U.S. patent application number 14/250776 was filed with the patent office on 2014-10-16 for sampling device.
This patent application is currently assigned to STOKES BIO LIMITED. The applicant listed for this patent is STOKES BIO LIMITED. Invention is credited to Kieran Curran, Damian Curtin, Julie Garvey, David McGuire.
Application Number | 20140308671 14/250776 |
Document ID | / |
Family ID | 43123650 |
Filed Date | 2014-10-16 |
United States Patent
Application |
20140308671 |
Kind Code |
A1 |
McGuire; David ; et
al. |
October 16, 2014 |
Sampling Device
Abstract
The present invention generally relates to devices, systems, and
methods for acquiring and/or dispensing a sample without
introducing a gas into a microfluidic system, such as a liquid
bridge system. An exemplary embodiment provides a sampling device
including: a sampling member for acquiring or dispensing a sample;
and a supply of a fluid that is immiscible with the sample; in
which the device is configured to provide a continuous flow of
immiscible fluid enveloping the sampling member.
Inventors: |
McGuire; David; (Limerick,
IE) ; Curran; Kieran; (Limerick, IE) ; Garvey;
Julie; (Limerick, IE) ; Curtin; Damian;
(Kerry, IE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
STOKES BIO LIMITED |
Limerick |
|
IE |
|
|
Assignee: |
STOKES BIO LIMITED
Limerick
IE
|
Family ID: |
43123650 |
Appl. No.: |
14/250776 |
Filed: |
April 11, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12468367 |
May 19, 2009 |
8697011 |
|
|
14250776 |
|
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Current U.S.
Class: |
435/6.12 ;
435/287.2 |
Current CPC
Class: |
B01L 3/0293 20130101;
C12Q 1/686 20130101; B01L 3/021 20130101; B01L 2200/0642 20130101;
C12Q 1/6806 20130101; B01L 7/52 20130101 |
Class at
Publication: |
435/6.12 ;
435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-18. (canceled)
19. A system for sample acquisition comprising: a sampling member;
a vessel for containing a sample and an overlay of a fluid that is
immiscible with the sample; wherein a distal end of the sampling
member is configured such that it is not removed above the
immiscible overlay between sample acquisitions.
20. The system according to claim 19, further comprising a robotics
system that controls movement of the sampling tube.
21. The system according to claim 20, further comprising a pump
connected to the sampling tube.
22. The system according to claim 19, further comprising a liquid
bridge that is in a fluid contact with the sampling tube.
23. A system for sample acquisition comprising: a sampling device
comprising an outer sheath and a plurality of tubes within the
sheath, wherein at least one of the tubes acquires a sample, and at
least one of the tubes expels a fluid that is immiscible with the
sample, wherein the at least one tube that acquires the sample is
extendable beyond a distal end of the sheath and retractable to
within the sheath; and a vessel for containing a sample and an
overlay of a fluid that is immiscible with the sample; wherein a
distal end of the outer sheath and the tube that acquires the
sample are configured to interact with the vessel to acquire the
sample without also acquiring a gas.
24. The system according to claim 23, wherein a distal portion of
the outer sheath is filled with the immiscible fluid, continuously
immersing a distal portion of the tube that acquires the sample in
the immiscible fluid.
25. The system according to claim 23, wherein a counter-flow exists
between the tube that expels the immiscible fluid and the tube that
acquires the sample.
26. The system according to claim 25, wherein the immiscible fluid
is continuously expelled by the at least one tube that expels the
immiscible fluid.
27. The system according to claim 26, wherein the immiscible fluid
is continuously taken in by the at least one tube that acquires the
sample.
28. The system according to claim 23, further comprising at least
one robotics system that controls movement of the sampling device,
and controls movement of the tube that acquires the sample.
29. The system according to claim 28, further comprising a first
pump connected to the at least one tube that acquires the sample,
and a second pump connected to the at least one tube that expels
the immiscible fluid.
30. The system according to claim 23, further comprising a liquid
bridge that is in fluid contact with the sampling device.
31. A method for acquiring a sample comprising: contacting a
sampling member to a vessel containing a sample, wherein the
sampling member is enveloped in a fluid that is immiscible with the
sample; acquiring the sample from the vessel, wherein the sample is
acquired without the introduction of a gas into the sampling
member.
32. The method according to claim 32, wherein there is a
counter-flow of the immiscible fluid from the exterior of the
sampling member to an interior of the sampling member.
33. The method according to claim 32, wherein the immiscible fluid
flows down an exterior of the sampling member, and is taken up an
interior of the sampling member.
34. The method according to claim 31, further comprising flowing
the sample to a liquid bridge.
35. The method according to claim 31, further comprising performing
PCR on the sample.
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to devices, systems,
and methods for acquiring and/or dispensing a sample without
introducing a gas into a microfluidic system, such as a liquid
bridge system.
BACKGROUND
[0002] Microfluidics is a technology involving micro-scale devices
that handle small volumes of fluids, e.g., microliter, nanoliter,
picoliter, or femtoliter volumes. Because microfluidics can
accurately and reproducibly control and dispense small volumes of
fluid, in particular volumes less than 1 .mu.l, application of
microfluidics provides significant cost-savings. The use of
microfluidics technology reduces cycle times, shortens
time-to-results, and increase throughput. Furthermore incorporation
of microfluidics technology enhances system integration and
automation.
[0003] An exemplary microfluidic device involves liquid bridge
technology. Liquid bridge technology involves sample droplet
formation utilizing immiscible fluids. A sample droplet is formed
at an end of an inlet port that extends into a chamber that is
filled with a carrier fluid. The carrier fluid is immiscible with
the sample droplet. The sample droplet grows until large enough to
span a gap to an outlet port in the chamber, forming an
axisymmetric liquid bridge. By adjusting the flow rate or by
introducing a second sample droplet to the first sample droplet, an
unstable funicular bridge is formed that subsequently ruptures from
the inlet port. After rupturing from the inlet port, the sample
droplet enters the outlet port, surrounded by the carrier fluid
from the chamber. The process then repeats itself.
[0004] Given the small dimensions of microfluidic systems that
utilize liquid bridge technology, introduction of gas into the
system presents significant operation problems. The most common
cause of gas introduction into a liquid bridge system is during
sample acquisition, i.e., interaction between a sample tip and a
vessel for acquiring the sample and introducing the sample into the
system. Once gas is introduced into the system, the system needs to
be shutdown and purged to remove the gas. Purging the system and
re-equilibrating the system for operation wastes time and valuable
resources.
[0005] There is an unmet need for devices and systems that can
acquire a sample and interface with a system without introducing a
gas into the system.
SUMMARY
[0006] The present invention generally relates to devices, systems,
and methods for acquiring and/or dispensing a sample without
introducing a gas into a microfluidic system, such as a liquid
bridge system. Devices and systems of the invention accomplish
sample acquisition without introduction of a gas by utilizing
counter-flow principles, thus providing a continuous flow of
immiscible fluid to envelop a sampling member. Accordingly, the
invention provides sample acquisition devices that can interact
with a vessel to introduce a sample into a microfluidic system,
e.g., a liquid bridge system, without introducing gas into the
system, thus avoiding the detrimental effects that a gas has on a
microfluidic system. Sampling devices and systems of the invention
improve microfluidic system efficiency by eliminating system
down-time that is involved with purging the microfluidic system to
remove unwanted gas, and re-equilibrating the system for
operation.
[0007] Numerous configurations of devices and systems that
accomplish sample acquisition and/or dispensing without
introduction of a gas are provided herein. One configuration of a
sampling device includes: a sampling member for acquiring or
dispensing a sample; and a supply of a fluid that is immiscible
with the sample; in which the device is configured to provide a
continuous flow of immiscible fluid enveloping the sampling member.
In this configuration, there is counter-flow of the immiscible
fluid from an exterior of the sampling member to an interior of the
sampling member.
[0008] The device can be configured for sample acquisition by
flowing the immiscible fluid down an exterior of the sampling
member, and taking in the immiscible fluid up an interior of the
sampling member. The device can be configured for sample dispensing
by flowing the immiscible fluid down an interior and an exterior of
the sampling member.
[0009] Another configuration of a sampling device includes: an
outer sheath; and a plurality of tubes within the sheath, in which
at least one of the tubes acquires a sample, and at least one of
the tubes expels a fluid that is immiscible with the sample,
wherein the at least one tube that acquires the sample is
extendable beyond a distal end of the sheath and retractable to
within the sheath. In this configuration, a distal portion of the
outer sheath is filled with the immiscible fluid, continuously
immersing the distal portion of the tube that acquires the sample
in the immiscible fluid.
[0010] In this configuration, there is counter-flow of the
immiscible fluid between the tube that expels the immiscible fluid
and the tube that acquires the sample. For example, the immiscible
fluid is continuously expelled from the tube that expels the
immiscible fluid, and the immiscible fluid is continuously taken in
by the tube that acquires the sample. The outer sheath of the
device is configured to interact with a vessel, and the tube that
acquires the sample is configured to interact with the sample in
the vessel.
[0011] Devices of the invention can be configured to be detachable
from, and adapted for coupling to, a pipette head of a pipette.
Devices of the invention can be configured to be detachable from,
and adapted for coupling to, a pipette head attachment assembly of
an autopipettor. Devices of the invention can be configured to
operate in fluid contact with a liquid bridge system.
[0012] An exemplary system for sample acquisition includes: a
sampling member; a vessel for containing a sample and an overlay of
a fluid that is immiscible with the sample; in which a distal end
of the sampling member is configured such that it is not removed
above the immiscible overlay between sample acquisitions. When the
sampling member needs to be removed from the vessel so that the
vessel can be removed from the system and another vessel can be
inserted, the system continuously expels immiscible fluid from the
sampling member as the sampling member is extracted from the vessel
and as the sampling member remains extracted from the vessel. Thus
the sampling member does not take in a gas during sample
acquisition, between sample acquisitions, and between vessel
changes.
[0013] The system can further include a robotics system that
controls movement of the sampling tube and a pump connected to the
sampling member. The system can also further include a liquid
bridge that is in fluid contact with the sampling member, a
thermocycler, and a detection system, such as an optics system.
[0014] Another exemplary system for sample acquisition includes: a
sampling device including an outer sheath and a plurality of tubes
within the sheath, in which at least one of the tubes acquires a
sample, and at least one of the tubes expels a fluid that is
immiscible with the sample, wherein the at least one tube that
acquires the sample is extendable beyond a distal end of the sheath
and retractable to within the sheath; and a vessel for containing a
sample and an overlay of a fluid that is immiscible with the
sample; in which a distal end of the outer sheath and the tube that
acquires the sample are configured to interact with the vessel to
acquire the sample without also acquiring a gas.
[0015] The system can further include a robotics system that
controls movement of the sampling device, and controls movement of
the sample acquisition tube. The system can further include a first
pump connected to the sample acquisition tube, and a second pump
connected to the at least one tube that expels the immiscible
fluid. The system can also further include a liquid bridge that is
in fluid contact with the sampling tube, a thermocycler, and a
detection system, such as an optics system.
[0016] The vessel can be a plate, for example a 96 well or 384 well
microtiter plate. The sample can be any chemical or biological
species. Certain samples include genetic material. Other samples
can include PCR reagents. The immiscible fluid is chosen based on
the nature of the sample. If the sample is hydrophilic in nature,
the immiscible fluid chosen is a hydrophobic fluid. An exemplary
hydrophobic fluid is oil, such as silicone oil. If the sample is
hydrophobic in nature, the immiscible fluid chosen is a hydrophilic
fluid.
[0017] The invention also provides a method for acquiring a sample
including; contacting a sampling member to a vessel containing a
sample, in which the sampling member is enveloped in a fluid that
is immiscible with the sample; and acquiring the sample from the
vessel, in which the sample is acquired without the introduction of
a gas into the sampling member. The method utilizes counter-flow of
the immiscible fluid. For example, the immiscible fluid flows down
an exterior of the sampling member, and is taken up an interior of
the sampling member.
[0018] The method can further include, flowing the sample to a
liquid bridge, flowing the sample to a thermocycler, analyzing the
sample, or performing PCR on the sample.
[0019] These and other aspects, features, and benefits according to
the invention will become clearer by reference to the drawings
described below and also the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is an embodiment of a sampling device, panel A
showing sample acquisition and panel B showing sample
dispensing.
[0021] FIG. 2 is another embodiment of a sampling device.
[0022] FIG. 3, panels A and B are drawings showing different
configurations of tubes for the device shown in FIG. 2.
[0023] FIG. 4, panels A to E are drawings depicting an embodiment
of a system having a sampling member and a vessel, and also depict
interaction of the sampling member and the vessel.
[0024] FIG. 5, panels A to E are drawings depicting an embodiment
of a system having a sampling member and a vessel, and also depict
interaction of the sampling member and the vessel.
DETAILED DESCRIPTION
[0025] The present invention generally relates to devices, systems,
and methods for acquiring and/or dispensing a sample without
introducing a gas into a microfluidic system, such as a liquid
bridge system. Numerous configurations of devices and systems that
accomplish sample acquisition and/or dispensing without
introduction of a gas into a microfluidic system are provided
herein.
[0026] FIG. 1 shows a configuration of a sampling device 100 for
sample acquisition and/or dispensing without introduction of gas
into a microfluidic system, e.g., a liquid bridge system. The
sampling device 100 includes a sampling member 101 for acquiring
(FIG. 1, panel A) or dispensing (FIG. 1, panel B) a sample. A
sampling member refers to any type of device used to acquire and/or
dispense a sample. Exemplary sampling members include tubes,
channels, capillaries, pipette tips, or probes. The sampling member
can be of any shape, for example, a cylinder, a regular polygon, or
an irregular polygon. The sampling member can be made of any
material suitable to interact with biological or chemical species.
Exemplary materials include TEFLON (commercially available from
Dupont, Wilmington, Del.), polytetrafluoroethylene (PTFE;
commercially available from Dupont, Wilmington, Del.), polymethyl
methacrylate (PMMA; commercially available from TexLoc, Fort Worth,
Tex.), polyurethane (commercially available from TexLoc, Fort
Worth, Tex.), polycarbonate (commercially available from TexLoc,
Fort Worth, Tex.), polystyrene (commercially available from TexLoc,
Fort Worth, Tex.), polyetheretherketone (PEEK; commercially
available from TexLoc, Fort Worth, Tex.), perfluoroalkoxy (PFA;
commercially available from TexLoc, Fort Worth, Tex.), or
fluorinated ethylene propylene (FEP; commercially available from
TexLoc, Fort Worth, Tex.).
[0027] Sampling device 100 further includes a supply of a fluid 106
that is immiscible with the sample. The supply of fluid can be
directly coupled to the sampling member. Alternatively, the supply
of fluid can be indirectly coupled to the sampling member, such as
by tubing or channels. Determination of the fluid to be used is
based on the properties of the sample. If the sample is a
hydrophilic sample, the fluid to used should be a hydrophobic
fluid. An exemplary hydrophobic fluid is oil, such as AS100
silicone oil (commercially available from Union Carbide
Corporation, Danbury, Conn.). Alternatively, if the sample is a
hydrophobic sample, the fluid to used should be a hydrophilic
fluid. One of skill in the art will readily be able to determine
the type of fluid to be used based on the properties of the
sample.
[0028] Sample device 100 is configured to provide a continuous flow
of immiscible fluid 102 enveloping the sampling member 101. This is
accomplished by utilizing counter-flow between the exterior 103 of
the sampling member 101 and the interior 104 of the sampling member
101. FIG. 1, panel A is a drawing depicting an embodiment in which
there is counter-flow of the immiscible fluid 102 from an exterior
103 of the sampling member 101 to an interior 104 of the sampling
member 101. In this configuration, the device can be utilized for
sample acquisition. FIG. 1, panel B is a drawing depicting an
embodiment in which the device 100 is configured for sample
dispensing by flowing the immiscible fluid 102 down an interior 104
and an exterior 103 of the sampling member 101.
[0029] Flow rates of the immiscible fluid are controlled by a fluid
controller, e.g., a PC running WinPumpControl software (Open Cage
Software, Inc., Huntington, N.Y.), connected to at least one pump.
An exemplary pump is shown in Davies et al. (WO 2007/091229). Other
commercially available pumps can also be used. Exemplary flow rates
range from about 1 .mu.l/min to about 100 .mu.l/min. An exemplary
flow rate is about 1 .mu.l/min, 3 .mu.l/min, 5 .mu.l/min, 10
.mu.l/min, 20 .mu.l/min, 30 .mu.l/min, 50 .mu.l/min, 70 .mu.l/min,
90 .mu.l/min, 95 .mu./min, or about 100 .mu.l/min. In certain
embodiments, the flow rate of immiscible fluid 102 down the
exterior 103 of the sampling member 101 is similar to or the same
as the flow rate of the immiscible fluid 102 up the interior 104 of
the sampling member 101. In certain embodiments, the flow rate of
immiscible fluid 102 down the exterior 103 of the sampling member
101 is slightly greater than the flow rate of the immiscible fluid
102 up the interior 104 of the sampling member 101. For example,
the flow rate of immiscible fluid 102 down the exterior 103 of the
sampling member 101 is about 10 .mu.l/min, while the flow rate of
the immiscible fluid 102 up the interior 104 of the sampling member
101 is about 8 .mu.l/min. Because the flow rate of the immiscible
fluid 102 down the exterior 103 of the sampling member 101 is about
the same as or greater than the flow rate of the immiscible fluid
102 up the interior 104 of the sampling member 101, the sampling
member 101 is continuously enveloped by the immiscible fluid 102.
Therefore, the sampling member 101 can acquire a sample without
introduction of a gas into a microfluidic system, e.g., a liquid
bridge system.
[0030] FIG. 2 shows a configuration of a sampling device 200 for
sample acquisition and/or dispensing without introduction of gas
into a microfluidic system, e.g., a liquid bridge system. The
sampling device 200 includes an outer sheath 201; and a plurality
of tubes within the sheath 201. In FIG. 2, device 200 is shown with
two tubes 203 and 204 that acquire a sample. However, device 200
can be configured with only a single tube for sample acquisition,
or can be configured with more than two tubes for sample
acquisition, e.g., 3 tubes, 4 tubes, 5 tubes, 10 tubes, 15 tubes,
20 tubes, 50 tubes, etc. In FIG. 2, device 200 is shown with one
tube 202 that expels a fluid that is immiscible with the sample
205. However, device 200 can be configured with more than one tube
that that expels a fluid that is immiscible with the sample, e.g.,
3 tubes, 4 tubes, 5 tubes, 10 tubes, 15 tubes, 20 tubes, 50 tubes,
etc. In device 200, the tubes that acquires the sample 203 and 204
are extendable beyond a distal end of the sheath and retractable to
within the sheath. FIG. 2 shows the sample acquisition tubes 203
and 204 retracted within the outer sheath 201.
[0031] FIG. 3, panel A shows a depiction of a of sampling device
200, having a center tube 301 that expels a fluid that is
immiscible with the sample, and four sample acquisition tubes 302
to 305 within outer sheath 306. FIG. 3, panel B shows a depiction
of a sampling device 200, having a tube 307 that expels a fluid
that is immiscible with the sample that is centered around 12
sample acquisition tubes 308 to 319, within outer sheath 320. The
tube that expels the immiscible fluid can have the same inner
diameter and outer diameter as the sample acquisition tubes.
Alternatively, the tube that expels the immiscible fluid can have a
different inner diameter and a different outer diameter than the
sample acquisition tubes. Exemplary dimensions of tubes 301 to 305
and 307 to 319 include an inner diameter of about 150 .mu.m and an
outer diameter of about 300 .mu.m. The diameter of the outer sheath
is dependant on the total number of tubes, and the configuration of
the tubes.
[0032] The outer sheath and the plurality of tubes can be of any
shape, for example, a cylinder, a regular polygon, or an irregular
polygon. The shape of the outer sheath is independent of the shape
of the plurality of tubes. The outer sheath and the plurality of
tubes can be made of any material suitable to interact with
biological or chemical species. Exemplary materials include TEFLON
(commercially available from Dupont, Wilmington, Del.),
polytetrafluoroethylene (PTFE; commercially available from Dupont,
Wilmington, Del.), polymethyl methacrylate (PMMA; commercially
available from TexLoc, Fort Worth, Tex.), polyurethane
(commercially available from TexLoc, Fort Worth, Tex.),
polycarbonate (commercially available from TexLoc, Fort Worth,
Tex.), polystyrene (commercially available from TexLoc, Fort Worth,
Tex.), polyetheretherketone (PEEK; commercially available from
TexLoc, Fort Worth, Tex.), perfluoroalkoxy (PFA; commercially
available from TexLoc, Fort Worth, Tex.), or Fluorinated ethylene
propylene (PEP; commercially available from TexLoc, Fort Worth,
Tex.).
[0033] Device 200 utilizes counter-flow between the tube 202 that
continuously expels a fluid that is immiscible with the sample 205,
and sample acquisition tubes 203 and 204 that continuously take in
immiscible fluid 205. Flow rates of the immiscible fluid are
controlled by a fluid controller, e.g., a PC running WinPumpControl
software (Open Cage Software, Inc., Huntington, N.Y.), connected to
at least one pump. An exemplary pump is shown in Davies et al. (WO
2007/091229). Other commercially available pumps can also be used.
Exemplary flow rates range from about 1 .mu.l/min to about 100
.mu.l/min. An exemplary flow rate is about 1 [l/min, 3 .mu./min, 5
.mu.l/min, 10 .mu.l/min, 20 .mu.l/min, 30 .mu.l/min, 50 .mu.l/min,
70 .mu.l/min, 90 .mu.l/min, 95 .mu.l/min, or about 100
.mu.l/min.
[0034] In certain embodiments, flow is controlled such that the
flow rate out of the tube 202 that continuously expels the
immiscible fluid 205 is the same or similar to the total intake
flow rate of sample acquisition tubes 203 and 204. For example, the
flow rate out of tube 202 can range from about 2 .mu.l/min to about
100 .mu.l/min, while the intake flow rate for each of sample
acquisition tubes 203 and 204 can range from about 1 .mu.l/min to
about 50 .mu.l/min. Exemplary flow rates are as follows: flow rate
of 2 .mu.l/min expelled from tube 202, with an intake flow rate for
each of sample acquisition tubes 203 and 204 of 1 .mu.l/min; flow
rate of 6 .mu.l/min expelled from tube 202, with an intake flow
rate for each of sample acquisition tubes 203 and 204 of 3
.mu.l/min; flow rate of 10 .mu.l/min expelled from tube 202, with
an intake flow rate for each of sample acquisition tubes 203 and
204 of 5 .mu.l/min; flow rate of 20 .mu.l/min expelled from tube.
202, with an intake flow rate for each of sample acquisition tubes
203 and 204 of 10 .mu.l/min; and flow rate of 100 .mu.l/min
expelled from tube 202, the intake flow rate for each of sample
acquisition tubes 203 and 204 is 50 .mu.l/min.
[0035] Alternatively, the flow rate out of tube 202 is greater than
the total intake flow rate of sample acquisition tubes 203 and 204.
For example, the flow rate out of tube 202 can range from about 5
.mu.l/min to about 100 .mu.l/min, while the intake flow rate for
each of sample acquisition tubes 203 and 204 can range from about 1
.mu.l/min to about 95 .mu.l/min. Exemplary flow rates are as
follows: flow rate of 6 .mu.l/min expelled from tube 202, with an
intake flow rate for each of sample acquisition tubes 203 and 204
of 2 .mu.l/mint: flow rate of 10 .mu.l/min expelled from tube 202,
with an intake flow rate for each of sample acquisition tubes 203
and 204 of 4 .mu.l/min; flow rate of 20 .mu.l/min expelled from
tube 202, with an intake flow rate for each of sample acquisition
tubes 203 and 204 of 8 .mu.l/min; and flow rate of 100 .mu.l/min
expelled from tube 202, with an intake flow rate for each of sample
acquisition tubes 203 and 204 of 48 .mu.l/min, in this regard, a
slightly greater amount of immiscible fluid is expelled into the
outer sheath than is taken in by the sample acquisition tubes.
Thus, a lower portion of the outer sheath 208 is continuously
filled with the immiscible fluid 205, and distal portions 206 and
207 of sample acquisition tubes 203 and 204 are continuously
immersed in the immiscible fluid.
[0036] The devices of the invention can be configured to be
detachable from, and adapted for coupling to, a pipette head of a
pipette. The devices of the invention can be configured to be
detachable from, and adapted for coupling to, a pipette head
attachment assembly of an autopipettor.
[0037] FIG. 4 depicts a system 400 including a sampling member 401
and a vessel 402, and shows interaction of the sampling member 401
and the vessel 402 for acquisition of samples. The vessel 402, can
be any type of vessel that is suitable for holding a sample.
Exemplary vessels include plates (e.g., 96 well or 384 well
plates), eppendorf tubes, vials, beakers, flasks, centrifuge tubes,
capillary tubes, cryogenic vials, bags, cups, or containers, The
vessel can be made of any material suitable to interact with
biological or chemical species. Exemplary materials include TEFLON
(commercially available from Dupont, Wilmington, Del.),
polytetrafluoroethylene (PTFE; commercially available from Dupont,
Wilmington, Del.), polymethyl methacrylate (PMMA; commercially
available from TexLoc, Fort Worth, Tex.), polyurethane
(commercially available from TexLoc, Fort Worth, Tex.),
polycarbonate (commercially available from TexLoc, Fort Worth,
Tex.), polystyrene (commercially available from TexLoc, Fort Worth,
Tex.), polyetheretherketone (PEEK; commercially available from
TexLoc, Fort Worth, Tex.), perfluoroalkoxy (PFA; commercially
available from TexLoc, Fort Worth, Tex.), or Fluorinated ethylene
propylene (FEP; commercially available from TexLoc, Fort Worth,
Tex.).
[0038] In this figure, the vessel is a plate. The plate has wells
403 and 404, and side walls that extend above the top of each well,
forming a recessed area 405 within the plate. The bottom portion of
each well is filled with samples 406 and 407, and the remaining
portion of each well 406 and 407 along with the recessed area 405
is filled with an overlay of a fluid that is immiscible with the
sample 408.
[0039] The system is primed by flowing the immiscible fluid out of
sampling member 401, until sampling member 401 is inserted into the
overlay of immiscible fluid 408. Once sampling member 401 is
inserted into the overlay of immiscible fluid 408, system pumps
reverse the flow of immiscible fluid, and the sampling member 401
takes in immiscible fluid from the overlay of immiscible fluid 408
(FIG. 4, panel A). The sampling member 401 is shown as a tube in
this figure, however, the sampling member can be any device that
can acquire a sample, such as a channel, a capillary, a pipette
tip, or a probe. The sampling member can be of any shape, for
example, a cylinder, a regular polygon, or an irregular polygon.
The sampling member can be made of any material suitable to
interact with biological or chemical species. Exemplary materials
include TEFLON (commercially available from Dupont, Wilmington,
Del.), polytetrafluoroethylene (PTFE; commercially available from
Dupont, Wilmington, Del.), polymethyl methacrylate (PMMA;
commercially available from TexLoc, Fort Worth, Tex.), polyurethane
(commercially available from TexLoc, Fort Worth, Tex.),
polycarbonate (commercially available from TexLoc, Fort Worth,
Tex.), polystyrene (commercially available from TexLoc, Fort Worth,
Tex.), polyetheretherketone (PEEK: commercially available from
TexLoc, Fort Worth, Tex.), perfluoroalkoxy (PFA; commercially
available from TexLoc, Fort Worth, Tex.), or Fluorinated ethylene
propylene (FEP; commercially available from TexLoc, Fort Worth,
Tex.).
[0040] Flow rates of the immiscible fluid are controlled by a fluid
controller, e.g., a PC running WinPumpControl software (Open Cage
Software, Inc., Huntington, N.Y.), connected to at least one pump.
An exemplary pump is shown in Davies et al. (WO 2007/091229). Other
commercially available pumps can also be used. Exemplary flow rates
range from about 1 .mu.l/min to about 100 .mu.l/min. An exemplary
flow rate is about 1 .mu.l/min, 3 .mu./min, 5 .mu.l/min, 10
.mu.l/min, 20 .mu.l/min, 30 .mu.I/min, 50 .mu.l/min, 70 .mu.l/min,
90 .mu.l/min, 95 .mu.l/min, or about 100 .mu.l/min. Because intake
of immiscible is at a low flow rate, for example 100 .mu.l/min, the
amount of immiscible fluid removed from the overlay of immiscible
fluid 408 in vessel 402 is negligible with respect to the amount of
time required to acquire each sample in the plate. In certain
embodiments, the system can include a supply of immiscible fluid in
fluid contact (e.g., by tubing) with the vessel 402 to replace the
immiscible fluid that is taken in by the sampling member 402 from
the overlay of immiscible fluid 408.
[0041] Now primed, the sampling member 401 is extended into well
403 to acquire an amount of sample 406 (FIG. 4, panel B). Samples
can be any type of biological or chemical species. In certain
embodiments, the sample is a gene or gene product from a biological
organism. Standard scientific protocols are available for
extraction and purification of mRNA and subsequent production of
cDNA. In other embodiments, the sample includes PCR reagents. A
typical Q-PCR reaction contains: fluorescent double-stranded
binding dye, Taq polymerase, deoxynucleotides of type A, C, G, and
T, magnesium chloride, forward and reverse primers and subject
cDNA, all suspended within an aqueous buffer. Reactants, however,
may be assigned into two broad groups: universal and reaction
specific. Universal reactants are those common to every Q-PCR
reaction, and include: fluorescent double-stranded binding dye, Taq
polymerase, deoxynucleotides A, C, G and T, and magnesium chloride.
Reaction specific reactants include the forward and reverse primers
and patient cDNA.
[0042] Once a sufficient amount of sample 406 has been acquired,
sampling member 401 is retracted from sample 406 in well 403 to the
overlay of immiscible fluid 408 (FIG. 4, panel C). Sampling member
401 remains in the overlay of immiscible fluid 408 and continues to
take in the immiscible fluid 408 (FIG. 4, panel C). Sampling member
401 proceeds to move through the recessed area 405 containing the
overlay of immiscible fluid 408 to the next well 404 containing a
sample 407 (FIG. 4, panel C). As sampling member 401 moves to the
next well 404, acquired sample 409 continues to move through
sampling member 401 (FIG. 4, panel C).
[0043] Once positioned above well 404, the sampling member 401 is
extended into well 404 to acquire an amount of sample 407 (FIG. 4,
panel D). Once a sufficient amount of sample 407 has been acquired,
sampling member 401 is retracted from sample 407 in well 404 to the
overlay of immiscible fluid. 408 (FIG. 4, panel D). Sampling member
401 remains in the overlay of immiscible fluid 408 and continues to
take in the immiscible fluid 408 (FIG. 4, panel D). Sampling member
401 proceeds to move through the recessed area 405 containing the
overlay of immiscible fluid 408 to the next well containing a
sample (FIG. 4, panel E). As sampling member 401 moves to the next
well, acquired sampled 410 continues to move through sampling
member 401 (FIG. 4, panel B). Acquired sample 409 and acquired
sample 410 are separated by the immiscible fluid 411.
[0044] The process repeats until the desired number of samples have
been acquired. Because sampling member 401 is continuously taking
in immiscible fluid 408 and is not removed above the overlay of
immiscible fluid 408, samples are acquired without the system
taking in any gas. Because samples within a vessel or within
separate vessels are separated by the immiscible fluid, there is no
carry-over or cross contamination between samples in a vessel and
between samples in different vessels.
[0045] The sampling member 401 is controlled by a robotics system.
The robotics system controls movement of the sampling member 401
between sample wells and during sample acquisition and/or
dispensing. At least one pump is connected to the sampling member
401. An exemplary pump is shown in Davies et al. (WO 2007/091229).
Other commercially available pumps can also be used. The pump is
controlled by a flow controller. e.g., a PC running WinPumpControl
software (Open Cage Software, Inc., Huntington, N.Y.), for
controlling direction of flow and flow rates. Sampling system 400
can be fluidly connected, e.g., tubes or channels, to an type of
analysis device. In certain embodiments, the sampling system 400 is
connected to a liquid bridge system, as shown in Davies et al. (WO
2007/091228). The liquid bridge system can be connected to a
thermocycler to perform PCR reactions on the acquired sample. An
exemplary thermocycler and methods of fluidly connecting a
thermocycler to a liquid bridge system are shown in Davies et al.
(WO 2005/023427, WO 2007/091230, and WO 2008/038259). The
thermocycler can be connected to an optical detecting device to
detect the products of the PCR reaction. An optical detecting
device and methods for connecting the device to the thermocycler
are shown in Davies et al. (WO 2007/091230 and WO 2008/038259).
[0046] FIG. 5 depicts a system 500 including a sampling device 501
and a vessel 502, and shows interaction of the sampling device 501
and the vessel 502 for acquisition and/or dispensing of samples
(FIG. 5, panel A). The sampling device 501 includes an outer sheath
507; and a plurality of tubes within the sheath. In FIG. 5, device
501 is shown with two tubes 504 and 505 that acquire a sample.
However, device 501 can be configured with only a single tube for
sample acquisition, or can be configured with more than two tubes
for sample acquisition, e.g., 3 tubes, 4 tubes, 5 tubes, 10 tubes,
15 tubes, 20 tubes, 50 tubes, etc. In FIG. 5, device 501 is shown
with one tube 503 that expels a fluid that is immiscible with the
sample. However, device 501 can be configured with more than one
tube that expels a fluid that is immiscible with the sample, e.g.,
3 tubes, 4 tubes, 5 tubes, 10 tubes, 15 tubes, 20 tubes, 50 tubes,
etc. In embodiments in which the vessel 502 is a plate, for example
a 96 well or 384 microtiter plate, the device 501 can be configured
with 24 tubes for sample acquisition. In this embodiment, the outer
diameter of the sample acquisition tubes is 0.3 mm and the diameter
of the outer sheath is 2.5 mm.
[0047] In device 501, the tubes that acquires the sample 504 and
505 are extendable beyond a distal end of the sheath and
retractable to within the sheath. FIG. 5, panel A shows the sample
acquisition tubes 504 and 505 retracted within the outer sheath
507.
[0048] The outer sheath and the plurality of tubes can be of any
shape, for example, a cylinder, a regular polygon, or an irregular
polygon. The shape of the outer sheath is independent of the shape
of the plurality of tubes. The outer sheath and the plurality of
tubes can be made of any material suitable to interact with
biological or chemical species. Exemplary materials include TEFLON
(commercially available from Dupont, Wilmington, Del.),
polytetrafluoroethylene (PTFE; commercially available from Dupont,
Wilmington, Del.), polymethyl methacrylate (PMMA; commercially
available from TexLoc, Fort Worth, Tex.), polyurethane
(commercially available from TexLoc, Fort Worth, Tex.),
polycarbonate (commercially available from TexLoc, Fort Worth,
Tex.), polystyrene (commercially available from TexLoc, Fort Worth,
Tex.), polyetheretherketone (PEEK; commercially available from
TexLoc, Fort Worth, Tex.), perfluoroalkoxy (PFA; commercially
available from TexLoc, Fort Worth, Tex.), or Fluorinated ethylene
propylene (FLP; commercially available from TexLoc, Fort Worth,
Tex.).
[0049] The vessel 502, can be any type of vessel that is suitable
for holding a sample. Exemplary vessels include plates (e.g., 96
well or 384 well plates), eppendorf tubes, vials, beakers, flasks,
centrifuge tubes, capillary tubes, cryogenic vials, bags, cups, or
containers. The vessel can be made of any material suitable to
interact with biological or chemical species. Exemplary materials
include TEFLON (commercially available from Dupont, Wilmington,
Del.), polytetrafluoroethylene (PTFE; commercially available from
Dupont, Wilmington, Del.), polymethyl methacrylate (PMMA;
commercially available from TexLoc, Fort Worth, Tex.), polyurethane
(commercially available from TexLoc, Fort Worth, Tex.),
polycarbonate (commercially available from TexLoc, Fort Worth,
Tex.), polystyrene (commercially available from TexLoc, Fort Worth,
Tex.), polyetheretherketone (PEEK; commercially available from
TexLoc, Fort Worth, Tex.), perfluoroalkoxy (PFA; commercially
available from TexLoc, Fort Worth, Tex.), or Fluorinated ethylene
propylene (FEP; commercially available from TexLoc, Fort Worth,
Tex.).
[0050] In this figure, the vessel is a plate having wells 508 and
509. The bottom portion of each well is filled with samples 510 and
511, and the remaining portion of each well 508 and 509 is filled
with an overlay of a fluid 512 that is immiscible with the samples
510 and 511. The immiscible fluid 512 is the same fluid that is
expelled by the immiscible fluid tube 503.
[0051] The system 500 is primed by continuously flowing the
immiscible fluid 512 out of the tube 503 that expels the immiscible
fluid, while sampling tubes 504 and 505 continuously intake the
immiscible fluid. Flow rates of the immiscible fluid are controlled
by a fluid controller, e.g., a PC running WinPumpControl software
(Open Cage Software, Inc., Huntington, N.Y.), connected to at least
one pump. An exemplary pump is shown in Davies et al. (WO
2007/091229). Other commercially available pumps can also be used.
Exemplary flow rates range from about 1 .mu.l/min to about 100
.mu.l/min. An exemplary flow rate is about 1 .mu.l/min, 3 .mu./min,
5 .mu.l/min, 10 .mu.l/min, 20 .mu.l/min, 30 .mu.l/min, 50
.mu.l/min, 70 .mu.l/min, 90 .mu.l/min, 95 .mu.l/min, or about 100
.mu.l/min.
[0052] In certain embodiments, flow is controlled such that the
flow rate out of the tube 503 that continuously expels the
immiscible fluid 512 is the same or similar to the total intake
flow rate of sample acquisition tubes 504 and 505. For example, the
flow rate out of tube 503 can range from about 2 .mu.l/min to about
100 .mu.l/min, while the intake flow rate for each of sample
acquisition tubes 504 and 505 can range from about 1 .mu.l/min to
about 50 .mu.l/min. Exemplary flow rates are as follows: flow rate
of 2 .mu.l/min expelled from tube 503, with an intake flow rate for
each of sample acquisition tubes 504 and 505 of 1 .mu.l/min; flow
rate of 6 .mu.l/min expelled from tube 503, with an intake flow
rate for each of sample acquisition tubes 504 and 505 of 3
.mu.l/min; flow rate of 10 .mu.l/min expelled from tube 503, with
an intake flow rate for each of sample acquisition tubes 504 and
505 of 5 .mu.l/min; flow rate of 20 .mu.l/min expelled from tube
503, with an intake flow rate for each of sample acquisition tubes
504 and 505 of 10 .mu.l/min; and flow rate of 100 .mu.l/min
expelled from tube 503, the intake flow rate for each of sample
acquisition tubes 504 and 505 is 50 .mu.l/min.
[0053] Alternatively, the flow rate out of tube 503 is greater than
the total intake flow rate of sample acquisition tubes 504 and 505.
For example, the flow rate out of tube 503 can range from about 5
.mu.l/min to about 100 .mu.l/min, while the intake flow rate for
each of sample acquisition tubes 504 and 505 can range from about 1
.mu.l/min to about 95 .mu.l/min. Exemplary flow rates are as
follows; flow rate of 6 .mu.l/min expelled from tube 503, with an
intake flow rate for each of sample acquisition tubes 504 and 505
of 2 .mu.l/min; flow rate of 10 .mu.l/min expelled from tube 503,
with an intake flow rate for each of sample acquisition tubes 504
and 505 of 4 .mu.l/min; flow rate of 20 .mu.l/min expelled from
tube 503, with an intake flow rate for each of sample acquisition
tubes 504 and 505 of 8 .mu.l/min and flow rate of 100 .mu.l/min
expelled from tube 503, with an intake flow rate for each of sample
acquisition tubes 504 and 505 of 48 .mu.l/min. In this regard, a
slightly greater amount of immiscible fluid is expelled into the
outer sheath than is taken in by the sample acquisition tubes.
Thus, a lower portion of the outer sheath 506 is continuously
filled with the immiscible fluid 512, and distal portions of sample
acquisition tubes 504 and 505 are continuously immersed in the
immiscible fluid.
[0054] The system is primed when a lower portion 506 of the outer
sheath 507 is filled with the immiscible fluid 512, and distal
portions of sample acquisition tubes 504 and 505 are continuously
immersed in the immiscible fluid 512.
[0055] Now primed, the sampling device 501 is extended into well
508 to acquire an amount of sample 510 (FIG. 5, panel B). The outer
sheath 507 is lowered into the overlay of immiscible fluid 512, and
does not contact sample no (FIG. 5, panel B). Sampling tubes 504
and 505 extend into the sample 510 (FIG. 5, panel B). Sample 510
can be any type of biological or chemical species. In certain
embodiments, the sample is a gene or gene product from a biological
organism. In other embodiments, the sample includes PCR reagents.
Once a sufficient amount of sample 510 has been acquired, sampling
tubes 504 and 505 are retracted from sample 510 in well 508, and
return to within the outer sheath 507 (FIG. 5, panel C).
[0056] Once sampling tubes 504 and 505 have retracted to within the
outer sheath 507, the outer sheath 507 retracts from the immiscible
fluid 512 in well 508 (FIG. 5, panel C). Sampling device 501 then
proceeds to move to the next well 509 containing a sample 511 (FIG.
5, panel C). As sampling device 501 moves to the next well 509,
acquired sample 513 continues to move through sampling device 501
(FIG. 5, panel C). Additionally, tube 503 continues to expel the
immiscible fluid 512, sampling tubes 504 and 505 continue to intake
the immiscible fluid 512, and the lower portion 506 of the outer
sheath 507 remains continuously filled with the immiscible fluid
(FIG. 5, panel C). Thus the distal portions of sampling tubes 504
and 505 remain continuously immersed in the immiscible fluid and do
not contact the atmosphere (FIG. 5, panel C). Thus, a sample is
acquired without the system taking in any gas.
[0057] Once positioned above the well 509, the sampling device 501
is extended into well 509 to acquire an amount of sample 511 (FIG.
5, panel D). The outer sheath 507 is lowered into the overlay of
immiscible fluid 512, and does not contact sample 511 (FIG. 5,
panel D). Sampling tubes 504 and 505 extend into the sample 511
(FIG. 5, panel D). Sample 511 can be any type of biological or
chemical species. In certain embodiments, the sample is a gene or
gene product from a biological organism. In other embodiments, the
sample includes PCR reagents. Once a sufficient amount of sample
511 has been acquired, sampling tubes 504 and 505 are retracted
from sample 511 in well 509, and return to within the outer sheath
507 (FIG. 5, panel D).
[0058] Once sampling tubes 504 and 505 have retracted to within the
outer sheath 507, the outer sheath 507 retracts from the immiscible
fluid 512 in well 509 (FIG. 5, panel E). Sampling device 501 then
proceeds to move to the next well containing a sample (FIG. 5,
panel E). As sampling device 501 moves to the next well, acquired
sample 514 continues to move through sampling device 501 (FIG. 5,
panel E). Acquired sample 513 and acquired sample 514 are separated
by the immiscible fluid 512. Additionally, tube 503 continues to
expel the immiscible fluid 512, sampling tubes 504 and 505 continue
to intake the immiscible fluid 512, and the lower portion 506 of
the outer sheath 507 remains continuously filled with the
immiscible fluid (FIG. 5, panel E). Thus the distal portions of
sampling tubes 504 and 505 remain continuously immersed in the
immiscible fluid and do not contact the atmosphere (FIG. 5, panel
E). Thus, samples are acquired without the system taking in any
gas. The process repeats until the desired number of samples have
been acquired. Because samples within a vessel or within separate
vessels are separated by the immiscible fluid, there is no
carry-over or cross contamination between samples in a vessel and
between samples in different vessels.
[0059] The sampling device 501 is controlled by at least one
robotics system. A first robotics system controls movement of the
sampling device 501 between sample wells and movement of the outer
sheath 507 during sample acquisition. A second robotics system
controls the sampling tubes 503 and 504 for extension from the
outer sheath 507 and retraction into the outer sheath 507. At least
one pump is connected to the tube 503 that expels the immiscible
fluid, and at least one pump is connected to the sample acquisition
tubes 503 and 504. An exemplary pump is shown in Davies et al. (WO
2007/091229). Other commercially available pumps can also be used.
The pump connected to tube 503 obtains the immiscible fluid from a
reservoir that is fluidly connected to the pump. The pumps are
controlled by a flow controller, e.g., a PC running WinPumpControl
software (Open Cage Software, Inc., Huntington, N.Y.), for control
of direction of flow and flow rates.
[0060] Sampling system 500 can be fluidly connected, e.g., tubes or
channels, to an type of analysis device. In certain embodiments,
the sampling system 500 is connected to a liquid bridge system, as
shown in Davies et al. (WO 2007/091228). The liquid bridge system
can be connected to a thermocycler to perform PCR reactions on the
acquired sample. An exemplary thermocycler and methods of fluidly
connecting a thermocycler to a liquid bridge system are shown in
Davies et al. (WO 2005/023427, WO 2007/091230, and WO 2008/038259).
The thermocycler can be connected to an optical detecting device to
detect the products of the PCR reaction. An optical detecting
device and methods for connecting the device to the thermocycler
are shown in Davies et al. (WO 2007/091230 and WO 2008/038259).
INCORPORATION BY REFERENCE AND EQUIVALENTS
[0061] References and citations to other documents, such as
patents, patent applications, patent publications, journals, books,
papers, web contents, have been made throughout this disclosure.
All such documents are hereby incorporated herein by reference in
their entirety for all purposes. Various modifications of the
invention and many further embodiments thereof, in addition to
those shown and described herein, will become apparent to those
skilled in the art from the full contents of this document,
including the references to the scientific and patent literature
cited herein.
* * * * *