
engaging with a chip carrier comprising an analysis chip.
[0036] FIGS. 8(A) and 8(B) provide a cross-sectional drawing
illustrating an extraction tool displacing a chip carrier within
the chamber of a sealed strip to detach the chip carrier.
[0037] FIGS. 9(A) and 9(B) provide a cross-sectional drawing
illustrating the extraction of a chip carrier from the chamber of a
sealed strip.
DETAILED DESCRIPTION OF THE INVENTION
I. General
[0038] The invention provides for the packaging, shipping,
handling, and storage of analysis chips, particularly analysis
chips for use in high-throughput optical systems. The invention
provides environmental packages having a plurality of chambers,
each containing an analysis chip that is sealed from the
environment. The environmental packages of the invention protect
the chips from environmental contamination, and also protect the
chips from damage on handling and shipping. In addition, the
packages of the invention provide for the facile removal of the
analysis chip, in particular with automated tools which can be
driven by a robotic system. The environmental packages allow for
each analysis chip to be individually sealed, such that the
extraction of one analysis chip does not expose the other chips in
the package to the environment.
[0039] The packages of the invention are provided as strips having
multiple chambers. Each chamber is separately environmentally
sealed such that one chip can be removed from its chamber without
compromising the chips in the other chambers. In some cases the
strip is a linear strip having, for example, 4, 6, 8, 9, or 12
chambers, each individually sealed. In some cases, the strip has
the chambers arranged in a radial arrangement. A radial
arrangement, for example with chambers arranged on a circle in a
strip also having a central portion, can be useful for introducing
the chambers into a robotic handling system. The radial arrangement
allows for turning of the strip to move a chamber into position for
interaction with the robotic system. The analysis chips are
generally attached, directly or indirectly, to a chip carrier. The
chip carrier generally has a structure which can immobilize the
chip within the chamber. The chip carrier has a structure which can
engage with an extraction tool, the structure, for example,
providing surfaces which the extraction tool can grip firmly,
allowing for the extraction of the chip from the strip. The chip
carrier can also facilitate transporting the chip from the
packaging strip to other stations wherein subsequent manipulations
including the addition of reagents and sample, and the placement of
the chip comprising the sample into an analysis portion of an
instrument.
[0040] The chambers are sealed in such a manner that the chambers
can effectively be opened to facilitate extraction of the chip
carrier holding the analysis chip. For example, one or more faces
of the chamber is sealed with a sheet or film which can be
punctured by a piercing tool to open the chamber. In one
embodiment, only the top of each of the chambers is sealed with a
sheet or film. The sheet or film is selected to have the physical
strength and integrity to environmentally protect the chip, while
also being susceptible to rupture by a piercing tool.
[0041] The chip carrier which holds the analysis chip is generally
secured within the chamber to prevent excessive movement within the
chamber during handling. The chip carrier can be physically
attached within the chamber. In one preferred embodiment, the chip
carrier is molded into a portion of the strip, e.g. the strip
housing, and is held in place by molded tabs. The tabs are
dimensioned to be strong enough to hold the chip carrier firmly in
place during shipping and handling, but to be weak enough that they
can be broken away to release the chip carrier for extraction. Thus
the molded tabs are break-away tabs that can be broken away by the
displacement of the chip carrier relative to the strip housing by
the extraction tool.
[0042] The analysis chip may be bonded directly to the chip
carrier, or the chip may be bonded through another element. The
chip can be provided, for example, as a chip assembly, wherein the
chip assembly is bonded to the chip carrier. The chip assembly can
comprise, for example a square or rectangular chip comprising an
array of optical confinement structures. Bonded to the top of this
chip is a well-forming piece having substantially the same square
or rectangular planar dimensions as the chip, and having one or
more holes through its center. When the well-forming piece is
bonded to the top of the chip, one or more wells are formed in
which the base of the well comprises the top of the chip, and the
walls of the well comprise the inside walls of the hole through the
well-forming piece. The well in the chip assembly provides
containment for fluids such as reagents and samples while they are
in contact with the chip. The thickness of the well-forming piece
is selected such that the volume of the well is sufficient to hold
the reagents and samples within the well. The well-forming piece
can also provide structural integrity for the chip. The chip can
comprise an array of zero mode waveguides (ZMWs) formed on a fused
silica chip, and the well-forming piece can comprise a thicker
fused silica chip to provide a reagent well and provide structural
rigidity.
[0043] One aspect of the invention is a method that allows for
environmentally protecting an analysis chip, then extracting the
chip from its packaging for addition of reagents and samples, and
for subsequent analysis. The method involves providing a packaging
strip having a plurality of environmentally protected chips
attached to chip carriers within the chambers of the strips. Each
individual chamber has at least one region sealed with a sealing
film. A piercing tool is used to pierce or puncture the sealing
film, and an extraction tool is used to detach the chip carrier and
extract it from the chamber. The extraction tool can then be used
to transport the chip carrier to another station for further
processing. In addition to piercing the sealing film, we have found
that it is useful for the piercing tool to have ridges for the
controlled cutting of the sealing film, and to be dimensioned such
that as the tool extends into the chamber, it pushes the sealing
film away providing an opening in the sealing film through which
the chip carrier can be extracted without contacting the sealing
film. For instance, the piercing tool can have a point in the
center, and four ridges extending out from the center. The ridges
act as cutting blades, cutting the sealing film to create four
flaps which can be displaced by another portion of the tool as it
extends into the chamber.
[0044] Once the piercing tool has opened the chamber, the
extraction tool engages the chip carrier, for example by gripping
the chip carrier. In one embodiment the chip carrier has a collar,
and the extraction tool closes on the outside of the collar or
expands from the inside of the collar to grip the chip carrier.
Once the extraction tool has engaged the chip carrier, the
extraction tool can be used to detach the chip carrier from the
chamber. Where the chip carrier is held in place by break-away tabs
such as molded break-away tabs, the extraction tool can displace
the chip carrier relative to the strip housing to break the tabs
and free the chip carrier. The detached chip carrier can then be
extracted from the chamber.
[0045] In a preferred embodiment, the piercing tool and extraction
tool are each part of a single piercing/extraction tool. Having a
single tool for both functions can increase the efficiency of
processing as the same tool that extends into the chamber to pierce
the film can also engage with the chip carrier and extract it from
the chamber without the need to remove one tool and insert another
one. In one embodiment, the piercing tool can extend from the
single tool to pierce and cut the sealing film, then be retracted
up into the tool to allow the extraction tool portion of the tool
to engage the chip carrier.
[0046] The packages and methods of the invention are particularly
suitable for implementation with automated robotic systems which
can be used to precisely hold the packing chip, to direct the
piercing and extraction tools, to transport the chip to chip
processing stations for the addition of reagents and samples, and
to transport the chip to the analysis portion of the instrument.
The extraction, transport, processing, and analysis can be carried
out within a single instrument.
[0047] The environmental packages and methods of the invention can
be used for a variety of types of analyses. A particularly valuable
application is single molecule sequencing. By way of example, a
complex of a template nucleic acid, a primer sequence and a
polymerase enzyme may be monitored, on a single molecule basis, to
observe incorporation of each additional nucleotide during template
dependent synthesis of the nascent strand. By identifying each
added base, one can identify the complementary base in the
template, and thus read off the sequence information for that
template. In the context of ZMWs, an individual
polymerase/template/primer complex may be provided within the
observation volume of the ZMW. As each of four labeled (e.g.,
fluorescent) nucleotides or nucleotide analogs is incorporated into
the synthesizing strand, the prolonged presence of the label on
such nucleotide or nucleotide analogs will be observable by an
associated optical detection system. Such sequencing processes and
detection systems are described in, e.g., Published U.S. Patent
Application No. 2003/0044781 and Published U.S. Patent Application
No. 2007/0036511, the full disclosures of which are incorporated
herein by reference in their entirety for all purposes.
[0048] The invention also provides for methods of producing a
sealed chip-carrying strip utilizing a molded strip housing having
a plurality of conduits having open tops and open bottoms, and
having molded within in each conduit a chip carrier. The chip
carriers are held within the conduits of the strip housing such
that they are accessible from both the top and the bottom. The chip
carriers are generally held in place using molded break-away tabs
to facilitate subsequent extraction of the chip carriers. To the
bottom of each of the chip carriers is bonded a chip assembly. The
top and bottom openings of the strip housing are then sealed to
environmentally enclose the chip. The sealing is done such that
either the top or the bottom or both the top and the bottom are
sealed with a sealing film. Generally the top opening is sealed
with a sealing film, allowing for the extraction of the chip
carrier by engaging with the top portion of the chip carrier.
Environmental Packaging Strip
[0049] The present invention provides an environmental packaging
strip that environmentally seals a plurality of analysis chips,
each in its own environmentally sealed chamber, each chamber partly
sealed with a sealing film.
[0050] FIG. 1 shows a cross-section of an embodiment of the
packaging strip of the invention. The strip has multiple chambers
100. The chambers in the figure are arranged linearly. Chambers in
other embodiments can be arranged in other configurations. Two
chambers 100 are shown. An analysis chip 154 is held within a
chamber 100. The analysis chip 154 is part of chip assembly 150
also comprising well-forming piece 152. The chip assembly 150 is
bonded to chip carrier 110. The chip assembly 150 can be bonded to
the chip carrier 110, for example, using an adhesive such as a UV
curable adhesive. Chip carrier 110 is held within the cavity with
molded break-away tabs 180. The walls of the chambers are formed by
molded strip housing 140. The strip housing 140 and chip carriers
110 can be produced as a single molded part. The strip has tab 142
for manual handling of the strip. The top of the chambers are
sealed with sealing film 120. On top of the chambers is the
optional strip capping piece 170. Strip capping piece 170 provides
some physical protection for the sealing film and finishes off the
top edges of the packaging strip. The strip capping piece 170 can
be a molded part which snaps onto the top of the strip. The bottom
of the chamber in this embodiment is also sealed with a sealing
film 130. Base piece 160 is attached to the bottom of the strip.
Base piece 160 can also be molded. Base piece 160 generally does
not have openings corresponding to the chambers. It provides
physical protection to the bottom strip and provides a base for
placing the strip down, and for placing the strips into holders and
storage trays. In the embodiment illustrated in FIG. 1, there is a
sealing strip on both the top and the bottom of each of the
chambers. In other embodiments, only one opening is sealed with a
film. For example, the top is sealed with a film, and the bottom is
sealed with a base piece which is attached in a manner that
provides environmental sealing.
[0051] FIG. 2 shows an exploded view illustrating the parts used
for producing a strip having 8 chips, each sealed within its own
chamber. Strip housing 210 is a molded part having 8 conduits which
when sealed become the chambers. Molded into the strip housing 210
are 8 chip carriers 230. The chip carriers 230 are molded into the
conduits such that they are accessible from both the top and the
bottom. Strip housing 210 has tabs 212 and 214 for manual handling
of the strip. The chip carriers are formed with a base that is
configured for bonding to chip assemblies. Chip assemblies 220,
each comprise analysis chips bonded to well-forming pieces to
produce wells on the top of each chip assembly, are attached to the
bases of the chip carriers within the conduits. The tops of the
conduits are sealed using sealing film 240, which can, for example,
be heat sealed onto the strip housing 210. Strip capping piece 220
can be attached to the top of the strip. Strip capping piece 220
has openings corresponding to the conduits to allow for the
extraction of the chip carriers from the chambers. Bottom sealing
film 250 is used to seal the bottoms of the conduits. Base piece
260, which is generally a solid piece, is attached to the bottom of
the strip to protect the bottom sealing film and provide a base for
the strip. As shown here, strip capping piece 220 and base piece
260 are molded parts that can be snapped on to attach to the strip
housing.
[0052] FIG. 3 shows a finished strip assembled from the parts shown
in FIG. 2. The strip has 8 chambers, each comprising one chip
carrier having one chip assembly bound to it. Once assembled, the
strip provides environmental protection for the chips until the
chips are extracted for further processing and analysis.
[0053] FIG. 4 shows a cross sectional view of a molded strip
housing 400 having a chip carrier 410 in each conduit, and having
bonded to each chip carrier a chip assembly 420. The view in FIG. 4
is looking down at the bottom of the molded strip housing 400. The
molded strip housing 400 has multiple conduits 440, each with a top
opening (below in the figure) and a bottom opening. In each
conduit, a chip carrier 410 is molded, being held in place with
tabs 450 which will hold the chip carrier in place during shipping
and handling, then can be broken away to detach and extract the
chip carrier in order to perform an analysis. The chip carriers 410
are configured to each have a base to which a chip assembly 420 can
be bonded. The chip carrier 410 has an opening surrounded by a
collar 460. The opening allows for the addition of reagents and
samples to the chip 422 while the chip 422 is bonded to the chip
carrier 410. Chip assembly 420 is made up of chip 422 bonded to
well-forming piece 424. The top and bottom openings of the conduits
are configured so that they can be sealed to produce chambers which
provide environmental protection for the chips 422, for example
with a sealing film.
[0054] The environmentally sealed strip has a plurality of
chambers. The number of chambers can be chosen depending on the use
or on the throughput of samples to be analyzed. The number of
chambers can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16 or
more. The number of chambers can be between about 2 and about 1000,
between about 4 and about 100, or between about 6 and about 24. The
chambers are generally arranged side by side in one layer. In some
embodiments the chambers are arranged in one dimension, such as
linearly. In some embodiments, the chambers are arranged in two
dimensions, for example in rows and columns. In some cases the
chambers are arranged in a radial arrangement, for example, one or
more concentric circles, or a radial arrangement. The sealed strip
can be dimensioned and have features for both for manual handling
by operators, and by automated robotic systems.
[0055] The chambers provide environmental protection for the chips.
Environmental protection includes protection from exposure to
gasses, liquids, and solids while the chips are stored,
transported, and handled. The level of protection from gasses can
be controlled by controlling the materials and dimensions of the
walls, top and bottom of the chamber. By choice of materials having
the appropriate transport properties, the chips can be protected,
for example, from gaseous species such as oxygen, moisture, ozone,
ammonia, acids or other volatile compounds. The sealing will also
protect the chips from atmospheric liquid and solid contaminants
such as dust and vapor, which can carry components that could be
harmful to the chip, or could interfere with the analysis. The
sealing can also protect the chips from extraneous biological
material including nucleic acids, proteins, and other cellular
materials. The chambers can also be used to protect the chips from
electromagnetic radiation such as visible and ultraviolet light. In
some cases, it is desirable that portions of the chambers be
transparent in order to provide visual observation within the
chamber. If desired, the chambers can be made with material having
ultraviolet absorbing components to protect the chips from
ultraviolet light while allowing visualization.
[0056] The sealing film is chosen to provide the level of
protection desired while also allowing for penetration and removal
of the chip carriers. Films containing metal foil can be useful,
for example for their low vapor transport properties. Such films
can also have good mechanical properties, and can be pierced and
cut in a reliable manner by the piercing tools described herein. In
some cases multilayer films having polymer and metal layers are
used.
[0057] The chip carrier of the invention provides a structure for
handling the analysis chip. The analysis chip can be thin, fragile,
and difficult to handle. The chip carrier provides a structure
which can engage with the tools for extracting the chip from the
chamber and for manipulating and transporting the chip to and from
various processing stations. The chip carrier can have specific
structures such as ridges, holes, pins, handles, and tabs designed
to interact with manipulation tools. The chip carrier is bonded to
the analysis chip either directly or indirectly through another
element. The chip carrier is generally bound to the chip such that
a portion of the chip is exposed for the addition of reagents and
samples. In some cases, the chip carrier comprises one or more
openings into which reagents and samples can be introduced. The
chip carrier can have one central opening for the addition of
reagents and samples. In some cases, the chip is bound to the
bottom of the chip carrier, where the chip carrier has an opening
at the top surrounded by a collar. The collar can provide a feature
for engagement with manipulating tools.
[0058] The chip carrier is generally constructed such that it can
be reversibly held within the chamber so that it will remain in
place during shipping and handling, but can be removed for
processing and analysis. The chip carrier can be held in place with
features that constrain the movement of the chip carrier within the
chamber. The chip carrier can be held within the chamber using
adhesives such as pressure sensitive, hot melt, or curable
adhesives. For example, a pressure sensitive adhesive can be used
which holds the chip carrier with enough force to keep it in place
during shipping and handling, but forms a bond that can be broken
by a tool holding the chip carrier. In some cases, it is preferred
that the chip carrier be held in place with break-away features
such as break-away molded tabs. The chip carrier can be formed in a
mold which also produces the strip housing, such that one molded
chip carrier is molded into place within each chamber. By designing
the molded tabs with the appropriate geometry, the chip carrier is
held in place during shipping and handling, and can be detached by
being displaced by an extraction tool. The number of tabs used to
hold the chip carrier in place can be chosen for the right balance
of retention and break-away properties as well as for ease of
manufacturing. The number of tabs can be 1, 2, 3, 4, 5, 6, 7, 8 or
more.
[0059] In some embodiments, the analysis chip is bonded to the
bottom or base of the chip carrier, the chip carrier is held in the
bottom portion of the chamber, and the chip carrier is extracted
through the top of the chamber. In some cases, the chip carrier is
held within the chamber such that it is below the level at which it
would be contacted by the sealing film as it is punctured and
displaced by the piercing tool prior to extraction. For example,
the chip carrier is held such that the top of the chip carrier is
below the sealing film at a distance that is one half of a
cross-sectional dimension of the chamber. Where the chamber has a
square cross-sectional profile, the top of the chip carrier is
disposed lower than one half of the distance of the length of the
side of the square. Where the chamber has a rectangular
cross-sectional profile, the top of the chip carrier is disposed
lower than one half of the distance of the length of a side of the
rectangle.
[0060] In some embodiments the chip carrier is made to have a base
which is dimensioned for the attachment of the analysis chip to the
base, for example using an adhesive. In some embodiments, the chip
is attached to the base of the chip carrier such that the bottom of
the chip extends below the chip carrier. This allows the bottom and
sides of the chip to be engaged to position the chip. It can be
difficult to precisely align the chip using the chip carrier as a
physical guide where very high precision is required. Aligning the
chip by physical contact to the analysis chip itself can be useful
where the chip has features, such as optical features, which must
be precisely aligned. For example, the attachment of the extraction
tool to the chip carrier, and physical alignment to a processing
station by the chip carrier can provide sufficient alignment
accuracy for operations such as the addition or removal of reagents
and samples, and physical contact with the chip itself can be used
to for the more demanding alignment of the chip, for example in an
analysis station.
[0061] The chip can be any type of analysis chip. The chip can
comprise a biopolymer array such as a hybridization chip for
binding specific nucleic acids such as those described in U.S. Pat.
Nos. 5,837,832 and 6,841,663. Suitable chips include chips for the
observation of single molecules such as described in U.S. Pat. No.
7,292,742. The chips can be used for nucleic acid sequencing, for
example as descried in U.S. Pat. Nos. 7,335,762 and 7,232,656.
[0062] The analysis chip can be constructed, in some cases, such
that sample is added to the top of the chip and the samples are
optically interrogated from below the chip. Such a chip can be a
chip having an array of optical confinements such as zero-mode
waveguides (ZMWs), in which the bottom of the chip comprises a
transparent material such as a glass or fused silica which has on
its top surface a cladding layer with an array of optical
confinement structures such as apertures through the cladding layer
to the transparent substrate.
[0063] In preferred aspects, a molecule to be analyzed, such as a
template/polymerase primer complex is provided, typically
immobilized, within an optically confined region, such as a zero
mode waveguide (ZMW), or proximal to the surface of a transparent
substrate, optical waveguide, or the like (see e.g., U.S. Pat. Nos.
6,917,726, and 7,170,050 and Published U.S. Patent Application No.
2007-0134128, the full disclosures of which are hereby incorporated
herein by reference in their entirety for all purposes). The
optically confined region is illuminated with an appropriate
excitation radiation for the fluorescently labeled nucleotides that
are to be used. Because the complex is within an optically confined
region, or very small illumination volume, only the reaction volume
immediately surrounding the complex is subjected to the excitation
radiation. Accordingly, those fluorescently labeled nucleotides
that are interacting with the complex, e.g., during an
incorporation event, are present within the illumination volume for
a sufficient time to identify them as having been incorporated.
[0064] ZMW arrays can be fabricated at ultra high density,
providing anywhere from 1000 ZMWs per cm.sup.2, to 1,000,000 ZMWs
per cm.sup.2, or more. Thus, at any given time, it may be desirable
to analyze the reactions occurring in from 100, 1000, 3000, 5000,
10,000, 20,000, 50,000, 100,000 or 1 Million, 10 Million or more
ZMWs or other reaction regions within a single analytical system or
even on a single substrate.
[0065] The analysis chip can be bonded to the chip carrier by any
suitable method. The analysis chip can be bonded to the chip
carrier using an adhesive such as a pressure sensitive adhesive, a
hot melt adhesive, or a curable adhesive such as a UV or visible
light curable adhesive.
[0066] The environmentally sealed strip can include a strip
housing. The strip housing is a part comprising a plurality of
conduits which, when sealed will become the environmentally sealed
chambers. The strip housing can be made by molding. The conduits
have top openings and bottom openings that are configured to be
sealed to form chambers. Either the top or bottom opening or both
the bottom and top openings are sealed with a sealing film. In some
embodiments the top opening is sealed with a sealing film, and the
bottom opening is sealed with a bottom piece that is more rigid
than the sealing film. In a preferred embodiment the chip carriers
are molded into the conduits when the strip housing is molded, and
are held in place by molded tabs which can be broken to extract the
chip carriers. The strip housing can be made of any suitable
material including molding plastics such as polycarbonates,
polyesters, polyacrylates, or polyolefins. In some cases, the strip
housing is molded out of a transparent material such as
polycarbonate.
Chip Assembly
[0067] In one aspect, the invention provides a chip assembly or
stacked chip comprising a chip having an array of optical
confinements and a well-forming piece. The chip comprises a
transparent substrate of fused silica having on its top surface a
layer of a cladding material. The cladding material can comprise a
metal such as aluminum. The chip has an array of optical
confinements, for example an array of nanoscale apertures through
the cladding layer. The chip is generally rectangular or square.
The dimensions of the chip can be from about 0.5 mm to about 100
mm, or from 5 mm to about 20 mm. The chip can be, for example, a
square chip about 9 mm on a side.
[0068] The well-forming piece comprises a fused silica chip having
one or more openings such that when the well-forming piece is
bonded to the analysis chip one or more wells is formed in which
the chip comprises the bases of the wells, and the walls of the
openings on the well-forming piece comprise the sides of the wells.
In some cases, the well-forming piece comprises one opening, and
the opening is in about the center of the chip. The well-forming
piece is generally thicker than the analysis chip. In some cases,
the well-forming piece is more than about 5 times thicker, more
than about 10 times thicker, more than about 20 times thicker, more
than about 50 times thicker, or more than about 100 times thicker
than the analysis chip. The well-forming piece can be about 0.5 mm
to about 20 mm, about 1 mm to about 10 mm, or about 1 mm to about 4
mm thick. The well-forming top-piece is generally made using a
material that has a similar thermal coefficient of expansion as the
material of the substrate of the chip. In some cases, the
well-forming piece comprises the same material as the substrate of
the chip. For example, both the substrate of the chip and the
well-forming piece can comprise fused silica.
[0069] The diameter of the well can range from, between about 1 mm
to about 9 mm, from about 2 mm to about 7 mm, or between about 4 mm
and about 6 mm.
[0070] In a preferred embodiment, the well-forming piece and the
chip are formed on wafers. Such wafers are used in semiconductor
and microfabrication processes. The wafer with the well-forming
pieces and the wafer with the analysis chips each comprise from 10
to about 1,000,000 or between about 100 and 100,000 analysis chips.
The well-forming piece has substantially the same number of
well-forming pieces configured to line up with each of the chips.
The two wafers are wafer bonded either directly or indirectly.
Suitable bonding methods include, fusion bonding, anodic bonding,
thermo-compression bonding, or adhesive bonding. After bonding of
the wafers, the wafers are diced to yield the plurality of analysis
chip assemblies.
[0071] We have discovered that where adhesives are used to bond the
well forming piece and the chip that in some cases the adhesive can
flow into the well region, and in some cases unexpectedly wick in
toward the center of the chip, interfering with the optical
confinements on the chip. In order to prevent the adhesive from
extending from the edges into the active portion of the chip, we
have found that we can control such wicking by incorporating
features onto the chip that divert the flow of adhesive. In one
embodiment, a moat or trench is created in the chip which extends
around the perimeter outside of the chip such that adhesive that
wicks in from the edges will be trapped in the moat. In one
embodiment the invention comprises a chip assembly comprising a
well-forming piece on top of a chip in which the well forming piece
has an opening which creates a well when the well forming piece is
bonded to the chip, and wherein the chip has a depressed region
which forms a moat or trench which extends around the outside of
the exposed area of the chip such that adhesive which may extend
into the exposed area of the chip from the edges of the well where
the well-forming piece and the chip are bonded. Where the
well-forming piece creates a well with a circular cross-section,
the moat will generally be a circular moat which is substantially
concentric with the well.
[0072] The width, height and distance of the trench from the well
wall can be any suitable dimensions for containing and arresting
the flow of adhesive. The trench dimensions are generally chosen to
provide a trench volume that is large enough to arrest adhesive
flow, but not so large that the trench impinges on the active
portion of the chip. The width of the trench can be, for example
from about 10 micron to about 200 micron, or from about 20 micron
to about 100 micron. The depth of the trench can be from about 5
micron to about 50 microns, from about 10 micron to about 20
microns, or about 5 micron to about 10 micron. The aspect ratio of
the width to the height of the trench can generally be from about
1:3 to about 3:1 or about 1:2 to about 2:1. The distance between
the inner wall of the top piece and the trench can be from about 50
micron to about 500 micron, or about 100 micron to about 300
micron.
[0073] FIG. 5 shows a cross section of a portion of a chip
assembly. Well-forming piece 500 is bonded on top of chip 510. The
portion shown in FIG. 5 is the edge of a well formed by having a
hole through the well-forming piece in which the exposed portion of
the chip comprises the bottom of the well. The well-forming piece
and the chip are bonded together using an adhesive 550. The chip
has a trench around the outside of the bottom of the well such that
if the adhesive wicks into the well, the trench will contain the
adhesive and prevent it from extending further across the chip
where it could interfere with features on the chip such as optical
confinements. For the embodiment shown in FIG. 5 the chip has a
plurality of optical confinements 540 which are formed as apertures
through a cladding layer 530. The trench 520 prevents the further
wicking of the adhesive 550 onto the region of the chip having the
optical confinements 540.
[0074] The individual chip assemblies can be fabricated using a
wafer bonding process. The wafers can be, for example, 6 in or 8 in
diameter wafers. The well-forming piece wafer and chip wafer are
patterned such that when the wafers are aligned, the holes in the
well-forming piece wafer aligns with the regions of optical
confinements on the chip wafer. The wafers are bonded by any
suitable means of wafer bonding. The wafers can be thermally
bonded, laser bonded, fusion bonded, anodically bonded,
thermo-compression bonded, or bonded with adhesives including
thermally cured, light cured, or UV cured adhesives. Where an
adhesive is used, the adhesive is generally applied to the bottom
of the well-forming-piece, for example by dipping, spray coating,
or micro-drop application. The well-forming piece wafer may have,
for example, hundreds to thousands of holes extending through it,
each hole forms one well on well-forming of the chip. After
bonding, the stacked wafers may undergo a grinding process to thin
the chip. Such grinding can be performed, for example, by chemical
mechanical polishing (CMP). The bonded wafer can then be diced to
produce the individual chip assemblies. The chip assemblies can
then be handled by pick and place machines to attach the chip
assemblies to the chip carriers. Techniques for carrying out these
processes are described, for example in "Introduction to
Microfabrication", Fransilla, Ed., Wiley, 2004.
[0075] In one embodiment, the thickness of the well-forming piece
is between about 1 mm and about 3 mm and the thickness of the chip
is between about 500 micron and about 900 microns. After bonding,
CMP is used to reduce the thickness of the chip to between about
200 microns and about 600 microns.
Method of Providing a Sealed Analysis Chip
[0076] In one aspect, the invention provides a method for providing
a sealed analysis chip from a sealed package for further processing
and placement into the analysis portion of an analysis instrument.
The methods of the invention comprise providing a plurality of
analysis chips, each within a sealed chamber sealed with a sealing
film. A piercing tool extends through the Sealing film, puncturing
the film and displacing the film to produce an opening through
which the analysis chip can be extracted. Each analysis chip within
the sealed chamber is attached to a chip carrier. An extraction
tool engages with the chip carrier, detaches the chip carrier from
the chamber, and extracts the chip carrier, thus extracting the
analysis chip which is bonded to it.
[0077] An embodiment of the method is illustrated in FIG. 6. A
single piercing extraction tool 600 is used to extract an analysis
chip from an environmentally sealed strip 610 by piercing and
displacing sealing foil 630 to gain entry into sealed chamber 620.
In FIG. 6(B) the piercing/extraction tool is extended from above
and down such that the piercing tool portion pierces and cuts the
sealing film 630. The piercing tool has four ridges, each of which
forms a cut which propagates as the tool extends into the cavity.
This creates four flaps, each corresponding to one of the sides of
the chamber. The flaps are pushed down and away by the tool such
that neither the chip carrier nor the analysis chip will come into
contact with the sealing film upon extraction. FIG. 6(C) shows the
piercing/extraction tool extended into the chamber. While in the
chamber, the piercing tool portion is retracted into the
piercing/extraction tool to allow the extraction tool portion to
engage with the chip carrier. In the illustrated embodiment, the
extraction tool portion closes onto the collar of the chip carrier,
gripping the chip carrier. While engaged with the chip carrier, the
extraction tool displaces the carrier relative to the strip,
detaching the chip carrier from the chamber. Once detached, the
chip carrier, bonded to the analysis chip is extracted from the
chamber. FIG. 6(D) shows the extraction tool holding the chip
carrier 650 that has been extracted from the chamber by lifting the
chip carrier up through the opening in the chamber produced by the
piercing tool. A flap 660 produced by the action of the piercing
tool can be seen in FIG. 6(D).
[0078] FIG. 7 shows a cross-sectional view of a single
piercing/extraction tool 700 extended into a chamber. In FIG. 7(A)
the piercing tool 710 is partially retracted, and the extraction
portion of the tool 720 has extended down adjacent to the collar
742 of the chip carrier 740, but has not yet engaged with the chip
carrier. The chip carrier is held in place within the chamber by
molded tabs. Attached to the chip carrier is the chip assembly 730
comprising the chip 732 bonded to the well-forming piece 734. In
FIG. 7(B) the piercing tool 710 has fully retracted, allowing the
extraction tool 720 to engage with the collar 742 of the chip
carrier 740. Here, the extraction tool comprises fingers which have
closed inward to grip the collar 742 of the chip carrier 740.
[0079] FIG. 8 has a cross-sectional view illustrating one
embodiment of the step of displacing the chip carrier to detach it
from the chamber. In FIG. 8(A) the extraction tool 850, which is a
part of the extraction/piercing tool 800, has gripped the chip
carrier 840 by the collar 842. The piercing tool 810 has been
retracted. The chip carrier 840 is held in place with molded tabs
820. In FIG. 8(B) the extraction tool 850 has been moved downward
relative to the strip housing 860. The displacement of the chip
carrier relative to the strip housing 860 results in the breaking
of the molded tabs 820, thereby detaching the chip carrier. The
detached chip carrier can then be extracted from the chamber.
[0080] FIG. 9 illustrates the extraction of the detached chip
carrier from the chamber. In FIG. 9A, the extraction/piercing tool
900 has been moved upward to lift the chip carrier 910 attached to
the analysis chip above the position it was in when it was attached
to the chamber. In FIG. 9(B), the extraction/piercing tool 900 has
lifted the chip carrier completely out of the chamber. The flaps
920 which were formed upon the piercing, cutting, and displacement
of the sealing film have been pushed down and away from the opening
of the chamber such that as the chip carrier is removed, there is
no contact between the chip carrier and the sealing film. The tool
can now move the chip carrier to a processing station where
reagents and or sample can be added to the chip assembly, and
further processing can be performed.
[0081] FIG. 9B also illustrates an embodiment in which the analysis
chip 940 and chip assembly 930 are attached to the base of the chip
carrier such that the bottom of the chip 940 extends below the
bottom of the chip carrier 910. Here, the edges of the chip 940 are
exposed below the chip carrier 910 in a manner such that the
extraction tool or another tool which engages with the chip carrier
can place the chip into a holder which physically references to one
or more edges of the chip. Physical alignment to the chip edges
provides greater positional accuracy to features on the chip than
is generally achieved by physical alignment using the chip
carrier.
[0082] The method can be used with any suitable environmentally
sealed strip including those described herein. The method can be
used with a strip having any suitable number of sealed chambers.
The number of chambers can be chosen depending on the use, for
example for throughput of samples that are to be analyzed. The
number of chambers can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 13, 14, 15, 16 or more. The number of chambers can be between
about 2 and about 1000, between about 4 and about 100, or between
about 6 and about 24. The chambers are generally arranged side by
side in one layer. The method can utilize chambers can be arranged
in one or two dimensions. In some cases the chambers are arranged
linearly. In other cases, the chambers can be arranged in rows and
columns. The chambers can also be arranged radially.
[0083] The piercing tool generally has one or more points to pierce
the film. In some cases, the piercing tool has one or more ridges
which act as blades to cut the sealing film as the piercing tool
extends into the chamber. The number of ridges can be 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12 or more than 12 ridges. In some cases,
the piercing tool has 3 to 8 ridges. In one preferred embodiment,
the number of ridges is four, preferably disposed in a cross
pattern such that when the tool is extended into the chamber, four
flaps are created which can be neatly pushed down and away from the
opening. The ridges can be disposed at an angle to the surface of
the sealing film such that as the tool is extended into the
chamber, a longer and longer cut is made. This approach provides
for the controlled cutting of the film along a series of cuts as
the tool progresses into the chamber. The blades will generally be
at an angle such that the central portion of the sealing film on
top of the chambers is contacted first, and the cut propagates from
the center toward the edges of the chamber. It is generally desired
that if the chambers have a square or rectangular cross-section
such that the sealing film is in the shape of a square or cross
section, that the cuts extend from the center to the corners of the
square or rectangle. Extending to the corners allows for flaps to
open a large portion of the area of the chamber.
[0084] In addition to cutting the sealing film, the piercing tool
of the invention can be dimensioned such that it also displaces the
sealing film, for example pushing the film down and away from the
opening in the chamber in a controlled manner. This function can be
accomplished by having a portion of the tool which has a width that
corresponds to the desired dimension for the opening in the sealed
film. This portion of the tool can also be a portion of the tool
that acts as the extraction tool. For example, the extraction tool
in a single piercing/extraction tool can be positioned above and at
a position wider than the piercing tool while the tool is extended
down into a chamber such that it has a width which displaces the
sealing film. The extraction tool can subsequently contract to
engage the chip carrier, the closed extraction tool now having
smaller outer cross sectional dimensions, such that it does not
contact the flaps of sealing film on the way out of the chamber
during extraction.
[0085] The extraction tool engages with the chip carrier to detach
and extract the chip carrier from the chamber. The extraction tool
can engage with the chip carrier by any suitable means. Features on
the extraction tool and chip carrier can be designed to mate in
such a way that the extraction tool can hold the chip carrier. For
example, one or more hook, screw, hole, pin, ridge, clip, catch,
hanger, nail, clasp, slide, ridge or other feature can be used. It
is generally desired that the carrier be reversibly engaged by the
extraction tool such that it can be subsequently be placed in
various stations for processing, inspection, and analysis. In such
cases a tool which is capable of gripping the chip carrier by one
or more feature on the chip carrier can be used. For example, the
extraction tool can have one or more moveable finger, member, or
appendage that can be moved to grasp the chip carrier can be used.
For example, the tool can have fingers which close in on the
outside of a collar to grasp the chip carrier. Alternately, the
tool can have fingers which expand out to hold the tool by pushing
against the inside of a collar.
[0086] The extraction tool can displace the chip carrier relative
to the strip housing in a number of ways. In some embodiments, the
chip carrier is displaced down or up. The chip carrier can also be
detached by a sideways or a twisting motion. It is generally
desired that the displacement distance be relatively small so as
not to contact the displaced chip carrier with other portions of
the strip housing. In some cases, the displacement is carried out
rapidly, for example, in order to snap a break away tab or release
an adhesive bond.
[0087] The extraction method can further comprise steps of moving
the chip carrier to one or more stations for further processing,
inspection, and/or analysis. After extraction, the extraction tool
generally transports the chip to a processing station or reaction
station where the chip carrier is placed. While in the processing
station, reagents are added to prepare the chip for analysis. For
example, the reagent that is added can include the addition of
reagents for immobilizing enzymes onto the chip. The reagents can
include sequencing reagents including the reagents required for
carrying out a nucleic acid synthesis reaction, for example by a
polymerase enzyme. Inspection steps can also be carried out. After
the chip carrier has been processed and inspected in one or more
process stations, the chip carrier with analysis chip is
transported to a station at which analysis is performed. For
example the analysis can comprise an optical analysis of multiple
regions on the chip simultaneously. The analysis can be the
sequencing of nucleic acids on the chip, for example, single
molecule DNA sequencing.
[0088] In some embodiments, the method is performed with an
environmentally sealed strip wherein the analysis chip is bonded to
the bottom of the chip carrier, the chip carrier is held toward the
bottom of the chamber, and the chip carrier is extracted through
the top of the chamber.
[0089] In some cases, the method is carried out such that the chip
carrier is held within the chamber such that it is below the level
at which it would be contacted by the sealing film as it is
punctured and displaced by the piercing tool prior to extraction.
For example, the chip carrier is held such that the top of the chip
carrier is below the sealing film at a distance that is one half of
a cross-sectional dimension of the chamber. Where the chamber has a
square cross-sectional profile, the top of the chip carrier can be
disposed lower than one half of the distance of the length of the
side of the square. Where the chamber has a rectangular
cross-sectional profile, the top of the chip carrier can be
disposed lower than one half of the distance of the length of a
side of the rectangle.
Method of Producing a Sealed Analysis Chip
[0090] In one aspect, the invention provides a method for producing
a sealed chip carrying strip comprising; providing a molded part
having a plurality of conduits extending therethrough, each conduit
having an open top, and open bottom, and side walls, wherein within
each conduit is a chip carrier having a top and a base that is held
in the conduit by break-away tabs; attaching a chip assembly
comprising an analysis chip to the base of the chip carrier; and
sealing the open bottom and the open top to environmentally seal
the analysis chips within the conduits; wherein at least one of the
open top and open bottom is sealed with a sealing film.
[0091] The process of the invention can be described by referring
to FIG. 2 which illustrates an embodiment of the process. The strip
housing 210 represents a molded part having a plurality of conduits
which extend through the housing. Here, each conduit has an open
top, an open bottom, and side walls. Within each conduit is a chip
carrier having a top and a base that is held in the conduit by
break-away tabs. This part can be produced in one molding step. The
chip assemblies 220 have an analysis chip on the bottom and a well
forming piece bonded to the top of the chip. The chip assemblies
220 are bonded to the bases of the chip carriers in the strip
housing. The chip assemblies can be bonded with an adhesive, can be
physically clipped or wedged in place, or, can be welded. After the
chip assemblies are attached, the chambers are sealed to provide
environmental protection. The sealing is generally carried out in a
controlled environment. The composition of the controlled
environment will depend on the makeup of the chip. The environment
will generally have controlled amounts of water and oxygen. In some
cases, the amount of water and oxygen will be minimized to control,
for example, corrosion and oxidation. In some cases, a controlled
amount of moisture is desirable. For example, in some cases,
enzymes, nucleotides, proteins or other biomolecules can be
included on the chip that is sealed within the chamber where the
presence of water is beneficial for the stability of the
biomolecules. In some cases, the sealing is carried out in an
environment comprising predominantly nitrogen or argon inert gasses
having low levels of oxygen.
[0092] The sealing film 240 is used to seal the top openings of the
conduits. The sealing can be accomplished using heat sealing. The
sealing can be accomplished using adhesives such as pressure
sensitive, hot melt, or light cured adhesives. The bottom openings
of the conduits are also sealed by the same or in a different
manner than the sealing of the top openings. As shown here, a
sealing film 250 is used to seal the bottom openings. In other
embodiments the bottom openings are sealed with rigid piece, for
example, piece that is more rigid than the sealing film. Where, as
shown in FIG. 2, a sealing film is used to seal the bottom
openings, a base piece 250 can be attached to the bottom of the
strip housing to provide a more durable strip package. The base
piece can be attached by any suitable means including adhesion. In
some cases, the base piece is snapped onto the strip housing. In
some embodiments, no sealing film is used to seal the bottom of the
chambers, and a rigid base piece is attached, for example by laser
welding or with adhesives such that an environmental seal of the
chambers is formed.
Instrument
[0093] In one aspect the invention provides an analysis instrument
for extracting a sealed analysis chip, subjecting the chip to
pre-analysis processing, and then performing an analysis using the
chip. The instrument has a station for receiving a sealed
environmental strip having sealed chambers. The chambers on the
strip are sealed with a sealing film. Inside each chamber in the
strip is a chip carrier which is bound to an analysis chip. The
instrument is an automated instrument under computer control. The
instrument has an automated chip extractor that has a piercing tool
and an extraction tool. The piercing tool is configured to pierce
the sealing layer a chamber; and generally to open a hole through
which the chip carrier holding the chip can be extracted. The
extraction tool is configured to engage with the chip carrier, and
to remove the chip carrier from the strip. The extraction tool also
transports the analysis chip to one or more other stations within
the instrument. Such stations include processing stations,
inspection stations, and analysis stations.
[0094] The processing stations carry out processes for preparing
the chip for analysis and for introducing the sample to the chip.
The processing stations can carry out reaction processes such as
the addition and removal of reagents, heating and cooling,
filtration, centrifugation, purification, and agitation. The
automated processing station can carry out many of the chemical,
biochemical, and molecular biological processes that can be carried
out manually in the laboratory. Automated sample processing
stations are known in the art. Such processing instrumentation can
be custom made. Systems and components are commercially available,
for example from the companies Caliper Corporation, Zymark,
ThermoFisher, and Tecan.
[0095] The analysis system can carry out any suitable type of
analysis using the chip. The instruments of the present invention
are particularly suited for highly multiplexed analyses in which a
liquid sample is in contact with the chip during the analysis. The
instruments are suited for optical analysis on a chip having
multiple analysis regions on its surface. The analysis can be the a
hybridization analysis for detecting binding of specific nucleic
acids, for example for determining genetic or gene expression
information such as described in U.S. Pat. Nos. 5,837,832 and
6,841,663. The analysis can comprise the observation of single
molecules such as described in U.S. Pat. No. 7,292,742. The
analytical instrument can be used for nucleic acid sequencing, for
example as descried in U.S. Pat. Nos. 7,335,762 and 7,232,656.
[0096] The piercing tool and extraction tool of the instrument can
be any suitable piercing or extraction tool including the piercing
and extraction tools described herein.
[0097] The sample strip accepted by the instrument has a plurality
of chambers. The number of chambers can be chosen depending on the
use, for example for throughput of samples that are to be analyzed.
The number of chambers can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13,
14, 15, 16 or more. The number of chambers can be between about 2
and about 1000, between about 4 and about 100, or between about 6
and about 24. The chambers are generally arranged side by side in
one layer. The chambers can be arranged in one or two dimensions.
In some cases the chambers are arranged linearly. In other cases,
the chambers can be arranged in rows and columns. The sealed strip
can be dimensioned for manual handling by operators, as well as by
automated robotic systems.
[0098] One aspect of the invention is an instrument configured to
carry out the methods described herein. In one embodiment, the
instrument is configured to method of providing an analysis chip
comprising: providing a strip having a plurality of sealed
chambers, each chamber having a bottom, a top, and sidewalls, the
top of each chamber sealed with a sealing film; wherein a chip
carrier comprising an analysis chip is disposed within each
chamber; piercing the sealing layer of at least one of the chambers
with a piercing tool; and extracting the chip carrier from the
chamber with an extraction tool.
[0099] In some cases, the chip carrier comprising the analysis chip
is extracted, moved to at least one processing station where the
analysis chip is processed, and then the chip is moved to an
analysis station where the chip is analyzed. In some embodiments,
the analysis chip is bonded to the base of the chip carrier such
that the analysis chip extends below the bottom of the chip
carrier. The exposed portion of the chip can be used to physically
align the chip within the analysis station. For example, one or
more edges of the chip can be used to physically align the analysis
chip in the analysis station in order to orient the chip with the
analysis equipment. This can be particularly useful for optical
systems for analyzing large numbers of samples on the chip.
* * * * *