U.S. patent application number 12/872997 was filed with the patent office on 2011-03-03 for flow cells and methods of filling and using same.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Mark ANDERSEN, John BRIDGHAM, David COX, Jian GONG, Kirk HIRANO, David LIU, Maryam SHARIATI, Sam WOO, Joon Mo YANG, Min YUE.
Application Number | 20110052446 12/872997 |
Document ID | / |
Family ID | 42984077 |
Filed Date | 2011-03-03 |
United States Patent
Application |
20110052446 |
Kind Code |
A1 |
HIRANO; Kirk ; et
al. |
March 3, 2011 |
FLOW CELLS AND METHODS OF FILLING AND USING SAME
Abstract
Various flowcell configurations and systems are provided as are
methods of making and using same. The flowcells, systems, and
methods of use can be useful in carrying out sequencing reactions
and next generation sequencing methods.
Inventors: |
HIRANO; Kirk; (Foster City,
CA) ; ANDERSEN; Mark; (Carlsbad, CA) ; GONG;
Jian; (San Marcos, CA) ; WOO; Sam; (Redwood
City, CA) ; COX; David; (Foster City, CA) ;
YANG; Joon Mo; (Redwood City, CA) ; YUE; Min;
(Belmont, CA) ; SHARIATI; Maryam; (Sunnyvale,
CA) ; BRIDGHAM; John; (Hillsborough, CA) ;
LIU; David; (Los Altos, CA) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
42984077 |
Appl. No.: |
12/872997 |
Filed: |
August 31, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61238633 |
Aug 31, 2009 |
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61238667 |
Aug 31, 2009 |
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61307623 |
Feb 24, 2010 |
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61307492 |
Feb 24, 2010 |
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61307641 |
Feb 24, 2010 |
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61307486 |
Feb 24, 2010 |
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Current U.S.
Class: |
422/68.1 ;
156/275.7 |
Current CPC
Class: |
Y10T 436/11 20150115;
C12Q 1/6869 20130101; B01L 2300/0867 20130101; B01J 2219/00466
20130101; B01J 2219/00527 20130101; B01J 19/0046 20130101; B01L
2300/0822 20130101; B01L 2300/0877 20130101; G01N 2035/00237
20130101; G02B 21/34 20130101; B01F 13/0222 20130101; B01L
2300/0819 20130101; B01L 3/50273 20130101; Y10T 436/143333
20150115; B01L 2200/0684 20130101; B01L 2200/0647 20130101; B01J
2219/00317 20130101; B01L 2200/16 20130101; G01N 2035/00148
20130101; B01L 3/502707 20130101; B01J 2219/00596 20130101; C12Q
1/6874 20130101; G01N 2035/1034 20130101; B01L 2200/027 20130101;
B01J 2219/00722 20130101; B01L 2400/0427 20130101; G01N 35/1002
20130101; B01J 2219/00576 20130101; B01L 2300/1822 20130101; B01J
2219/00648 20130101; B01F 13/0059 20130101; B01J 2219/00659
20130101; B01L 2300/0887 20130101; G01N 35/00069 20130101; G01N
35/1097 20130101 |
Class at
Publication: |
422/68.1 ;
156/275.7 |
International
Class: |
G01N 33/00 20060101
G01N033/00; B32B 37/12 20060101 B32B037/12 |
Claims
1. A flowcell device comprising: a substrate; one or more flowcells
formed in the substrate, each flowcell comprising a detection
window; a plurality of reagent supply reservoirs formed in the
substrate; a plurality of fluid delivery pathways each in fluid
communication with at least one of (a) the one or more flowcells,
and (b) at least one of the plurality of reagent supply reservoirs,
wherein each fluid delivery pathway is configured to be controlled
by electro-wetting circuitry; and an electrical connector
configured to provide electrical connections from a control unit to
the electro-wetting circuitry.
2. The flowcell device of claim 1, wherein the substrate comprises
a plurality of layers and the detection window is transparent and
comprises one or more of the layers.
3. A system comprising the flowcell device of claim 1 and a control
unit, wherein the control unit comprises an electrical connector
configured to interface with the electrical connector of the
flowcell device.
4. The system of claim 3, wherein the control unit further
comprises a detector configured to detect a reaction in each of the
one or more flowcells when the electrical connector of the flowcell
device is interfaced with the electrical connector of the control
unit.
5. A flowcell system, comprising: a flowcell formed in a substrate;
a first port in fluid communication with the flowcell; a second
port in fluid communication with the flowcell; a reagent supply
block comprising a plurality of reagent containers and a plurality
of reagents disposed respectively in the plurality of reagent
containers; an injector system comprising one or more injectors
disposed in an x-y-z movable stage, each injector comprising an
injector tip; and a control system configured to move the injector
system to the reagent supply block such that each injector tip can
withdraw a reagent from the reagent supply block, and configured to
move the injector system to the flowcell such that each injector
tip can inject the reagent withdrawn from the reagent supply block
through at least one of the first port and the second port and into
the flowcell.
6. The flowcell system of claim 5, wherein the one or more
injectors comprises a single injector.
7. The flowcell system of claim 5, wherein the one or more
injectors comprises a plurality of injectors.
8. The flowcell system of claim 5, further comprising flexible
tubing in fluid communication with the first port and the
flowcell.
9. The flowcell system of claim 5, wherein the first port comprises
a port formed in the substrate.
10. The flowcell system of claim 5, wherein the first port
comprises a zero volume injector port.
11. The flowcell system of claim 5, wherein each of the first port
and the second port is in valved fluid communication with (i) a
source of vacuum, (ii) a source of air or argon gas, (iii) a source
of wash solution, and (iv) a waste reservoir.
12. The flowcell system of claim 11, wherein each of the first port
and the second port is in fluid communication with a rotary valve
that in turn is in fluid communication with each of (i) the source
of vacuum, (ii) the source of air or argon gas, (iii) the source of
wash solution, and (iv) the waste reservoir.
13. A method of making a flowcell, comprising: forming
channel-defining features on a surface of a substrate; and
adhesively bonding a cover layer to the channel-defining features
to form a flowcell comprising at least one channel, wherein the
adhesively bonding comprises curing a uv-curable adhesive.
14. The method claim 13, wherein the uv-curable adhesive comprises
a mercapto-ester adhesive.
15. The method claim 13, wherein the channel-defining features are
formed from a uv-curable adhesive.
16. The method claim 15, wherein the channel-defining features
comprise a mercapto-ester adhesive.
17. The method claim 13, wherein the adhesively bonding comprises
spin-coating a uv-curable adhesive layer onto the cover layer and
then contacting the uv-curable adhesive layer with the
channel-defining features.
18. The method claim 13, wherein the adhesively bonding comprises:
positioning the cover layer adjacent the channel-defining features
to form a capillary gap there between; filling the capillary gap
with a uv-curable adhesive, by capillary action; and curing the
uv-curable adhesive.
19. The method of claim 13, wherein the forming channel-defining
features on a surface of a substrate comprises: applying a layer of
uv-curable adhesive to the surface of the substrate, masking
portions of the layer of uv-curable adhesive layer; curing portions
of the uv-curable adhesive layer that are not masked, by exposure
to uv-radiation; and cleaning off portions of the uv-curable
adhesive layer that were not masked and that were not cured,
leaving channel-defining features formed on the surface of the
substrate.
Description
RELATED APPLICATIONS
[0001] This application claims priority pursuant to 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application Ser. No.
61/238,633, filed on Aug. 31, 2009, entitled "Enhanced Systems and
Methods For Sequence Detection," U.S. Provisional Patent
Application Ser. No. 61/238,667, filed on Aug. 31, 2009, entitled
"Enhanced Flowcell and Reagent Delivery For Sequence Detection,"
U.S. Provisional Patent Application Ser. No. 61/307,623, filed on
Feb. 24, 2010, entitled "Methods of Bead Manipulation and Forming
Bead Arrays," U.S. Provisional Patent Application Ser. No.
61/307,492, filed on Feb. 24, 2010, entitled "Flowcells and Methods
of Filling and Using Same," U.S. Provisional Patent Application
Ser. No. 61/307,641, filed on Feb. 24, 2010, entitled "Flowcells
and Methods of Filling and Using Same," and U.S. Provisional Patent
Application Ser. No. 61/307,486, filed on Feb. 24, 2010, entitled
"Flowcell, Flowcell Delivery System, Reagent Delivery System, and
Method For Sequence Detection," the entirety of each of these
applications being incorporated herein by reference thereto.
FIELD
[0002] The present disclosure is directed towards molecular
sequencing, in particular towards low-volume flowcell design and
reagent delivery optimization.
BACKGROUND
[0003] There is a desire to make fluid handling systems more
efficient in the use of reagents. There is also a desire to
decrease volume requirements in fluid processing flowcells. To
accomplish these objectives, requirements for fluid dispensing
systems become more demanding. Some systems provide poor precision
in low volume regimes, large dead volumes, and are of large overall
size and plumbing lengths. With reduction in dimensions, however,
it would become difficult to robustly and uniformly fill a flowcell
without causing voids and bubbles. Furthermore, as surface area of
the flowcell deposition surface increases, yield goes down and the
cost of making the deposition substrate increases.
[0004] Lamination methods to make a flowcell using pressure
sensitive adhesive (PSA) can be used at room-temperature, but
factors, such as non-uniform PSA thicknesses and rough edges cut by
die-cutting, may affect washing performance
SUMMARY
[0005] Various embodiment of a flowcell device are provided herein.
For example, in one embodiment, the device can include a substrate
with one or more flowcells formed in the substrate and each
flowcell comprising a detection window. Additionally, the device
can include a plurality of reagent supply reservoirs formed in the
substrate with a plurality of fluid delivery pathways each in fluid
communication with at least one of (a) the one or more flowcells,
and (b) at least one of the plurality of reagent supply reservoirs,
wherein each fluid delivery pathway is configured to be controlled
by electro-wetting circuitry. The device can also include an
electrical connector configured to provide electrical connections
from a control unit to the electro-wetting circuitry. The substrate
can include a plurality of layers. Further, the detection window
can be transparent and include one or more of the layers.
[0006] Various embodiments of an analysis system are also provided
herein. For example, the system can include an embodiment of the
flowcell device and a control unit, wherein the control unit
comprises an electrical connector configured to interface with the
electrical connector of the flowcell device. The control unit can
further include a detector configured to detect a reaction in each
of the one or more flowcells when the electrical connector of the
flowcell device is interfaced with the electrical connector of the
control unit.
[0007] In another embodiment, a flowcell system is provided which
includes a flowcell formed in a substrate, a first port in fluid
communication with the flowcell, and a second port in fluid
communication with the flowcell. Additionally, the system can
include a reagent supply block comprising a plurality of reagent
containers and a plurality of reagents disposed respectively in the
plurality of reagent containers, and an injector system comprising
one or more injectors disposed in an x-y-z movable stage with each
injector comprising an injector tip. The system further includes a
control system configured to move the injector system to the
reagent supply block such that each injector tip can withdraw a
reagent from the reagent supply block, and configured to move the
injector system to the flowcell such that each injector tip can
inject the reagent withdrawn from the reagent supply block through
at least one of the first port and the second port and into the
flowcell.
[0008] The one or more injectors can include a single injector or a
plurality of injectors. The system can further include flexible
tubing in fluid communication with the first port and the flowcell.
In one embodiment, the first port includes a port formed in the
substrate. The first port can include a zero volume injector port.
Additionally, each of the first port and the second port can be in
valved fluid communication with (i) a source of vacuum, (ii) a
source of air or argon gas, (iii) a source of wash solution, and
(iv) a waste reservoir. In one embodiment, each of the first port
and the second port is in fluid communication with a rotary valve
that in turn is in fluid communication with each of (i) the source
of vacuum, (ii) the source of air or argon gas, (iii) the source of
wash solution, and (iv) the waste reservoir.
[0009] Various embodiments of a method of making a flowcell are
also provided herein. For example, in one embodiment, the method
includes forming channel-defining features on a surface of a
substrate, and adhesively bonding a cover layer to the
channel-defining features to form a flowcell comprising at least
one channel, wherein the adhesively bonding comprises curing a
uv-curable adhesive. In one embodiment, the uv-curable adhesive can
include a mercapto-ester adhesive. The channel-defining features
can be formed from, for example, a uv-curable adhesive, and the
channel-defining features can include a mercapto-ester
adhesive.
[0010] In one embodiment, the adhesively bonding step includes
spin-coating a uv-curable adhesive layer onto the cover layer and
then contacting the uv-curable adhesive layer with the
channel-defining features. Additionally, in one embodiment, the
adhesively bonding step can include positioning the cover layer
adjacent the channel-defining features to form a capillary gap
there between, filling the capillary gap with a uv-curable
adhesive, by capillary action, and curing the uv-curable adhesive.
In one embodiment, the forming channel-defining features on a
surface of a substrate step includes applying a layer of uv-curable
adhesive to the surface of the substrate, masking portions of the
layer of uv-curable adhesive layer, curing portions of the
uv-curable adhesive layer that are not masked, by exposure to
uv-radiation, and cleaning off portions of the uv-curable adhesive
layer that were not masked and that were not cured, leaving
channel-defining features formed on the surface of the
substrate.
[0011] Various embodiments of a method of making a flowcell are
provided herein. In one embodiment, the method includes laminating
a die-cut or laser-cut adhesive film onto a surface of a substrate
to form channel-defining features on the surface of the substrate,
and laminating a cover layer to the channel-defining features to
form a flowcell comprising at least one channel In one embodiment,
the die-cut or laser-cut adhesive film comprises a die-cut pressure
sensitive adhesive layer. In one embodiment, the die-cut or
laser-cut adhesive film includes a laser-cut polyimide film.
Additionally, in one embodiment, the cover layer can include a
cyclo-olefin polymer material.
[0012] In one embodiment, the method can further include forming
one or more fluidic inlets to the at least one channel, wherein the
one or more fluidic inlets are formed through the substrate. The
method can further include disposing reagents in the flowcell and
contacting the cover layer with a heating surface of a thermal
cycler. In one embodiment, the method can further include disposing
reagents in the flowcell and contacting the substrate with a
heating surface of a thermal cycler.
[0013] Various embodiments of a method filling a flowcell are also
provided herein. In one embodiment, the method includes introducing
a reagent into a first end of a flowcell, using a pressurized pump,
the flowcell comprising a channel defined at least in part by a
glass slide and a thermal black. While introducing the reagent, the
method can also include drawing a vacuum on an opposite end of the
flowcell to pneumatically facilitate filling the flowcell with the
reagent.
[0014] In one embodiment, the flowcell can include a tapered
configuration having a narrower end at the first end and a wider
end at the opposite end. The flowcell can include a support between
the thermal block and the glass slide, the support being configured
to maintain a desired spacing between the thermal block and the
glass slide.
[0015] Various embodiment of a flowcell are also provided. In one
embodiment, the flowcell includes a first substrate having a first
surface, the first substrate comprising a thermal block, an
alternating electrode band layer having a surface facing a surface
of the thermal block, and a polymeric coating on a surface of the
alternating band layer opposite the surface facing the thermal
block, wherein the polymeric coating defines the first surface. The
flowcell can also include a second substrate comprising a second
surface facing the first surface, the second substrate comprising a
glass slide having a reaction surface comprising a plurality of
reaction sites thereon, the reaction surface defining the second
surface, and a flow channel defined between the first surface and
the second surface. In one embodiment, the reaction surface
comprises a plurality of beads fixed to a surface of the glass
slide. In one embodiment, the flowcell further comprises a printed
circuit board between the thermal block and the alternating
electrode band layer.
[0016] Various embodiments of a flowcell system are also provided
herein. For example, in one embodiment the system includes a
flowcell, a flowcell supply pathway in fluid communication with the
flowcell, and a flowcell supply pathway degassing system configured
to degas gas from the flowcell supply pathway before the gas
reaches the flowcell.
[0017] In one embodiment, the system can further include a detector
positioned along the flowcell supply pathway and configured to
detect gas in the flowcell supply pathway. The system can further
include an aspirator tip in fluid communication with the flowcell
supply pathway and configured to aspirate gas in the flowcell
supply pathway before the gas reaches the flowcell. In one
embodiment, the flowcell system can further include a porous
hydrophobic material defining at least a portion of the flowcell
supply pathway and configured to degas gas in the flowcell supply
pathway by gas venting, before the gas reaches the flowcell. In one
embodiment, the flowcell system can further include an air bubble
injector in fluid communication with the flowcell supply pathway
and configured to inject air bubbles as spacers between reagent
volumes flowing through the flowcell supply pathway.
[0018] Various embodiments of a method of mixing different reagents
together in a flowcell supply pathway are also provided herein. In
one embodiment, the method includes injecting an air bubble into a
flowcell supply pathway of an embodiment of a flowcell system
disclosed herein. The method further includes injecting different
reagents into the flowcell supply pathway, behind the air bubble,
then injecting another air bubble into the flowcell supply pathway,
behind the different injected reagents, and moving the air bubbles
and the different injected reagents through the flowcell supply
pathway to cause mixing of the different injected reagents in the
flowcell supply pathway.
[0019] Various embodiments of a flowcell system are also provided
herein. In one such embodiment, the system includes a flowcell, a
flowcell supply pathway in fluid communication with the flowcell,
and a mixing chamber disposed along the flowcell supply pathway and
in fluid communication with a plurality of different reagent
supplies. The system can also include a flowcell syringe pump
configured to move fluid from the mixing chamber into and through
the flowcell.
[0020] In one embodiment, the flowcell system can further include
an injection manifold assembly comprising the plurality of
different reagent supplies, and a feed line in fluid communication
with the mixing chamber, wherein each of the different reagent
supplies of the plurality of different reagent supplies is in
valved communication with the feed line. In one embodiment, the
system, can also include a multiplex manifold assembly comprising
the plurality of different reagent supplies, a feed line in fluid
communication with the mixing chamber, and a pressure source,
wherein each of the different reagent supplies of the plurality of
different reagent supplies is in valved communication with the feed
line, and the pressure source is configured to force the different
reagent supplies of the plurality of different reagent supplies
into the feed line.
[0021] In one embodiment, a flowcell system is provided which
includes a flowcell, a flowcell supply pathway in fluid
communication with the flowcell, and a reagent supply disposed
along the flowcell supply pathway and in fluid communication with
the flowcell supply pathway. The system can also include a system
fluid supply disposed along the flowcell supply pathway and in
fluid communication with the flowcell supply pathway, and a
regulated air pressure source configured to pump reagent from the
reagent supply into and through the flowcell supply pathway and
configured to pump system fluid from the system fluid supply into
and through the flowcell supply pathway.
[0022] In one embodiment, the system can include a syringe pump
configured to pull system fluid from the system fluid supply and
move the system fluid into the flowcell supply pathway. The system
can also include a second reagent supply disposed along the
flowcell supply pathway and in fluid communication with the
flowcell supply pathway, a first syringe pump configured to pull
system fluid from the system fluid supply and move reagent from the
reagent supply into the flowcell supply pathway, and a second
syringe pump, different than the first, configured to pull system
fluid from the system fluid supply and move second reagent from the
second reagent supply into the flowcell supply pathway.
[0023] These and other embodiments are described in greater detail
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The present disclosure will be more fully understood from
the following detailed description taken in conjunction with the
accompanying drawings, in which:
[0025] FIG. 1 is a side view of a detection system that can be used
to detect signals generated in a flowcell, according to various
embodiments of the present teachings;
[0026] FIG. 2 is a substrate with electrodes configuration for an
electro-wetting fluid manipulation system according to various
embodiments of the present teachings, wherein electrodes define a
deposition region and a number of buses to transport reagents of
interest from reservoirs to specific deposition regions;
[0027] FIG. 3 is a top view of an integrated card device according
to various embodiments of the present teachings, wherein reagent
reservoirs, fluid delivery and mixing paths, and flowcells are all
integrated into the device;
[0028] FIG. 4 is an exploded view of a system according to various
embodiments of the present teachings, showing a sample slide, a
plurality of reagents in a chiller block, and a multi-injector
apparatus;
[0029] FIG. 5 is an exploded view of a system according to various
embodiments of the present teachings, showing a sample slide, a
plurality of reagents in a chiller block, and a zero-volume
multi-injector apparatus;
[0030] FIG. 6 is an exploded view of a system according to various
embodiments of the present teachings, showing a sample slide, a
plurality of reagents in a chiller block, and a single injector
apparatus;
[0031] FIG. 7 is an exploded view of a system according to yet
other various embodiments of the present teachings, showing a
sample slide, a plurality of reagents in a chiller block, and a
single injector apparatus;
[0032] FIG. 8 is a diagram of a system according to various
embodiments of the present teachings, showing a sample slide and a
single injector fluid manipulation system comprising rotator
valves;
[0033] FIG. 9 is a side view of a channel layer for a microchannel
device according to yet other various embodiments of the present
teachings;
[0034] FIG. 10 is a flow diagram of two steps of a manufacturing
method that uses the channel layer of FIG. 9 and a spin-coated
adhesive cover layer, wherein the diagram shows side views of a
cover layer, an adhesive-coated cover layer, and an assembled
microchannel device according to various embodiments of the present
teachings;
[0035] FIG. 11 is a flow diagram of two steps of a manufacturing
method that uses the channel layer of FIG. 9 and a cover layer
adhered thereto by an adhesive applied by a capillary application
method, wherein the diagram shows side views of a cover layer, an
adhesive-application step, and an assembled microchannel device
according to various embodiments of the present teachings;
[0036] FIG. 12 is a flow diagram of three steps of a manufacturing
method to make a channel layer according to various embodiments of
the present teachings, wherein an adhesive is applied by
spin-coating and uv light and a mask are used to form a channel
layer for a microchannel device;
[0037] FIG. 13 is a flow diagram of a step of spin-coating a glass
slide with a uv-curable adhesive to form a cover layer according to
various embodiments of the present teachings;
[0038] FIG. 14 is a side view of an assembled microchannel device
comprising the channel layer of FIG. 12 and the cover layer of FIG.
13, according to various embodiments of the present teachings;
[0039] FIG. 15 is a flow diagram of two steps of a lamination
manufacturing method useful to make an assembled microchannel
device according to various embodiments of the present
teachings;
[0040] FIG. 16 is a side view of a laminating step of a
manufacturing method to form an assembled microchannel device
according to various embodiments of the present teachings;
[0041] FIG. 17 is a side view of a fluidics interface device
comprising a slide and a zirconium coating, according to various
embodiments of the present teachings;
[0042] FIG. 18 is a side view of the fluidics interface device of
FIG. 17, showing the device positioned with respect to a heating
apparatus and an optics system, according to various embodiments of
the present teachings;
[0043] FIG. 19 is a side view of the fluidics interface device of
FIG. 17, showing the device positioned with respect to a heating
apparatus and an optics system, according to other embodiments of
the present teachings;
[0044] FIG. 20 is a side view of a flowcell comprising a tapered
assembly configured to be filled using a vacuum assist system,
according to various embodiments of the present teachings;
[0045] FIG. 21 is a side view of a flowcell comprising a slide
support and configured to be filled using a vacuum assist system,
according to various embodiments of the present teachings;
[0046] FIG. 22 is a side view of a flowcell assembly configured to
be pressure filled using a fluidic pump, according to various
embodiments of the present teachings;
[0047] FIG. 23 is a side view of a flowcell assembly configured to
be filled using electro-wetting, according to various embodiments
of the present teachings;
[0048] FIG. 24 is a side view of a degassing system for degassing a
reagent pathway for a flowcell, according to various embodiments of
the present teachings;
[0049] FIG. 25 is a schematic diagram of a degassing system
comprising a pathway along a porous member, to a flowcell,
according to various embodiments of the present teachings;
[0050] FIG. 26 is a side view of an air bubble aspirator system for
aspirating an air bubble from a reagent pathway for a flowcell,
according to various embodiments of the present teachings;
[0051] FIG. 27 is a schematic diagram of an air bubble aspirating
system comprising an air bubble detector and aspirator along a
reagent pathway to a flowcell, according to various embodiments of
the present teachings;
[0052] FIG. 28 is a schematic diagram of a reagent injection
pumping system for pumping reagents to a flowcell, according to
various embodiments of the present teachings;
[0053] FIG. 29 is a schematic diagram of a multiplexed reagent
injection pumping system for pumping reagents to a flowcell,
according to various embodiments of the present teachings;
[0054] FIG. 30 is a schematic diagram of a pressure-driven air
bubble injection system to promote flow mixing in a flowcell,
according to various embodiments of the present teachings;
[0055] FIG. 31 is a schematic diagram of a reagent injection
pumping system for pumping reagents to a flowcell, according to
various embodiments of the present teachings;
[0056] FIG. 32 is a schematic diagram of a reagent injection
pumping system for pumping reagents to a flowcell, according to
various embodiments of the present teachings.
DETAILED DESCRIPTION
[0057] According to various embodiments of the present teachings, a
fluid handling system is provided that comprises a multi-axis
pipetting robot, a syringe pump, a distribution valve, tubing, a
Peltier-based reagent chiller, and a Peltier flowcell heater. The
components can be off-the-shelf and fluidic processing protocols
using the system can be flexible for a variety of reagents, can be
flexible with respect to changing assays, and can provide robust
mixing and a relatively low cost for hardware. In some embodiments,
many existing components of the Life Technologies SOLiD platform
(Life Technologies, Carlsbad, Calif.) can be used. In some
embodiments, a flowcell is provided for the Life Technologies SOLiD
platform. In one embodiment, an embodiment of a flowcell as
described in Applicant's co-pending U.S. Patent Application Ser.
No. ______, filed on Aug. 31, 2010, entitled "Low-Volume Sequencing
System and Method of Use," the entirety of which is incorporated
herein by reference thereto.
[0058] According to various embodiments, the flowcell can comprise
a system wherein molecules of interest are attached to a
transparent substrate and are observed with an objective. The
surface that has the attached molecules can at least partially
define an enclosed volume into which various reagents can be
introduced to make contact with the molecules of interest. FIG. 1
is a side view of a detection system that can be used to detect
signals generated in the flowcell.
[0059] In FIG. 1, a flowcell 40 is shown which comprises a fluidic
chamber 42 in which reagents can be reacted with molecules of
interest attached to the inner surface 44 of a transparent bead
substrate 46, for example, a glass slide. In the embodiment shown,
the molecules of interest can be colonized on beads 48 that have
been deposited on and fixed to inner surface 44. A gasket seal 50
can be provided to seal fluidic chamber 42. A thermal block 52 can
be provided, for example, as shown, to partially define fluidic
chamber 42.
[0060] Flowcell 40 can be mounted vertically as shown, or
horizontally, or in a diagonal orientation. A pressure driven flow
system can be used to fill the flowcell 40, and gravity can be used
to assist in filling and draining the pressure driven flow system.
The depth of the flowcell can be less than about 300 times the
depths of the viewed features and 10 or less multiples of the
diffusion layers. Dimensions can be minimized to minimize the
volume of reagents that are used. In some embodiments, the depth of
the flowcell is reduced relative to the depth of existing SOLiD
platform flowcells. The flowcells and flowcell systems according to
the present teachings overcome challenges that would otherwise
arise with a reduced flowcell depth. In some embodiments, signals
from multiple fields of view are detected one field at a time. The
signals can be those emitted for a volume of one of a multiple
fields of view.
[0061] According to various embodiments, a massively high
throughput system is provided that comprises a large surface area.
In some embodiments, a system is provided that scans an entire
field after each stepwise addition of nucleic acid bases in a
sequencing reaction. The scanning can occur in a fixed location and
a synchronous addition of bases can be implemented.
[0062] In some embodiments, a system is provided to control the
hydrophobic and hydrophilic nature of surfaces of the flowcell by
means of an electro-wetting system and process. As exemplified in
FIG. 2, a substrate 54 comprising a multiple-electrode
configuration. An electro-wetting fluid manipulation system
according to various embodiments of the present teachings is
provided, wherein electrodes define a plurality of deposition
regions 56 and a number of buses 58 to transport reagents of
interest from reservoirs to specific deposition regions 56.
Depending on electrode density, smaller portions of deposition
regions 56 can be divided to form sub-portions 60 of a size and
shape desired.
[0063] According to various embodiments, a hybrid system is
provided wherein a mixture of small electrodes and electrode pads
of larger surface areas can be combined to create preconfigured
regions. The large electrodes can be used to define deposition
regions, and the small electrodes can each be used as a bus to
transport reagents of interest from a reservoir to a specific
deposition region.
[0064] In some embodiments, a method is provided that uses
electro-wetting properties and an arrangement as exemplified in
FIG. 2 to uniformly fill and evacuate a wide channel having a
shallow depth. The flowcell electro-wetting system can be provided
that utilizes capacitive charging forces to uniformly fill and
evacuate imaging areas with controlled precision.
[0065] In some embodiments, a system is provided that comprises one
electrode and a ground point or plane. Charging can be used to
change the nature of wetting of a flowcell for facilitating
filling. In a one-dimensional system, alternating electrode bands
can be provided to enable charging of flowcell reagents for
controlled filling and evacuation. In a two-dimensional embodiment,
even greater flexibility in terms of flowcell filling can be
provided. Specific flowcell regions can be targeted and customized
deposition geometries can be enabled. The protocols can be
optimized to have imaging, for example, scanning, synchronized with
targeted flowcell filling. Such a desired configuration can enable
synchronous scanning of bases in a single field of view in
embodiments where the system divides detection of the flowcell into
multiple fields of view. Reagent delivery can be targeted to a
single field of view at a time. Each field of view can be
fluidically isolated for reagent delivery. Chip surfaces can be
decreased in size for better yield, within practical limits. By
utilizing a system of targeted reagent delivery system and method
as described herein, high throughput levels can be achieved.
[0066] In some embodiments, methods to improve reaction and
exchange efficiency in a flowcell are provided. Electrodynamic
movement of fluid can be used to improve the efficiency. During a
period of contact between a reagent and molecules of interest
within the flowcell, for example, during incubation or a nucleic
acid incorporation event, mixing can be facilitated by alternating
electrode electro-wetting charges to create movement of the
reagents and thus improve reactivity by promoting greater uniform
exposure of the molecules of interest to the reagents. Exposure to
the residual reagents left behind after evacuation can be minimized
as well. In some embodiments, this reduces the amount of reagents
and time needed for subsequent wash steps.
[0067] According to various embodiments, an integrated on-chip
system is provided that comprises an electro-wetting configuration
of electrodes and deposition regions. The electro-wetting system
can be scaled to incorporate all the fluid handling subsystems at a
low cost. An exemplary embodiment is shown in FIG. 3, which is a
top view of an integrated chip 64. The chip configuration shown
illustrates exemplary components of a chip comprising reagent
reservoirs in the form of wells 66, fluid delivery paths 68, fluid
mixing areas 69, and flowcells 70 all integrated into a card-shaped
substrate 72. Chip 64 can comprise a cover layer 74, for example, a
transparent film, on top of substrate 72. Chip 64 can be configured
to be plugged into an electrical control and detection system (not
shown). This configuration can enable a zero dead volume system
that can manipulate nanoliter-sized volumes. Chip 64 can be loaded
with stored bulk reagents that are deposited into wells 66, and the
reagents can then be distributed, as needed, in any desired
quantity. A simple electrical edge connector 76 can be used for the
electrical interfaces with the electrical control and detection
system. Registration points, holes, or other features such as a
registration corner 78 can be provided to optically couple the
flowcells to the detector, camera, and/or scanner.
[0068] In some embodiments, the integrated chip and system can
enable very low overall reagent consumption and can enable precise
sub-microliter dispensing of expensive sequencing probe reagents.
The system and method provide efficient exchange rates and
reactivity of molecules. The overall foot print can be compact and
small. Very little, if any, tubing is required and the chip can be
easy to manufacture. The system and method provide small dead
volumes and little wasted reagents, and enable high throughput for
synchronous single molecule sequencing platforms. Also, the system
and method enable a reuse of small sample volumes in the microliter
size range. The concepts can be applied to the Life Technologies
(Carlsbad, Calif.) SOLiD platform and fluidic handling subsystems
including next generation SOLiD and single molecule sequencing
systems.
[0069] According to various embodiments, yet another flowcell
system configuration is provided. The flowcell system exhibits
improved fluid handling relative to existing flowcell systems, such
that the flowcell can be better washed, for example, between
reagent delivery steps. The system provides a minimum volume
pathway of reagents from a sample tray, for example, from an
autosampler tray, to a flowcell slide, and thus reduces reagent
usage and washing effort. In some embodiments, reagents can
efficiently be added from an autosampler tray to the slide and can
be efficiently returned back to the autosampler tray.
[0070] In some embodiments, a current autosampler for a Life
Technologies (Carlsbad, Calif.) SOLiD platform is modified to
attach a single injector or multi-injector to the xyz arm. Reagents
can be aspirated from the autosampler tray vial using an existing
syringe pump. In some embodiments, the sample can be either
aspirated into the syringe barrel or into a sample loop having a
hold volume large enough to retain the desired reagent volume. The
device can be used to replace the current plumbing system in the
SOLiD platform. The injector configuration can eliminate any need
for reagent to traverse from a reagent vial, through an autosampler
needle, through a syringe valve block, and through a long length of
tubing on its way to the slide. Retrieval of the reagent from slide
back to the autosampler tray can be provided by reversing the
addition process.
[0071] The shorter pathway can be much less complicated to clean
between reagent delivery steps, relative to longer existing
pathways. Also, the injector/sample loop plumbing can be cleaned
using wash solutions pumped from a large wash solution reservoir
and into a liquid waste station. An exemplary configuration is
shown schematically in FIG. 8, described in more detail below,
which is a diagram of a system according to various embodiments of
the present teachings, showing a sample slide and a single injector
fluid manipulation system comprising rotator valves.
[0072] In some embodiments, the injectors can be configured to
deliver wash solutions via a large reservoir to wash the slide. The
injectors can be pressurized or pump-assisted. The single injector
or multi-injector can deliver air and/or argon, for example, to
assist in removing liquids using a vacuum source. Also, the slide
can be washed without the injector using a source of wash solution
connected to the slide. While washing the slide is described
herein, it is to be understood that it is the flow slide defined by
the slide, which is cleaned and loaded with reagents.
[0073] The injector or injectors can be mated to and/or otherwise
align with ports of the slide so that delivery of reagents can be
made from the top or the bottom of the slide. The configuration can
enable a reduction in bubbles formed during filling the slide and
can improve washing of the slide.
[0074] FIG. 4 is an exploded view of a system 80 according to
various embodiments of the present teachings. System 80 comprises a
flowcell slide apparatus 82, a plurality of reagents in a
multi-compartment chiller block 84, and a multi-injector apparatus
86. Flowcell slide apparatus 82 comprises plumbing described below
and a slide 83. Slide 83 defines a flowcell having an inlet 88 and
an outlet 90. Flexible tubes 92 and 94 can be provided in fluid
communication with inlet 88 and outlet 90, respectively. Flexible
tubes 92 and 94 can enable sample to move as the tubes and flowcell
are filled. Injector ports 96 and 98 and respective vales 100 and
102 are provided to respectively control filling and evacuating of
the flowcell defined by slide 83. Injector ports 96 and 98 can
comprise zero volume ports that are, for example, fixed and in
close proximity to the multi-injector apparatus 86 so
multi-injector apparatus 86 needs only to move a short distance to
be aligned for injection. Inlet 88 and outlet 90 can be provided
with valves as shown. Waste reservoirs 104 and 106 can be provided
in fluid communication with injector ports 96 and 98
respectively.
[0075] As shown in FIG. 4, the plurality of reagents in the
multi-compartment chiller block 84 can be segregated in a plurality
of respective compartments 108. Multi-injector apparatus 86 can
comprise a plurality of injector tips 110 that are configured to
move reagents from chiller block 84 to ports 96 and 98 of slide
apparatus 82. Injector tips 100 can be mounted in an xyz stage 112.
Injector tips 110 can be in fluid communications with a conduit 114
configured to interface with a syringe, a conduit 116 configured to
interface with a wash solution reservoir, a conduit 118 configured
to interface with a source of air and/or argon, and a conduit 120
configured to interface with a vacuum source.
[0076] FIG. 5 is an exploded view of a system 122 according to
various embodiments of the present teachings, showing a zero-volume
injector port slide apparatus 124, a plurality of reagents in a
chiller block 84, and a multi-injector apparatus 86. Zero-volume
injector port slide apparatus 124 comprises plumbing described
below and a slide 83. Slide 83 defines a flowcell having an inlet
88 and an outlet 90. Injector ports 96 and 98 and respective vales
101 and 103 are provided to respectively control filling and
evacuating of the flowcell defined by slide 83. Injector ports 96
and 98 can comprise zero volume ports that are, for example, fixed
and in close proximity to the multi-injector apparatus 86 so
multi-injector apparatus 86 needs only to move a short distance to
be aligned for injection. Waste reservoirs 104 and 106 can be
provided in fluid communication with injector ports 96 and 98
respectively.
[0077] As shown in FIG. 5, the plurality of reagents in the
multi-compartment chiller block 84 can be segregated in a plurality
of respective compartments 108. Multi-injector apparatus 86 can
comprise a plurality of injector tips 110 that are configured to
move reagents from chiller block 84 to ports 96 and 98 of slide
apparatus 124. Injector tips 100 can be mounted in an xyz stage
112. Injector tips 110 can be in fluid communications with a
conduit 114 configured to interface with a syringe, a conduit 116
configured to interface with a wash solution reservoir, a conduit
118 configured to interface with a source of air and/or argon, and
a conduit 120 configured to interface with a vacuum source.
[0078] FIG. 6 is an exploded view of a system 126 according to
various embodiments of the present teachings. System 126 comprises
an xyz movable flowcell slide apparatus 128, a plurality of
reagents in a multi-compartment chiller block 84, and a single
injector apparatus 130. Flowcell slide apparatus 128 comprises
plumbing described below and a slide 83. Slide 83 defines a
flowcell having an inlet 88 and an outlet 90. Flexible tubes 92 and
94 can be provided in fluid communication with inlet 88 and outlet
90, respectively. Flexible tubes 92 and 94 can enable sample to
move as the tubes and flowcell are filled. Injector ports 96 and 98
and respective vales 100 and 102 are provided to respectively
control filling and evacuating of the flowcell defined by slide 83.
Injector ports 96 and 98 can comprise zero volume ports that are,
for example, fixed and in close proximity to the single injector
apparatus 130 so single injector apparatus 130 needs only to move a
short distance to be aligned for injection. Waste reservoirs 104
and 106 can be provided in fluid communication with injector ports
96 and 98 respectively.
[0079] As shown in FIG. 6, the plurality of reagents in the
multi-compartment chiller block 84 can be segregated in a plurality
of respective compartments 108. Single injector apparatus 130 can
comprise a first injector tip 132 that is configured to move
reagents from chiller block 84 to ports 96 and 98 of slide
apparatus 128. First injector tip 132 and second injector tip 134
can be mounted in an xyz stage 136 and can be in fluid
communications with a first conduit 138 and a second conduit 140,
respectively. First conduit 138 can be in fluid communication with
a sample loop and a syringe. Second conduit 140 can be configured,
for example, with a rotary valve, to be in selective fluid
communication with a wash solution reservoir 142, a source of air
and/or argon 144, a vacuum source 146, and a waste reservoir 148.
Valves can provided between conduit 140 and each of the sources
with which it is in fluid communication. Injector tips 132 and 134
can be at different heights. Injector tips 132 and 134 can share
the same stage.
[0080] FIG. 7 is an exploded view of a system 150 according to yet
other various embodiments of the present teachings, showing a
sample slide apparatus 152, a plurality of reagents in a chiller
block 86, and a single injector apparatus 154. Sample slide
apparatus 152 comprises plumbing described below and a slide 83.
Slide 83 defines a flowcell having an inlet 88 and an outlet 90.
Conduits 156 and 158 can be provided in fluid communication with
inlet 88 and outlet 90, respectively. Conduits 156 and 158 can
enable sample to move as the flowcell is filled. Injector ports 96
and 98 and respective vales 100 and 102 are provided to
respectively control filling and evacuating of the flowcell defined
by slide 83. Injector ports 96 and 98 can comprise zero volume
ports that are, for example, fixed and in close proximity to the
multi-injector apparatus 86 so single injector apparatus 154 and/or
slide 83 need only to move a short distance to be aligned for
injection. Waste reservoirs 104 and 106 can be provided in fluid
communication with injector ports 96 and 98, respectively, for
example, through respective rotary valves. One or more vacuum
source 120 can be provided in fluid communication with injector
ports 96 and 98, respectively, for example, through respective
rotary valves. One or more air and/or argon sources can be provided
in fluid communication with injector ports 96 and 98, respectively,
for example, through respective rotary valves. One or more wash
solution reservoirs can be provided in fluid communication with
injector ports 96 and 98, respectively, for example, through
respective rotary valves.
[0081] As shown in FIG. 7, the plurality of reagents in the
multi-compartment chiller block 84 can be segregated in a plurality
of respective compartments 108. Single injector apparatus 154 can
comprise an injector tip 132 that is configured to move reagents
from chiller block 84 to ports 96 and 98 of sample slide apparatus
152. Injector tip 132 can be mounted in an xyz stage 136 and can be
in fluid communications with a conduit 138. Conduit 138 can be in
fluid communication with a sample loop and a syringe.
[0082] FIG. 8 which shows an exploded schematic view of a system
according to various embodiments of the present teachings, showing
a sample slide 160 in a system comprising many of the features
described above with reference to FIGS. 4-7. An autosampler 161 is
also shown along with a sample tray 169 from which sample can be
withdrawn and injected into sample slide 160. Autosampler 161 can
comprise a single injector needle injector 165 operated by a
syringe 163 and a rotary valve 167. FIG. 8 depicts a rotary valve
162 in fluid communication with inlet 88 and many other sources as
described herein. FIG. 8 also depicts a rotary valve 164 in fluid
communication with outlet 90 and many other sources as described
herein. Rotary valves 162, 164, and 167 can each independently be
in fluid communications with a conduit configured to interface with
a syringe 163, a conduit configured to interface with a wash
solution reservoir 116, a conduit configured to interface with a
source of air and/or argon 118, and a conduit configured to
interface with a vacuum source 120. Rotary valves 162, 164, and 167
can each independently be in fluid communication with a conduit
configured to interface with an auxiliary source 170, for example,
a source of buffer, other reagent, pressure differential, or the
like. Rotary valves 162, 164, and 167 can each independently be in
fluid communication with a conduit configured to interface with a
waste conduit or reservoir 104, 106, or 172, respectively.
[0083] The systems shown in FIGS. 4-8 minimize contamination and
loss of reagents caused by dead volume. The systems also enable an
efficient washing of a flowcell and minimize reagent volume usage
by using short pathways and by returning uncontaminated reagent for
reuse.
[0084] According to various embodiments of the present teachings, a
fabrication method is provided to make a flowcell. The
configuration minimizes cost per run for sequencing reactions using
the flowcell. The configuration also reduces run time of a
sequencing reaction using the flowcell, for example, when used in
conjunction with the SOLiD platform available from Life
Technologies of Carlsbad, Calif.
[0085] According to various embodiments, a system comprising a
microfluidics channel is provided that can manipulate a very small
volume of liquid, for example, a volume of 25 uL or less, for
example, of about 10 uL or less. The system provides fast delivery
of reagents and sample to a flowcell and efficiently positions
reagents in the flowcell. In some embodiments, a uv-curable
adhesive is used in the construction of a flowcell according to the
present teachings. In an exemplary embodiment, NORLAND adhesive, a
uv-curable adhesive from Norland Products, is used in manufacturing
a multi-layered flowcell according to the present teachings. The
refractive index of the NORLAND adhesive can approximate that of
silica glass, and it has very low auto-fluorescence. Exemplary
adhesives are NORLAND's optical adhesives, NOA 60 (a mercapto-ester
adhesive), NOA 61 (a mercapto-ester adhesive), NOA 63 (a
mercapto-ester adhesive), NOA 65 (a mercapto-ester adhesive), and
NOA 68 (a mercapto-ester and tetrahydrofurfuryl methacrylate
adhesive). UV-curable glues with these properties can be used in
various embodiments. After fully curing, NORLAND adhesive exhibits
no detrimental effect on enzymes or oligonucleotides used, for
example, in a PCR or other DNA sequencing reaction. The adhesive
can be PCR-compatible. Various NORLAND adhesives generally used for
glass bonding, plastics bonding, and/or glass-plastic bonding, can
be used.
[0086] In an exemplary embodiment, as shown in FIG. 9, a channel
layer 180 can be bonded to a cover layer by spin-coating of the
adhesive, by capillary action placement of the adhesive, by another
adhesive moving force, or by a combination thereof. In FIG. 9, it
can be seen that channel layer 180 comprises a substrate 182 and
channel defining structures, here in the form of walls 184. In some
embodiments, NORLAND adhesive itself can be used to build up a
structure that defines a feature of channel layer 180, a cover
layer, an intermediate layer, or a combination thereof. The
viscosity of the NORLAND is small enough to enable the adhesive to
be moved by capillary action into a very small gap, for example,
into a sub-micrometer (.mu.m) gap. Channel layer 180 can be
fabricated from glass and by a method comprising glass etching,
channel build up by SU-8, a combination thereof, and the like.
Channel layer 180 can be fabricated from plastic and by a method
comprising machining, molding, a combination thereof, and the
like.
[0087] FIG. 10 shows a method of spin-coating a uv-curable
adhesive, for example, a NORLAND adhesive, into a layer 186 on a
glass slide 188 to form a cover layer 190. Subsequently, cover
layer 190 is bonded to channel layer 180 to form a multi-channel
flowcell 192. Bonding can be by contacting channel layer 180 with
cover layer 190 and curing adhesive layer 186 with ultraviolet (uv)
light.
[0088] FIG. 11 shows a method of applying a uv-curable adhesive,
for example, a NORLAND adhesive, as a droplet 187. The viscosity
and properties of the adhesive enable it to be moved by capillary
action into a sub-micrometer gap formed between channel layer 180
and a glass slide 188 that have been brought together. For this
purpose, an exemplary NORLAND adhesive that can be used is NOA 89
(a low viscosity, mercapto-ester and 1,6 hexanediol diacrylate
adhesive). The capillary action causes the adhesive to move in the
direction shown by the arrowhead between glass slide 188 and
channel layer 180. The adhesive can form a layer that can be cured
to bond glass slide 188 to channel layer 180 to form a
multi-channel flowcell 194.
[0089] Bonding a channel layer to a cover layer can be achieved by
any suitable method, but a spin coating method is depicted in FIG.
10 and a capillary action spreading method is depicted in FIG. 11.
As shown in FIGS. 12-14, the spinning speed for the adhesive
coating can be used to determine the height of the channel, for
example, to define a microfluidic channel.
[0090] FIGS. 12-14 show a method of manufacturing a multi-channel
flowcell 200 shown in FIG. 14. Flowcell 200 comprises a channel
layer 202 made the method depicted in FIG. 12, and a cover layer
204 made by the method depicted in FIG. 13. As shown in FIG. 12, an
ordered array, for example, available as a plastic or cyclic
olefin, or a glass slide 206 comprising a metal-oxide layer, is
spin-coated with a uv-curable adhesive to form an adhesive layer
208 on glass slide 206. A mask 210 is applied to adhesive layer 208
and the resulting structure 212 is exposed to uv-light 214. The
uncured adhesive is then removed leaving channels defined by the
cured adhesive.
[0091] As shown in FIG. 13, cover layer 204 is formed by
spin-coating an adhesive layer 216 onto a glass slide 218. Cover
layer 204 is then contacted with channel layer 202 and uv light 220
is used to cure adhesive layer 216.
[0092] Yet other embodiments of the present teachings are shown in
FIGS. 15-19. A fabrication method to construct a flowcell, and a
flowcell system, are provided. The flowcell reduces the cost per
run for a sequencing reaction, reduces the run time for a
sequencing reaction, and is cheaper, simpler, and more reliable
than existing flowcells. In some embodiments, lamination of
pressure sensitive adhesive (PSA) at room-temperature provides a
simple and fast method of making a microfluidics channel structure,
for example, a flowcell.
[0093] As shown in FIG. 15, an ordered array, for example,
available as a plastic or cyclic olefin, or a glass slide 206
comprising a metal oxide coating, is laminated with an adhesive
layer or film, such as a cut adhesive layer, for example, a die-cut
or laser-cut adhesive layer 222. Die-cutting or laser-cutting can
be used to define channel walls 224 in the resulting flowcell
structure. Die-cut or laser-cut adhesive layer 222 can be very
thin, for example, less than 100 .mu.m in thickness. Exemplary
materials for die-cut or laser-cut adhesive film 222 include, but
are not limited to, pressure sensitive adhesive materials,
polyimide materials, and the like. Adhesive film 22 can then be
laminated with a cover layer 226 to form a flowcell 228. Cover
layer 226 can comprise a polymeric material, or the like, for
example, a cyclic olefin polymer material, or the like, and can be
very thin, for example, less than 100 .mu.m in thickness.
[0094] FIG. 16 shows a method of laminating cover layer 226 to a
channel layer 230 by using the nip 232 between two rotating nip
rollers 234 and 236.
[0095] A fluidics interface can be provided for a flowcell such as
flowcell 228 shown in FIG. 15. As mentioned, the thickness of a PSA
layer can be used to determine the height of a microfluidics
channel through flowcell 228, which can be, for example, less than
100 .mu.m. FIG. 17 shows that fluidics inlets and outlets to the
microfluidic channels of a flowcell can be formed by making
through-holes 238 in glass slide 206 or substrate layer, of
flowcell 228 shown in FIG. 15, to form an assembly 240. In the case
of plastics, through holes 238 can be drilled, molded, or otherwise
machined, and in the case of a glass substrate ultrasonic drilling
can be used to provide a very clean and smooth through-hole.
[0096] Fluidic inlet and/or outlet through holes 238 can be
interfaced with an instrument, for example, with a reagent supply
system, by o-ring, septum, an elastic material, a combination
thereof, and the like. The fluidics interface can comprise a gasket
layer with thru-holes formed therein. The gasket can be clamped
against the flowcell, the instrument, or both. In some embodiments,
the gasket fluidics interface is encapsulated in a fluidics
interface assembly and fluidics interface assembly can comprise a
gasket layer on one side against the slide or substrate, and a
fluidics interface with the robot tip at the other side.
[0097] FIGS. 18 and 19 depict how the laminated package can be
interfaced with thermal and optical systems in the instrument, and
show the pros and cons of each orientation. In the configuration
shown in FIG. 18, an optical pathway is directed toward the
brighter side of beads disposed in the flowcell. In FIG. 18, the
flatness of cover film 226 does not affect the flatness of image
taken of the flowcell. In the embodiment of FIG. 19, the relatively
thin layer of cover film 226, compared to the thickness of
substrate 206, provides better thermal conduction compared to the
embodiment of FIG. 18.
[0098] In yet other embodiments of the present teachings, efficient
use is made of reagents to minimize amounts needed. The fluid
handling system manipulates many reagents and takes into
consideration accuracy, precision, residual carryover,
contamination, cost, speed, head pressure, storage conditions and
temperature, dispensing resolution, mixing capability, chemistry
protocols, and uniform filling of the flowcells without voids.
Exemplary configurations and approaches for implementation of a
high performance fluid handling system that takes these factors
into consideration are shown in connection with FIGS. 20-32.
[0099] FIGS. 20-22 show embodiments of the present teachings that
enable effective flowcell filling. In some embodiments, a pneumatic
system is used with or without a pumping system. FIG. 20 shows a
flowcell system 250 comprising a tapered design and that uses
vacuum assist to load reagents. Flowcell system 250 comprises a
flowcell 252 defined between a glass slide 254 and a thermal block
256. Dispensed reagent 258 is pulled into flowcell 252 through a
syringe pump fill port 260 at the bottom of flowcell 252, and
vacuum 262 applied to the top of flowcell 252 facilitates liquid
loading of flowcell 252.
[0100] FIG. 21 shows a flowcell system 251 comprising a
configuration that uses vacuum assist to load reagents. Flowcell
system 251 comprises a flowcell 253 defined between a glass slide
254 and a thermal block 256. Dispensed reagent 258 is pulled into
flowcell 253 through a syringe pump fill port 260 at the bottom of
flowcell 253, and vacuum 262 applied to the top of flowcell 253
facilitates liquid loading of flowcell 253. A support 264 is
provided in a central portion of flowcell 253 to maintain the shape
of flowcell 253 during vacuum loading of sample and reagents.
Support 264 can be configured to maintain a separation distance
between glass slide 254 and thermal block 256.
[0101] FIG. 22 shows a flowcell system 266 comprising a flexible
slide 268 and a valve or port configuration that enables pressure
to be built up inside a flowcell 270 defined between flexible slide
268 and a thermal block 256. A fluidic pump filling system 272 can
be provided, for example, having an opening to flowcell 270 at a
bottom or bottom portion of flowcell 270. Another opening 274 can
be provided, for example, at a top or top portion of flowcell 270,
for example, for introducing sample or reagent and/or for plugging
flowcell 270 so that pressure can be built up inside by fluidic
pump filling system 272. Relaxation of flexible slide 268 after
pressurization can be used as a motive force enabling movement of
reagents into and through flowcell 270.
[0102] In some embodiments, a system 280 and method of filling a
flowcell can involve an electro-wetting configuration as shown in
FIGS. 23A-23C. System 280 offers fast fluid handling performance
and enables a flow through system. System 280 comprises a glass
slide 282 having a plurality of reaction sites and/or beads 284
deposited thereon and defining a surface of a flowcell 286.
Flowcell 286 is also defined by a polytetrafluoroethylene coating
288 on an electrode layer 290 comprising a plurality of electrode
bands 292 (FIG. 23C). FIG. 23C is an enlarged view of portion 2300
shown in FIG. 23B. Electrode bands 292 can be deposited on a PC
board 292 that is in contact with a thermal block 294. With the
flow through system, disposed reagents 296 can follow each other
through flowcell 286 with an air gap between each different
reagent, eliminating the need to evacuate and/or drain flowcell 286
before each subsequent reagent loading step. The new reagents
replace the previous reagents with very little or no carryover. The
method reduces the number of wash steps and makes efficient use of
reagents.
[0103] According to various embodiments, a fluid handling system is
provided that efficiently uses reagents by enabling a relatively
fast diffusion of reagents wherein the diffusion layer thickness is
smaller compared to the height of a flowcell. The present teachings
maximize the efficient incorporation of reagents. Alternating
electrode bands 292 of electrode layer 290 enable electro-wetting
allows and a recirculation loop that assists in diffusion of
reagent molecules to analytes of interest at the reaction sites
and/or beads. Electrode bands or other electro-wetting
configurations of electrodes can be used to enable an effective
means of agitation of reagents within the flowcell. In some
embodiments, reagents are exposed to ambient conditions that allow
for evaporation of reagents effectively increasing the
concentration of the reagents being consumed.
[0104] According to various embodiments, challenges with current
designs are overcome by providing an air gap between reagents
sequentially loaded into a flowcell. In some embodiments, as shown
in FIGS. 24 and 25, a degassing system 300 is provided that
comprises an air bubble 302 or air gap that moves fluids through a
flowcell entry pathway 304 formed, at least in part, by a manifold
body 320. Manifold body 320 can comprise or be in contact with a
porous hydrophobic membrane 322, for example, a gas venting
membrane. Pressure driven flow 306 can be provided by a source to
drive various reagents, for example, reagents 308, 310, and 312,
through flowcell entry pathway 304 on their way to a flowcell 305.
The degassing system enables the use of air bubbles and air spacers
while also enabling a method to degas the air so that it does not
reach the flowcell. FIG. 25 shows a degasser 322 comprising a long
pathway along a porous hydrophobic membrane 322.
[0105] According to various embodiments, FIGS. 26 and 27
demonstrate a degassing system 330 that comprises an air bubble
aspirator 332 configured to aspirate air bubbles between reagent
aliquots sequentially loaded and flowed through a flowcell entry
pathway 334 on their way to a flowcell 335. In some embodiments,
degassing system 330 comprises an air bubble 302 or air gap that
moves fluids through flowcell entry pathway 334 formed, at least in
part, by a manifold body 336. Manifold body 336 can comprise or be
in contact with air bubble aspirator 332. Pressure driven flow 306
can be provided by a source to drive various reagents, for example,
reagents 308, 310, and 312, through flowcell entry pathway 334 on
their way to flowcell 335. FIG. 27 shows air bubble aspirator 332
used in conjunction with, and timed with, a detector 338 along
flowcell entry pathway 334. The aspirating system enables the use
of air bubbles and air spacers along a flowcell entry pathway while
also enabling a method to degas the air so that it is removed and
does not reach the flowcell.
[0106] In some embodiments, the system can automatically remove air
bubbles that may have inadvertently been introduced, in addition to
removing intentionally introduced spacing air gaps. The system can
also detect and remove air bubbles resulting from the breakup of
intentionally injected air gaps. Gentle aspiration can be used to
achieve a robust removal of air bubbles while minimizing reagent
mixing in the flowcell.
[0107] The present teachings provide a system and method for
sensing and monitoring reagent streams to and/or from a flowcell.
The streams can be monitored for air bubbles and the system can be
configured to aspirate air bubbles from the stream. The system and
method can completely remove air bubbles while minimizing the
amount of excess reagents aspirated and preventing reagent
carryover.
[0108] Further embodiments of the present teachings include those
depicted with reference to FIGS. 28-32. In some embodiments,
reagent indexing and delivery is provided by a pipetting robot that
is versatile, accurate, and efficient with small volume dispensing.
In some embodiments, however, a system is configured for moving
reagents with an automated injection system. The systems described
with reference to FIGS. 28-32 incorporate aspects of an injection
manifold system with the flexibility and accuracy of a syringe
pump. The system can replace conventional pipetting and instead can
use an injection valve manifold. In some embodiments, 3-port, 2-way
valves are provided that are connected in series on a manifold that
comprises a common port connecting the valves. The syringe pump
allows for versatility in dispensing volumes. Two exemplary
embodiments are shown in FIGS. 28 and 29.
[0109] FIG. 28 is a schematic diagram of a reagent injection
pumping system for pumping reagents to a flowcell, according to
various embodiments of the present teachings. FIG. 28 shows a
system comprising reagent injection pumping with dedicated valves.
As shown in FIG. 28, a system 350 is provided wherein an injection
manifold assembly 352 allows for zero dead volume, thus preserving
reagents, and a syringe pump 354 allows for precise metering of
reagents. One valve 353 can be provided for each different reagent.
In some embodiments, reagents that are to be mixed can be grouped
into the same manifold such that carryover and cross contamination
are of very little or no concern. In embodiments where carryover is
of concern, a wash cycle can be added to clean the fluid lines.
System 350 can comprise a mixing chamber 356, a flowcell 358, and a
waste reservoir 360. In some embodiments, the pumping method is
indirect, so the reagents do not actually traverse the pump and
distribution valve and the system avoids problems of large dead
volumes associated with the use of too many valves.
[0110] FIG. 29 is a schematic diagram of a multiplexed reagent
injection pumping system for pumping reagents to a flowcell,
according to various embodiments of the present teachings. FIG. 29
shows a system comprising reagent injection pumping and multiplexed
valves. The embodiment enables a low cost approach to minimizing
the number of valves by multiplexing reagents. FIG. 29 shows an
exemplary schematic of a multiplexed valve array system 370
comprising an injection manifold assembly 372 that allows for zero
dead volume, thus preserving reagents, and a syringe pump 374
allows for precise metering of reagents. Valves 376 can be
provided, one for each reagent column and one for each reagent row.
Reagents that are to be mixed can be grouped into the same manifold
such that carryover and cross contamination are of very little or
no concern. System 370 can comprise a mixing chamber 378, a
flowcell 380, and a waste reservoir 382. In some embodiments, the
pumping method is indirect, so the reagents do not actually
traverse the pump and distribution valve and the system avoids
problems of large dead volumes associated with the use of too many
valves.
[0111] In some embodiments, a multiplexed system is provided
wherein there is one valve for each row of reagents and reagent
columns are addressed by another set of valves. For example, to
address 25 reagents, 10 valves are needed (five for five rows and
five for five columns) The column valves can be configured to
enable pressurization or vacuum release, enabling a particular
reagent to be pulled out preferentially to other reagents in the
row.
[0112] In some embodiments, effective reagent mixing can be
performed by adding all the reagents of interest into a mixing
chamber. Then the contents of the chamber can be exposed to several
cycles of aspiration and dispensing. Another method to mix reagents
is to introduce reagents into a channel under laminar flow
conditions trapped between air bubbles. As the slug moves along the
channel under pressure driven flow, the contents are mixed due to
the fluidic drag of the side walls. FIG. 30 illustrates the
effect.
[0113] FIG. 30 is a schematic diagram of a pressure-driven air
bubble injection system, and an enlarged close-up of a portion
thereof, useful to promote flow mixing along a pathway to a
flowcell, according to various embodiments of the present
teachings. As shown in FIG. 30, a flowcell entry pathway 390 is in
fluid communication with an air bubble injector 392 comprising a
valve 394. Three reagent sources 396, 398, and 400 provide three
different respective reagents to flowcell entry pathway 390. After
an injection of a first air bubble 402 into entry pathway 390, all
three reagents are then injected to entry pathway 390 followed by
injection of a second air bubble 404. Movement of air bubbles 402
and 404, and the reagents between the air bubbles, through entry
pathway 390, causes mixing currents 406 as demonstrated in the
enlarged close-up portion of FIG. 30. Mixing currents 406 can
promote mixing of the three reagents as they travel through entry
pathway 390 on their way toward a flowcell (not shown).
[0114] Among the advantages achieved by a system as shown in FIG.
30, the components are simple, the system is compact and has a
small overall footprint, there is an ability to address many
reagents, there are small reagent delivery volumes that can be
dispensed precisely, there is less tubing than existing fluid
supply pathways, the system is easy to manufacture, there is less
dead volume and wasted reagents compared to known systems, there
are shorter fluidic pathways that require less washing than longer
pathways, and there is an increase in intermolecular interaction
efficiency. Other advantages include no crystallization of solids
on sliding seals, less time to test effects of chemicals on pumps,
less volume to switch fluids such as rinse solutions and reagents,
and less cross-contamination of fluids. These teachings can be
applied to platforms that use fluidic handling subsystems, for
example, including the SOLiD platform from Life Technologies of
Carlsbad, Calif.
[0115] In yet another embodiment of the present teachings, FIG. 31
shows a schematic diagram of a reagent injection pumping system for
pumping reagents to a flowcell. A system 410 is shown wherein a
regulated air pressure source 412 provides regulated air pressure
to a deionized system fluid source 414 and to a chemical source 416
such as a source of a reagent. Flow restrictors 418 and 420 can be
provided as well as injection valves 422 and 424, to control
injection of air, reagents, and system fluid along a pathway 426 to
a flowcell. A syringe pump 428 can also be used to control the flow
of fluids in the correct direction and rate, through pathway
426.
[0116] FIG. 32 is a schematic diagram of a reagent injection
pumping system for pumping reagents to a flowcell, according to yet
other embodiments of the present teachings. A system 430 is shown
wherein a regulated air pressure source 432 provides regulated air
pressure to a deionized system fluid source 434 and to chemical
sources 436, 438, 440, and 442, such as sources of different
respective reagents. Flow restrictors can be provided as well as
injection valves to control injection of air, reagents, and system
fluid along pathways to a flowcell. A plurality of syringe pumps
448 can also be used to control the flow of fluids in the correct
direction and rate, through the pathways. The system can comprise a
source of elution buffer 450 driven by a syringe pump 458 to a
dispensing pipette 460.
[0117] One skilled in the art will appreciate further features and
advantages of the present disclosure based on the above-described
embodiments. Accordingly, the disclosure is not to be limited by
what has been particularly shown and described, except as indicated
by the appended claims. All publications and references cited
herein are expressly incorporated herein by reference in their
entirety.
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