U.S. patent application number 12/703046 was filed with the patent office on 2010-09-02 for relating to devices.
Invention is credited to Xiaojia Chen, Matthew Estes, Andrew Hopwood, Cedric Hurth, Pieris Koumi, John Lee-Edghill, Ralf Lenigk, Nina Moran, Alan Nordquist, Jianing Yang, Frederic Zenhausern.
Application Number | 20100221726 12/703046 |
Document ID | / |
Family ID | 42111529 |
Filed Date | 2010-09-02 |
United States Patent
Application |
20100221726 |
Kind Code |
A1 |
Zenhausern; Frederic ; et
al. |
September 2, 2010 |
RELATING TO DEVICES
Abstract
A method of analysis, instrument for analysis and device for use
in such an instrument are provided, which perform a number of
processes need to reach a useful result in the context of a wide
variety of samples. The sequence of those processes being
optimised. A device, instrument using the device and method of use
are also provided which offer reliable performance of a heating
based process, with minimal condensation and/or sample loss
issues.
Inventors: |
Zenhausern; Frederic;
(Fountain Hills, AZ) ; Nordquist; Alan; (Payson,
AZ) ; Lenigk; Ralf; (Chandler, AZ) ; Hurth;
Cedric; (Tempe, AZ) ; Yang; Jianing; (Tempe,
AZ) ; Chen; Xiaojia; (Chandler, AZ) ; Estes;
Matthew; (Tempe, AZ) ; Lee-Edghill; John;
(Birmingham, GB) ; Moran; Nina; (Birmingham,
GB) ; Hopwood; Andrew; (Birmingham, GB) ;
Koumi; Pieris; (Birmingham, GB) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Family ID: |
42111529 |
Appl. No.: |
12/703046 |
Filed: |
February 9, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61151117 |
Feb 9, 2009 |
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61151104 |
Feb 9, 2009 |
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61151107 |
Feb 9, 2009 |
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61151111 |
Feb 9, 2009 |
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Current U.S.
Class: |
435/6.12 ;
204/450; 435/287.2 |
Current CPC
Class: |
G01N 2201/062 20130101;
B01L 7/52 20130101; C25B 9/00 20130101; G01N 27/44721 20130101;
B01L 3/502738 20130101; G01N 21/01 20130101; B01L 7/525 20130101;
G01N 2021/6484 20130101; G01N 21/645 20130101; G01N 2201/0846
20130101; B01L 3/502715 20130101; C12Q 1/6806 20130101; G01N 1/10
20130101; Y10T 436/143333 20150115; G01N 27/44791 20130101; B01L
3/502761 20130101; G01N 21/6428 20130101 |
Class at
Publication: |
435/6 ;
435/287.2; 204/450 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34; B01D 57/02 20060101
B01D057/02 |
Claims
1. A device, for processing a sample, the device including: one or
more sample processors, wherein the sample processors include a
sample preparation step, sample extraction step, sample retention
step, elution step, amplification step, denaturing step, detection
step and results output step.
2. A device according to claim 1 in which the device is a
cartridge.
3. A device according to claim 1 in which the device includes a
sample processor for one or more of a sample receiving step,
purification step, washing step, PCR step, investigation step,
analysis step.
4. A device according to claim 1 in which the sample receiving step
includes the transfer of a sample from outside the device to inside
the device, the sample receiving step receiving the sample from a
collection device or from a storage device.
5. A device according to claim 1 in which the sample preparation
step includes contacting the sample with one or more reagents.
6. A device according to claim 1 the sample retention step includes
contacting the sample with one or more reagents and/or components
which retain the sample component(s) relative to one or more waste
components in the sample.
7. A device according to claim 6 in which the sample component(s)
are retained on one or more magnetic beads.
8. A device according to claim 6 in which the waste component(s)
are removed from the retained sample components by being washed
away from the retention step using one or more further reagents
and/or components.
9. A device according to claim 6 in which the retained and/or
selected sample is eluted with the eluent conveying the retained
and/or selected sample to the next step.
10. A device according to claim 6 in which the elution step removes
one or more components of the sample from a first form into a
second form, the first form being bound to a surface or substrate,
the second form being in a liquid.
11. A device according to claim 1 in which the amplification step
includes contacting the sample with one or more reagents and/or
components to cause amplification and/or contacting the sample with
conditions to cause amplification.
12. A device according to claim 1 in which the denaturing step
prepares the sample for electrophoresis.
13. A device according to claim 1 in which the electrophoresis step
includes transferring the sample to a start location for
electrophoresis, the start location being in a channel, one or more
voltage conditions being used to transfer the sample to the start
location, one or more further voltage conditions may be used to
provide the separation.
14. A device according to claim 1 in which the analysis step
establishes one or more of the characteristics of the sample by
seeking a response from the device to an operation, the operation
being the application of light.
15. A device according to claim 1 in which the analysis step
establishes the relative position of the elements having a
characteristic.
16. A device according to claim 1 in which the results output step
displays the one or more results from the analysis step and/or a
processed form thereof.
17. A device according to claim 1 in which the results output step
transmits the one or more results from the analysis step and/or a
processed form thereof to a remote location.
18. A device according to claim 1 in which the results output step
is followed by a further processing step, the further processing
interpreting the results to provide further results, the further
processing step being provided at a location remote from the
instrument.
19. An instrument for analysing a sample, the instrument including:
a device having one or more sample processors; electronics for
operating the sample processors, wherein the sample processors
include a sample preparation step, sample extraction step, sample
retention step, elution step, amplification step, denaturing step,
detection step and results output step, the device being provided
according to claim 1.
20. A method for analysing a sample, the method including providing
an instrument including: a device having one or more sample
processors; electronics for operating the sample processors,
wherein the sample processors include a sample preparation step,
sample extraction step, sample retention step, elution step,
amplification step, denaturing step, detection step and results
output step; operating one or more of the sample processors.
21. A method according to claim 20, wherein the device is a
cartridge.
22. A method according to claim 20 wherein the device includes a
sample processor for one or more of a sample receiving step,
purification step, washing step, PCR step, investigation step,
analysis step.
23. A device, for processing a sample, the device including: one or
more sample processors, wherein one of the sample processors is
provided using a chamber with an inlet and an outlet, a valve being
connected to the inlet to provide a first sealing location, a
further valve connected to the outlet to provide a second sealing
location, one or more interconnected channels and chambers being
provided between the first sealing location and the second sealing
location, the channel or channels and chamber or chambers provided
between the first sealable location and the second sealable
location having a first extent, a heating device being provided to
heat the chamber, the heating device having a second extent
parallel to the first extent of the channel or channels and chamber
or chambers, wherein the second extent is at least 75% of the first
extent.
24. A device according to claim 23, wherein a channel connected to
the inlet, the chamber and a channel connected to the outlet are
provided between the first sealable location and the second
sealable location.
25. A device according to claim 23, wherein the channel or channels
and chamber or chambers provided between the first sealable
location and the second sealable location have an extent in a first
plane and the heating device has an extent parallel to the first
plane.
26. A device according to claim 23 in which the extent of the
heating device is greater than 90%.
27. An instrument including a device according to claim 23.
28. A method for analysing a sample, the method including providing
an instrument including: a device having one or more sample
processors; electronics for operating the sample processors,
wherein one of the sample processors is provided using a chamber
with an inlet and an outlet, a valve being connected to the inlet
to provide a first sealing location, a further valve connected to
the outlet to provide a second sealing location, one or more
interconnected channels and chambers being provided between the
first sealing location and the second sealing location, the channel
or channels and chamber or chambers provided between the first
sealable location and the second sealable location having a first
extent, a heating device being provided to heat the chamber, the
heating device having a second extent parallel to the first extent
of the channel or channels and chamber or chambers, wherein the
second extent is at least 75% of the first extent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Utility Application which claims
benefit of Ser. No. 61/151,117, filed Feb. 9, 2009 in the United
States, and of Ser. No. 61/151,107, filed Feb. 9, 2009 in the
United States, and of Ser. No. 61/151,111, filed Feb. 9, 2009 in
the United States, and of Ser. No. 61/151,104, filed Feb. 9, 2009
in the United States, and which application(s) are incorporated
herein by reference. A claim of priority to all, to the extent
appropriate is made.
BACKGROUND OF THE INVENTION
[0002] The invention concerns improvements in and relating to
analysis, particularly, but not exclusively, in relation to
biological samples, such as DNA.
SUMMARY OF THE INVENTION
[0003] According to a first aspect of the invention, there is
provided an instrument for analysing a sample, the instrument
including:
[0004] a device having one or more sample processors;
[0005] electronics for operating the sample processors.
[0006] According to a second aspect of the invention, there is
provided a device, for processing a sample, the device
including:
[0007] one or more sample processors.
[0008] According to a third aspect of the invention, there is
provided a method of producing a device, the method including:
[0009] forming one or more sample processors;
[0010] providing electronics for operating the sample
processors.
[0011] The instrument may provide an integrated set of process
steps and/or sample processors. The process steps and/or sample
processors may include a sample receiving step and/or sample
preparation step and/or sample extraction step and/or sample
retention step and/or purification step and/or washing step and/or
elution step and/or amplification step and/or PCR step and/or
denaturing step and/or investigation step and/or detection step
and/or electrophoresis step and/or analysis step and/or results
output step.
[0012] The device may be a cartridge. The device is preferably a
single use device. The device is preferably only used to process
and/or provide the results for one sample. The device is preferably
disposable.
[0013] The device may have an orientation of use, for instance in
the instrument.
[0014] The sample receiving step may be provided on the device.
[0015] The sample receiving step may include an inlet to the
device. The sample receiving step may include a chamber, preferably
into which the sample is received. The chamber may have an inlet in
the upper portion of the chamber, for instance the upper 20%. The
chamber may have a gas outlet, for instance a vent. The gas outlet
may be provided in the upper portion of the chamber, for instance
the upper 20%.
[0016] The chamber may be connected to a pump, for instance an
electrochemical pump. The pump may be a first pump provided on the
device. The first pump may provide the drive to move one or more
fluids and/or liquids through the chamber and/or first chamber
and/or second chamber and/or third chamber and/or fourth chamber
and/or into a fifth chamber. The inlet from the pump may be
provided in the upper section of the chamber, for instance the
upper 20%, preferably upper 5%.
[0017] The chamber may have a sample outlet. The sample outlet may
be provided in the lower portion of the chamber, for instance the
lower 20%, more preferably lower 10% and ideally the lowest part of
the chamber. The outlet may be in the bottom wall of the
chamber.
[0018] The sample receiving step may have a first state in which it
is isolated from one of more of the other steps in the cartridge.
The one or more other steps may be a sample preparation step and/or
sample extraction step and/or sample retention step and/or
purification step and/or washing step and/or elution step and/or
sample amplification step and/or denaturation step and/or detection
step and/or electrophoresis step and/or analysis step. The sample
receiving step may have the first state during loading of the
sample. The sample receiving step may be provided with a valve. The
valve may be provided at the sample outlet and/or on the channel
leading from the sample receiving step and/or leading from the
sample outlet. The valve may be a closed state to open state
valve.
[0019] The outlet channel may be connected to the next step, for
instance to the sample preparation step.
[0020] The sample preparation step may be provided on the device.
The sample preparation step may be provided on the same device as
the sample receiving step.
[0021] The sample preparation step may include an inlet, preferably
a channel. The channel may be connected to the sample receiving
step. The sample preparation step may include a first chamber,
preferably into which the sample passes.
[0022] The first chamber may have an inlet in the upper portion of
the first chamber, for instance the upper 20%.
[0023] The first chamber may have a circular cross-section. The
cross-section may be relative to a horizontal axis. The first
chamber may be provided with a buffer. The buffer may be provided
to control conditions for a subsequent process and/or reaction, for
instance in one or more further chambers and/or channels. The first
chamber may be provided with a one or more particles. The particles
may be beads. One or more of the particles may be magnetic. The one
or more particles may have a magnetic material within a surface
layer or layers. The particles may be provided with one or more
reagents or materials which releasable bind and/or link and/or
combine with a part of the sample, for instance DNA.
[0024] The first chamber may have a sample outlet. The sample
outlet may be provided in the lower portion of the chamber, for
instance the lower 20%, more preferably lower 10% and ideally the
lowest part of the chamber. The outlet may be in the bottom wall of
the chamber.
[0025] References to vertical within the document may mean within
25.degree. of the vertical, preferably within 10.degree. and
ideally within 5.degree., and potentially be completely vertical.
References to horizontal within the document may mean within
25.degree. of the horizontal, preferably within 10.degree. and
ideally within 5.degree., and potentially be completely
horizontal.
[0026] Reference to the number of a chamber, such as to a fourth
chamber do not mean or imply that the chamber has to be preceded by
such a number of chambers. The number is merely used to clarify one
chamber from another.
[0027] The sample outlet may connect to a channel. Preferably the
channel has a plurality of sections. The channel may have a
vertical section and/or a horizontal section and/or a second
vertical section and/or a second horizontal section and/or a third
vertical section. The channel may have a vertical section and a
horizontal section and a second vertical section and/or a second
horizontal section and/or a third vertical section. The channel may
connect to a second chamber.
[0028] The second chamber may have an inlet in the upper portion of
the second chamber, for instance the upper 20%.
[0029] The second chamber may have a elongate cross-section. The
second chamber may have a cross-section formed by a semicircle at
each end and a rectilinear section joining them The cross-section
may be relative to a horizontal axis. The second chamber may be
provided with a one or more particles. The particles may be beads.
One or more of the particles may be magnetic. The one or more
particles may have a magnetic material within a surface layer or
layers. The particles may be provided with one or more reagents or
materials which releasable bind and/or link and/or combine with a
part of the sample, for instance DNA. The second chamber may be
provided with a buffer. The buffer may be provided to control
conditions for a subsequent process and/or reaction, for instance
in one or more further chambers and/or channels.
[0030] The second chamber may have a sample outlet. The sample
outlet may be provided in the lower portion of the second chamber,
for instance the lower 20%, more preferably lower 10% and ideally
the lowest part of the second chamber. The outlet may be in the
bottom wall of the chamber.
[0031] The sample outlet may connect to a channel. Preferably the
channel has a plurality of sections. The channel may have a
vertical section and/or a horizontal section and/or a second
vertical section and/or a second horizontal section and/or a third
vertical section and/or a third horizontal section and/or fourth
vertical section. The channel may have a vertical section and a
horizontal section and a second vertical section and a second
horizontal section and a third vertical section and/or a third
horizontal section and/or fourth vertical section. The channel may
connect to a third chamber.
[0032] The third chamber may have an inlet in the upper portion of
the third chamber, for instance the upper 25%.
[0033] The third chamber may have a non-linear cross-section. The
third chamber may have a cross-section formed by a semicircles or
part semicircles at one or both ends. A rectilinear section may
join the semicircles or part semicircle together. The cross-section
may be relative to a horizontal axis. The third chamber may be
provided with a one or more particles. The particles may be beads.
One or more of the particles may be magnetic. The one or more
particles may have a magnetic material within a surface layer or
layers. The particles may be provided with one or more reagents or
materials which releasable and/or reversibly bind and/or link
and/or combine with a part of the sample, for instance DNA.
[0034] The third chamber may have a gas outlet, for instance a
vent. The gas outlet may be provided in the upper portion of the
chamber, for instance the upper 20%, preferably upper 10% and
ideally upper 5%. The gas outlet may be provided in the top wall of
the third chamber. The gas outlet may be provided in a recess at
the top of the third chamber. The gas outlet may lead to the
outside of the device, for instance through a vent. A valve may be
provided between the third chamber and the vent. The valve may be
an open state to closed state valve.
[0035] The third chamber may have a sample outlet. The sample
outlet may be provided in the lower portion of the third chamber,
for instance the lower 10%, more preferably lower 5% and ideally
the lowest part of the third chamber. The outlet may be in the
bottom wall of the chamber.
[0036] The sample outlet may connect to a channel. Preferably the
channel has a plurality of sections. The channel may have a
vertical section and/or a horizontal section and/or a second
vertical section and/or a second horizontal section and/or a third
horizontal section and/or a third vertical section. The channel may
have a vertical section and a horizontal section and a second
vertical section and a second horizontal section and/or a third
vertical section and/or a third horizontal section. The channel may
connect to one or more further chambers, such as a fourth
chamber.
[0037] The sample preparation step or a part thereof may have a
first state in which it is isolated from one of more of the other
steps in the cartridge and/or from one or more other parts of the
sample preparation step. The one or more other steps may be a
sample receiving step and/or sample extraction step and/or sample
retention step and/or purification step and/or washing step and/or
elution step and/or sample amplification step and/or
electrophoresis step and/or analysis step. The sample preparation
step or part thereof may have the first state during contacting of
the sample with the buffer and/or particles and/or first chamber
and/or second chamber and/or third chamber. The sample preparation
step or a part thereof may be provided with a valve. The valve may
be provided at the sample outlet, preferably from the third chamber
and/or on the channel leading from the sample preparation step to a
further step and/or on the channel leading from the part of the
sample preparation step to the next part of the sample preparation
step and/or on the channel leading from the sample outlet. The
valve may be a closed state to open state valve.
[0038] The sample preparation step and/or a part of the sample
preparation step, particularly the part that follows the part
described above, may include a fourth chamber.
[0039] The fourth chamber may have an inlet in the upper portion of
the fourth chamber, for instance the upper 25%. The inlet may be in
a corner of the fourth chamber.
[0040] The fourth chamber may have a non-linear cross-section. The
fourth chamber may have a cross-section formed by a horizontal top
wall, horizontal or inclined lower wall and transition end walls
joining the top and lower walls. The transition end walls may be
curved. The cross-section may be relative to a horizontal axis. The
fourth chamber may be provided with a gas, such as air, preferably
prior to the sample arrival.
[0041] The fourth chamber may have a sample outlet. The sample
outlet may be provided in the lower portion of the fourth chamber,
for instance the lower 10%, more preferably lower 5% and ideally
the lowest part of the fourth chamber. The outlet may be in the
bottom wall of the chamber or preferably in a corner of the
chamber, ideally the corner opposing the inlet.
[0042] The sample outlet may connect to a channel. Preferably the
channel has a plurality of sections. The channel may have a
vertical section and/or a horizontal section and/or a second
vertical section. The channel may have a vertical section and a
horizontal section and a second vertical section. The channel may
connect to a fifth chamber.
[0043] The fifth chamber may have an inlet in the upper portion of
the fifth chamber, for instance the upper 25%. The inlet may be in
a corner of the fifth chamber.
[0044] The fifth chamber may have a non-linear cross-section. The
fifth chamber may have a cross-section formed by a horizontal top
wall, horizontal or inclined lower wall and transition end walls
joining the top and lower walls. The transition end walls may be
curved. The cross-section may be relative to a horizontal axis. The
fifth chamber may be provided with a gas, such as air, preferably
prior to the sample arrival.
[0045] The fifth chamber may have a sample outlet. The sample
outlet may be provided in the lower portion of the fifth chamber,
for instance the lower 10%, more preferably lower 5% and ideally
the lowest part of the fifth chamber. The outlet may be in the
bottom wall of the chamber or preferably in a corner of the
chamber, ideally the corner opposing the inlet.
[0046] The sample outlet may connect to a channel. Preferably the
channel has a plurality of sections. The channel may have a
vertical section and/or a horizontal section and/or a second
vertical section and/or second horizontal section and/or third
vertical section and/or third horizontal section and/or fourth
vertical section. The channel may have a vertical section and a
horizontal section and a second vertical section and/or second
horizontal section and/or third vertical section and/or third
horizontal section and/or fourth vertical section. The channel may
connect to a sixth chamber.
[0047] The sixth chamber may have an inlet in the lower portion of
the sixth chamber, for instance the lower 20%, preferably lower 10%
and ideally lower 5%. The inlet may be in the bottom wall of the
sixth chamber.
[0048] The sixth chamber may have a non-linear cross-section. The
sixth chamber may have a cross-section formed by a horizontal
bottom wall, horizontal top wall and side walls that diverge
between the bottom and the top. The corners may be provided with
curved transition walls. The sixth chamber may be provided with
air.
[0049] The sixth chamber may have a gas outlet, for instance a
vent. The gas outlet may be provided in the upper portion of the
chamber, for instance the upper 20%, preferably upper 10% and
ideally upper 5%. The gas outlet may be provided in the top wall of
the sixth chamber. The gas outlet may lead to the outside of the
device, for instance through a vent. A valve may be provided
between the sixth chamber and the vent. The valve may be an open
state to closed state valve
[0050] The sixth chamber may be connected to a pump, for instance
an electrochemical pump. The pump may be a second pump provided on
the device. The second pump may provide the drive to move one or
more fluids and/or liquids through the sixth chamber and/or seventh
chamber and/or into a waste chamber. The pump may provide gas to
one or more of the chambers, particularly the sixth chamber. The
gas may promote mixing within the one or more chambers,
particularly the sixth chamber. The inlet from the pump may be
provided in the upper section of the sixth chamber, for instance
the upper 20%, preferably upper 5%.
[0051] The channel connecting the pump to the sixth chamber may be
provided with a valve. The valve may be an open state to closed
state valve. The channel may include a first vertical section
and/or first horizontal section and/or second vertical section
and/or second horizontal section and/or third horizontal section
and/or fourth vertical section.
[0052] The sixth chamber may have a sample outlet. The sample
outlet may be provided in the lower portion of the sixth chamber,
for instance the lower 10%, more preferably lower 5% and ideally
the lowest part of the sixth chamber. The outlet may be in the
bottom wall of the chamber.
[0053] The sample outlet may connect to a channel. Preferably the
channel has a plurality of sections. The channel may have a
vertical section and/or a horizontal section and/or a second
vertical section and/or a second horizontal section and/or a third
vertical section. The channel may have a vertical section and a
horizontal section and a second vertical section and a second
horizontal section and/or a third vertical section. The channel may
connect to one or more further chambers, such as a seventh
chamber.
[0054] The sample preparation step or a part thereof may have a
first state in which it is isolated from one of more of the other
steps in the cartridge and/or from one or more other parts of the
sample preparation step. The one or more other steps may be a
sample receiving step and/or sample retention step and/or
purification step and/or washing step and/or elution step and/or
sample amplification step and/or electrophoresis step and/or
analysis step. The sample preparation step or part thereof may have
the first state during contacting of the sample with the fourth
chamber and/or fifth chamber and/or sixth chamber and/or during
mixing of the sample, buffer and particles and/or during heating of
the sixth chamber. The sample preparation step or a part thereof
may be provided with a valve. The valve may be provided at the
sample outlet, preferably from the sixth chamber and/or on the
channel leading from the sample preparation step to a further step
and/or on the channel leading from the part of the sample
preparation step to the next part of the sample preparation step
and/or on the channel leading from the sample outlet. The valve may
be a closed state to open state valve.
[0055] The seventh chamber may have an inlet in the upper portion
of the seventh chamber, for instance the upper 20%, preferably
upper 10% and ideally upper 5%. The inlet may be in the top wall of
the seventh chamber.
[0056] The seventh chamber may have a non-linear cross-section. The
seventh chamber may have a cross-section formed by a horizontal
bottom wall, horizontal top wall and side walls that diverge
between the bottom and the top. The corners may be provided with
curved transition walls. The top wall may include a recess, such as
a semi-circular recess. The seventh chamber may be provided with
air.
[0057] The seventh chamber may have a gas outlet, for instance a
vent. The gas outlet may be provided in the upper portion of the
chamber, for instance the upper 20%, preferably upper 10% and
ideally upper 5%. The gas outlet may be provided in the top wall of
the seventh chamber. The gas outlet may lead to the outside of the
device, for instance through a vent. A valve may be provided
between the seventh chamber and the vent. The valve may be an open
state to closed state valve. The channel leading to the valve may
connect to the seventh chamber in a recess, such as a semi-circular
recess, provided in the upper section of the seventh chamber.
[0058] The seventh chamber may have a second gas outlet, for
instance a vent. The second gas outlet may be provided in the upper
portion of the chamber, for instance the upper 20%, preferably
upper 10% and ideally upper 5%. The second gas outlet may be
provided in the top wall of the seventh chamber. The second gas
outlet may lead to the outside of the device, for instance through
a second vent. A second valve may be provided between the seventh
chamber and the second vent. The second valve may be an open state
to closed state valve. The second channel leading to the second
valve may connect to the seventh chamber in a recess, such as a
semi-circular recess, provided in the upper section of the seventh
chamber.
[0059] The seventh chamber may be connected to a pump, for instance
an electrochemical pump. The pump may be a second pump provided on
the device. The seventh chamber may be connected to the pump by a
first route through the sixth chamber and/or by a second route
through an eighth chamber and/or wash chamber.
[0060] The sample preparation step or a part thereof may have a
first state in which it is isolated from one of more of the other
steps in the cartridge and/or from one or more other parts of the
sample preparation step. The one or more other steps may be a
sample receiving step and/or sample extraction step and/or sample
retention step and/or purification step and/or washing step and/or
elution step and/or sample amplification step and/or
electrophoresis step and/or analysis step. The sample preparation
step or part thereof may have the first state during contacting of
the sample with the seventh chamber. The sample preparation step or
a part thereof may be provided with one or more valves, preferably
to provide the isolation.
[0061] A valve may be provided at the sample outlet, preferably
from the seventh chamber and/or on the channel leading from the
sample preparation step to a further step and/or on the channel
leading from the part of the sample preparation step to the next
part of the sample preparation step and/or on the channel leading
from the sample outlet. The valve may be a closed state to open
state valve.
[0062] One or more valves may be provided on the channel connecting
the second pump to the seventh chamber by a second route. The one
or more valves may include an open state to closed state valve or
valves and/or a closed state to open state valve or valves.
[0063] One or more valves may be provided on the channel connecting
the third pump to the seventh chamber. The one or more valves may
include an open state to closed state valve or valves and/or a
closed state to open state valve or valves.
[0064] One or more valves may be provided on the channel connecting
the seventh chamber to an tenth chamber and/or waste chamber. The
one or more valves may include an open state to closed state valve
or valves and/or a closed state to open state valve or valves.
[0065] The seventh chamber may be connected to an eighth chamber
and/or wash chamber, for instance by a channel.
[0066] The inlet to the eighth chamber may be provided in the upper
portion of the eighth chamber, for instance the upper 20%,
preferably upper 10% and ideally upper 5%. The inlet may be in the
top wall of the eighth chamber. The outlet may be provided in the
corner of the eighth chamber.
[0067] The channel connecting the eighth chamber and/or wash
chamber to the seventh chamber may have a plurality of sections.
The channel may have a vertical section and/or a horizontal section
and/or a second vertical section and/or a second horizontal section
and/or a third vertical section and/or a third horizontal section.
The channel may have a vertical section and a horizontal section
and a second vertical section and a second horizontal section
and/or a third vertical section and/or third horizontal
section.
[0068] The channel may include one or more valves. The one or more
valves may include an open state to closed state valve or valves
and/or a closed state to open state valve or valves.
[0069] The eighth chamber and/or wash chamber may have an inlet in
the upper portion of the eighth chamber, for instance the upper
20%.
[0070] The eighth chamber may be connected to a pump, for instance
an electrochemical pump. The pump may be a second pump provided on
the device. The second pump may provide the drive to move one or
more fluids and/or liquids through the eighth chamber and/or
seventh chamber and/or sixth chamber and/or into a tenth chamber.
The inlet from the pump may be provided in the upper section of the
chamber, for instance the upper 20%, preferably upper 5%.
[0071] The eighth chamber may have an outlet. The outlet may be
provided in the lower portion of the chamber, for instance the
lower 20%, more preferably lower 10% and ideally the lowest part of
the chamber. The outlet may be in the bottom wall of the
chamber.
[0072] The sample preparation step or a part thereof may have a
first state in which it is isolated from one of more of the other
steps in the cartridge and/or from one or more other parts of the
sample preparation step. The one or more other steps may be a
sample receiving step and/or sample extraction step and/or
purification step and/or washing step and/or elution step and/or
sample amplification step and/or electrophoresis step and/or
analysis step. The sample preparation step or part thereof may have
the first state during contacting of the sample with the seventh
chamber and/or contact between the eighth chamber and/or wash
chamber and the seventh chamber. The sample preparation step or a
part thereof may be provided with one or more valves, preferably to
provide the isolation.
[0073] The seventh chamber may be connected to a tenth chamber
and/or waste chamber, for instance by a channel. The outlet from
the seventh chamber may be provided in the lower portion of the
seventh chamber, for instance the lower 20%, preferably lower 10%
and ideally lower 5%. The outlet may be in the bottom wall of the
seventh chamber. The outlet may be provided in the corner of the
seventh chamber.
[0074] The channel connecting the seventh chamber to the tenth
chamber and/or waste chamber may have a plurality of sections. The
channel may have a vertical section and/or a horizontal section
and/or a second vertical section and/or a second horizontal section
and/or third vertical section and/or third horizontal section
and/or fourth vertical section and/or fourth vertical section
and/or fifth vertical section. The channel may have a vertical
section and a horizontal section and a second vertical section and
a second horizontal section and third vertical section and/or third
horizontal section and/or fourth vertical section and/or fourth
vertical section and/or fifth vertical section.
[0075] The channel may include one or more valves. The one or more
valves may include a closed state to open state valve or valves,
preferably provided on the channel above.
[0076] The one or more valves may include an open state to closed
state valve or valves, preferably provided on a parallel channel
section. The parallel channel section may include a first vertical
section and/or first horizontal section and/or second vertical
section and/or second horizontal section and/or third vertical
section and/or third horizontal section and/or fourth vertical
section. The parallel channel section may be connected to the
second vertical and/or third vertical sections of the channel it is
provided as a parallel channel to.
[0077] The tenth chamber and/or waste chamber may have a
rectilinear cross-section, potentially with rounded corners.
[0078] The tenth chamber and/or waste chamber may have an inlet in
the upper portion of the chamber, for instance the upper 20%.
[0079] The tenth chamber and/or waste chamber may be connected to a
pump, for instance an electrochemical pump. The pump may be a
second pump provided on the device. The second pump may provide the
drive to move one or more fluids and/or liquids through the eighth
chamber and/or seventh chamber and/or sixth chamber and/or into a
tenth chamber.
[0080] The tenth chamber and/or waste chamber may have a gas
outlet, for instance a vent. The gas outlet may be provided in the
upper portion of the chamber, for instance the upper 20%,
preferably upper 10% and ideally upper 5%. The gas outlet may be
provided in the top wall of the tenth chamber. The gas outlet may
lead to the outside of the device, for instance through a vent. A
valve may be provided between the tenth chamber and the vent. The
valve may be an open state to closed state valve
[0081] The sample preparation step or a part thereof may have a
first state in which it is isolated from one of more of the other
steps in the cartridge and/or from one or more other parts of the
sample preparation step. The one or more other steps may be a
sample receiving step and/or sample extraction step and/or sample
retention step and/or sample amplification step and/or
electrophoresis step and/or analysis step. The sample preparation
step or part thereof may have the first state during contacting of
the sample with the seventh chamber and/or contact between the
ninth chamber and/or elution chamber and the seventh chamber and/or
tenth chamber and/or waste chamber. The sample preparation step or
a part thereof may be provided with one or more valves, preferably
to provide the isolation.
[0082] The seventh chamber may be connected to the ninth chamber
and/or elution chamber, for instance by a channel. The inlet to the
seventh chamber may be provided in the upper portion of the seventh
chamber, for instance the upper 20%, preferably upper 10% and
ideally upper 5%. The inlet may be in the top wall of the seventh
chamber. The inlet may be provided in the corner of the seventh
chamber.
[0083] The channel connecting the ninth chamber and/or elution
chamber to the seventh chamber may have a plurality of sections.
The channel may have a vertical section and/or a horizontal section
and/or a second vertical section and/or a second horizontal
section. The channel may have a vertical section and a horizontal
section and a second vertical section and a second horizontal
section.
[0084] The channel may include one or more valves. The one or more
valves may include an open state to closed state valve or valves
and/or a closed state to open state valve or valves.
[0085] The ninth chamber and/or elution chamber may have a circular
cross-section. The cross-section may be relative to a horizontal
axis. The ninth chamber and/or elution chamber may be provided with
an eluent. The eluent may be provided to control conditions for a
subsequent process and/or reaction, for instance in one or more
further chambers and/or channels, such as the seventh chamber.
[0086] The ninth chamber and/or elution chamber may have an inlet
in the upper portion of the ninth chamber and/or elution chamber,
for instance the upper 20%.
[0087] The ninth chamber and/or elution chamber may be connected to
a pump, for instance an electrochemical pump. The pump may be a
third pump provided on the device. The third pump may provide the
drive to move one or more fluids and/or liquids through the ninth
chamber and/or elution chamber and/or seventh chamber and/or other
step and/or amplification chamber. The inlet from the pump may be
provided in the upper section of the chamber, for instance the
upper 20%, preferably upper 5%.
[0088] The ninth chamber and/or elution chamber may have an outlet.
The outlet may be provided in the lower portion of the chamber, for
instance the lower 20%, more preferably lower 10% and ideally the
lowest part of the chamber. The outlet may be in the bottom wall of
the chamber.
[0089] The sample preparation step or a part thereof may have a
first state in which it is isolated from one of more of the other
steps in the cartridge and/or from one or more other parts of the
sample preparation step. The one or more other steps may be a
sample receiving step and/or sample extraction step and/or sample
retention step and/or washing step and/or sample amplification step
and/or electrophoresis step and/or analysis step. The sample
preparation step or part thereof may have the first state during
contacting of the sample with the seventh chamber and/or contact
between the ninth chamber and/or elution chamber and the seventh
chamber. The sample preparation step or a part thereof may be
provided with one or more valves, preferably to provide the
isolation.
[0090] The seventh chamber may have a sample outlet. The sample
outlet may be provided in the lower portion of the seventh chamber,
for instance the lower 10%, more preferably lower 5% and ideally
the lowest part of the seventh chamber. The outlet may be in the
bottom wall of the chamber and/or in a corner of the chamber.
[0091] The sample outlet may connect to a channel. Preferably the
channel has a plurality of sections. The channel may have a
horizontal section and/or a vertical section and/or a second
horizontal section and/or a second vertical section and/or third
horizontal section and/or third vertical section and/or fourth
horizontal section and/or fourth vertical section and/or fifth
horizontal section. The channel may have a horizontal section and a
vertical section and a second horizontal section and a second
vertical section and third horizontal section and/or third vertical
section and/or fourth horizontal section and/or fourth vertical
section and/or fifth horizontal section. The channel may connect to
one or more further chambers, such as an eleventh chamber, and/or
to an amplification step.
[0092] The sample preparation step or a part thereof may have a
first state in which it is isolated from one of more of the other
steps in the cartridge and/or from one or more other parts of the
sample preparation step. The one or more other steps may be a
sample receiving step and/or sample retention step and/or
purification step and/or washing step and/or electrophoresis step
and/or analysis step. The sample preparation step or part thereof
may have the first state during contacting of the sample with the
seventh chamber and/or further chamber and/or amplification step.
The sample preparation step or a part thereof may be provided with
one or more valves. The valve may be provided at the first and/or
second gas outlets for the seventh chamber and/or channel to the
second pump and/or channel to the sixth chamber and/or channel to
the tenth chamber.
[0093] The sample amplification step may be provided on the device.
The sample amplification step may be provided on the same device as
the sample receiving step and/or sample preparation step.
[0094] The sample amplification step may include a first inlet,
preferably a channel. The channel may be connected to the sample
receiving step and/or sample preparation step.
[0095] The sample amplification step may include a second inlet,
preferably a channel. The channel may be connected to a pump, for
instance the fourth pump on the device.
[0096] The sample amplification step may include a first outlet,
preferably a channel. The channel may be connected to a sample
storage step or location, for instance a chamber.
[0097] The sample amplification step may include a second outlet,
for instance a channel. The channel may be connected to a further
step, for instance a denaturing step and/or electrophoresis step
and/or analysis step.
[0098] The first inlet and/or second outlet may be provided on the
inlet channel for the amplification step. The first inlet and/or
second outlet may share a section of channel and have separate
channel sections.
[0099] The first outlet and/or second inlet may be provided on the
outlet channel for the amplification step. The first outlet and/or
second inlet may share a section of channel and have separate
channel sections.
[0100] The sample amplification step may include a chamber, for
instance an eleventh chamber. The chamber is preferably connected
to the channel. The chamber preferably receives the sample. The
sample may be a washed sample. The sample may be a purified sample.
The sample may be less than the whole of the sample provided to the
device.
[0101] The chamber may be provided with a curved base. The base may
be semi circular in cross-section. The base may be a part of a
cylinder or hemisphere or proportion thereof. The chamber may be
provided with a curved top. The top may be semi circular in
cross-section. The top may be a part of a cylinder or hemispherical
or a portion thereof.
[0102] The top may be a larger volume than the bottom. The top
hemisphere or portion thereof may be larger then the lower
hemisphere or portion thereof.
[0103] A transition surface may extend between the base of the
chamber and the top of the chamber.
[0104] The chamber may include a support location for one or more
particles, such as a bead. The one or more particles may provide
one or more or all the reagents for a reaction, particularly an
amplification, such as PCR. The support location may define a
position of rest for the one or more particles. Preferably in the
position of rest, the one or more particles do not block or obscure
an inlet to and/or outlet from the chamber. Preferably in the
position of rest at least 50%, preferably at least 60% and more
preferably at least 70% of the surface area of the one or more
particles are exposed to the chamber.
[0105] Preferably an inlet for a sample and/or an inlet from a
previous chamber is provided in a side wall of the chamber. The
inlet may be provided in the mid section of the height of the
chamber, preferably the middle 20%, more preferably the middle
10%.
[0106] Preferably the outlet for the sample and/or outlet to a
receiving location and/or other chamber is provided in a side wall
of the chamber. The outlet may be provided in the mid section of
the height of the chamber, preferably the middle 20%, more
preferably the middle 10%.
[0107] The inlet and the outlet are preferably provided opposite
one another. The inlet and the outlet are preferably provided at
the same height in the chamber.
[0108] The chamber may have an orientation of use. A chamber may be
provided with a horizontal base and/or a horizontal top. The base
and/or top, may be horizontal +/-10.degree., preferably
+/-5.degree. and more preferably +/-3.degree..
[0109] The chamber may be provided with one or more side walls. The
side wall(s) may be vertical +/-10.degree., preferably +/-5.degree.
and more preferably +/-3.degree..
[0110] The chamber may include a support location for one or more
particles, such as a bead. The one or more particles may provide
one or more or all the reagents for a reaction, particularly an
amplification, such as PCR. The support location may define a
position of rest for the one or more particles. Preferably in the
position of rest, the one or more particles do not block or obscure
an inlet to and/or outlet from the chamber.
[0111] Preferably in the position of rest at least 50%, preferably
at least 60% and more preferably at least 70% of the surface area
of the one or more particles are exposed to the chamber.
[0112] Preferably an inlet for a sample and/or an inlet from a
previous chamber is provided in the top of the chamber or in the
upper section of the chamber. The upper section may be the upper
20%, more preferably the upper 10%.
[0113] Preferably the outlet for the sample and/or outlet to a
receiving location and/or other chamber is provided in the top of
the chamber. The upper section may be the upper 20%, more
preferably the upper 10%. The inlet and the outlet may be the
same.
[0114] Preferably the chamber is provided with a chamber filling
outlet. Preferably fluid enters the chamber via the inlet and flows
out of the chamber through the chamber filling outlet during the
filling of the chamber. The chamber filling outlet is preferably
provided in the base or lower section of the chamber, for instance
the lower 20% or more preferably 10%.
[0115] The channel connected to the inlet to the chamber may be
provided with a valve. The channel connected to the outlet from the
chamber may be provided with a valve. One or more of the valves may
be open state to closed state valves, particularly for the first
inlet and/or second outlet channels. One or more of the valves may
be closed state to open state valves, particularly for the second
inlet and/or first outlet channels. One or more of the valves may
be provided closer to the chamber than the split into the first
inlet and second outlet and/or second inlet and first outlet
sections of channel. If the valves are provided further from the
chamber than the split into the first inlet and second outlet
and/or second inlet and first outlet sections of channel, then a
separate valve may be provided for each channel section.
[0116] The valve connected to the inlet may provide a first sealing
location. The valve connected to the outlet may provide a second
sealing location. One or more interconnected channels and chambers
may be provided between the first sealing location and the second
sealing location. Preferably the channel connected to the inlet,
the chamber and the channel connected to the outlet are provided
between the first sealable location and the second sealable
location.
[0117] The section of the device including the first sealable
location, second sealable location and channels and chambers
provided there between may have an extent, preferably in a first
plane. The section of the device including the first sealable
location, second sealable location and channels and chambers
provided there between may have a planar form and/or planar
exterior surface extending in a first plane.
[0118] A heating device may be provided to heat the chamber. The
heating device may have an extent parallel to the first plane. The
heating device may have an extent parallel to the first plane of
the planar form of the section and/or planar exterior surface. The
extent of the heating device may be greater than 75% of the extent
of the channels and chambers between the first sealable location
and the second sealable location. The extent of the heating device
may be greater than 75% of the extent of the channels and chambers
between the first sealable location and the second sealable
location, considered in terms of the area those extend to in the
first plane. The extent of the heating device may be greater than
80%, 90% or even 95%, possibly even 98% or 100% of such extents.
The heating device may be incident with at least 75% of the extent
of the channels and chambers provided between the first sealable
location and the second sealable location, when the extent of those
channels and chambers is projected perpendicular to the first
plane. The extent of the heating device may be greater than 80%,
90% or even 95%, possibly even 98% or 100% of such extents.
[0119] The chamber may have an orientation of use. A chamber may be
provided with a horizontal base and/or a horizontal top. The base
and/or top, may be horizontal +/-10.degree., preferably
+/-5.degree. and more preferably +/-3.degree..
[0120] The chamber may be provided with one or more side walls. The
side wall(s) may be vertical +/-10.degree., preferably +/-5.degree.
and more preferably +/-3.degree..
[0121] The junction between the base and the side walls may be
curved. The junction between the top and the side walls may be
curved. The junction between the top and the side walls may be
provided by an intermediate wall. The intermediate wall may be
inclined relative to the top and/or side walls.
[0122] The chamber may include a support location for one or more
particles, such as a bead. The one or more particles may provide
one or more or all the reagents for a reaction, particularly an
amplification, such as PCR. The support location may define a
position of rest for the one or more particles. Preferably in the
position of rest, the one or more particles do not block or obscure
an inlet to and/or outlet from the chamber. Preferably in the
position of rest at least 50%, preferably at least 60% and more
preferably at least 70% of the surface area of the one or more
particles are exposed to the chamber. The support location may be
provided by the base of the chamber.
[0123] Preferably an inlet for a sample and/or an inlet from a
previous chamber is provided in the top of the chamber or in the
upper section of the chamber. The upper section may be the upper
20%, more preferably the upper 10%.
[0124] The inlet may be provided in a corner of the chamber.
[0125] Preferably the outlet for the sample and/or outlet to a
receiving location and/or other chamber is provided in the top of
the chamber. The upper section may be the upper 20%, more
preferably the upper 10%. The inlet and the outlet may be provided
at the same height.
[0126] The outlet may be provided in a corner of the chamber.
[0127] An inlet channel may be provided which leads to the inlet.
An outlet channel made be provided which leads away from the
outlet. A by-pass channel may be provided for the chamber. The by
pass channel may connect a part of the inlet channel to a part of
the outlet channel.
[0128] The by-pass channel may be a continuation of the channel
from which the inlet channel and/or outlet channel branch. The
by-pass channel and channel may have a common axis.
[0129] The by-pass channel may be a branch from the channel from
which the inlet channel branches. The by-pass channel and/or inlet
channel may be provided with an axis which is not a continuation of
the axis of the channel from which they branch. Preferably, the
by-pass channel is provided with an axis which is not a
continuation of the axis of the channel from which it branches,
with still more preferably the inlet channel being provided with on
a common axis to that of the portion of the channel which adjoins
it.
[0130] The by-pass channel may be a branch from the channel from
which the outlet channel branches. The by-pass channel and/or
outlet channel may be provided with an axis which is not a
continuation of the axis of the channel from which they branch.
Preferably, the by-pass channel is provided with an axis which is
not a continuation of the axis of the channel from which it
branches, with still more preferably the outlet channel being
provided with on a common axis to that of the portion of the
channel which adjoins it.
[0131] Preferably one or more dimensions of the outlet channel are
smaller than the corresponding dimension of the inlet channel. The
value of the one or more dimensions may be considered at the
location within the inlet channel and/or outlet channel where that
dimension has its lowest value. The one or more dimensions may
include one or more or all of the width and/or height and/or
cross-sectional area. The cross-sectional area may be measured
perpendicular to the direction of flow in the inlet channel and/or
outlet channel and/or perpendicular to the alignment or axis of the
inlet channel and/or outlet channel.
[0132] The resistance to fluid flow provided by the outlet and/or
outlet channel may be greater than the resistance to fluid flow
provided by the inlet and/or inlet channel. The resistance to fluid
flow provided by the outlet and/or outlet channel may be greater
than the resistance to fluid flow provided by the by-pass
channel.
[0133] The path of least resistance for the fluid may be through
the inlet and into the chamber until the fluid reaches the outlet
and/or outlet channel. The path of least resistance for the fluid
may be through the by-pass channel once the fluid has reached the
outlet and/or outlet channel.
[0134] The fluid flow may switch from the inlet channel to the
by-pass channel when a predetermined volume of fluid is provided in
the chamber.
[0135] The chamber may have an orientation of use. A chamber may be
provided with a curved base. The base may be semi circular. The
base may be a hemisphere or proportion thereof. The chamber may be
provided with a top wall, such as a planar top wall. The top wall
may be provided in one or more portions. The plane of one or more
of those portions may be different to the plane of one or more of
the other portions.
Preferably the Planes are Parallel.
[0136] An inclined transition surface may extend between the base
of the chamber and the side walls of the chamber. The side wall may
connect to the top of the chamber. The side walls may be vertical
in the orientation of use.
[0137] The chamber may include a support location for one or more
particles, such as a bead. The one or more particles may provide
one or more or all the reagents for a reaction, particularly an
amplification, such as PCR. The support location may define a
position of rest for the one or more particles. Preferably in the
position of rest, the one or more particles do not block or obscure
an inlet to and/or outlet from the chamber. Preferably in the
position of rest at least 50%, preferably at least 60% and more
preferably at least 70% of the surface area of the one or more
particles are exposed to the chamber.
[0138] Preferably an inlet for a sample and/or an inlet from a
previous chamber is provided in a side wall of the chamber. The
inlet may be provided in the lower section of the height of the
chamber, preferably the lower 30%, more preferably the lower
10%.
[0139] Preferably the outlet for the sample and/or outlet to a
receiving location and/or other chamber is provided in a top wall
of the chamber. The outlet may be provided in the top section of
the height of the chamber, preferably the top 20%, more preferably
the top 10%.
[0140] The inlet and the outlet are preferably provided opposite
one another. The inlet and the outlet are preferably provided at
different heights in the chamber.
[0141] The sample amplification step or a part thereof may have a
first state in which it is isolated from one of more of the other
steps in the cartridge and/or from one or more other parts of the
sample amplification step. The one or more other steps may be a
sample receiving step and/or sample preparation step and/or sample
retention step and/or purification step and/or washing step and/or
elution step and/or electrophoresis step and/or analysis step. The
sample amplification step or part thereof may have the first state
during contacting of the sample with the chamber, particularly the
chamber in which amplification is provided.
[0142] The sample denaturing step may be provided on the device.
The sample denaturing step may be provided on the same device as
the sample receiving step and/or sample preparation step and/or
sample amplification step. The sample denaturing step may include a
chamber.
[0143] The chamber may be connected to the amplification step,
preferably by a channel. The channel may be connected to the second
outlet from the amplification step. The inlet to the chamber may be
provided in the upper portion of the chamber, for instance the
upper 20%, preferably upper 10% and ideally upper 5%. The inlet may
be in the top wall of the chamber. The inlet may be provided in the
corner of the chamber.
[0144] The channel connecting the amplification step and/or
amplification chamber to the chamber may have a plurality of
sections. The channel may have a horizontal section and/or a
vertical section and/or a second horizontal section and/or a second
vertical section. The channel may have a horizontal section and a
vertical section and a second horizontal section and/or a second
vertical section.
[0145] The channel may include one or more valves. The one or more
valves may include an open state to closed state valve or valves
and/or a closed state to open state valve or valves.
[0146] The chamber may have a non-linear cross-section. The chamber
may have a cross-section formed by a horizontal top wall, inclined
lower wall and end walls joining the top and lower walls. The
transition end walls may be curved or linear. The cross-section may
be relative to a horizontal axis.
[0147] The chamber may have a sample outlet. The sample outlet may
be provided in the lower portion of the chamber, for instance the
lower 10%, more preferably lower 5% and ideally the lowest part of
the chamber. The outlet may be in the bottom wall of the chamber or
preferably in a corner of the chamber, ideally the corner opposing
the inlet.
[0148] The chamber may be connected to a pump, for instance an
electrochemical pump. The pump may be a fourth pump provided on the
device. The fourth pump may provide the drive to move one or more
fluids and/or liquids through the amplification step and/or
amplification chamber and/or chamber and/or one or more further
chambers and/or denaturing step.
[0149] The connection to the pump may be via the amplification step
and/or amplification chamber.
[0150] In one embodiment, the amplification chamber is connected to
the denaturing chamber, preferably with no further chambers
provided there between.
[0151] The channel leading from the amplification chamber to the
denaturing chamber may split into two channels. One of the two
channels may lead, preferably past a valve, to the denaturing
chamber. The denaturing chamber may have a vent channel which
preferably extends past a valve. One of the two channels may lead,
preferably past a valve to an archive chamber. The archive chamber
may have a vent channel which preferably extends past a valve.
[0152] The denaturing chamber may have an outlet channel which
leads to the analysis step and/or electrophoresis step.
[0153] In a second embodiment, the pump may be connected to a
channel which leads to an inlet for a further chamber. The further
chamber may contain one or more reagents or materials, for instance
for denaturing the sample. The further chamber may have an outlet
leading to a channel and/or to the amplification step and/or to the
amplification chamber.
[0154] Particularly in a second embodiment, the chamber may be
connected to a second chamber, preferably by a channel. The inlet
to the second chamber may be provided in the upper portion of the
second chamber, for instance the upper 20%, preferably upper 10%
and ideally upper 5%. The inlet may be in the top wall of the
second chamber. The inlet may be provided in the corner of the
second chamber.
[0155] Particularly in a second embodiment, the channel connecting
the chamber to the second chamber may have a plurality of sections.
The channel may have one or more horizontal sections and/or one or
more vertical sections.
[0156] Particularly in a second embodiment, the second chamber may
have a non-linear cross-section. The chamber may have a
cross-section formed by a horizontal top wall, inclined lower wall
and end walls joining the top and lower walls. The transition end
walls may be curved or linear. The cross-section may be relative to
a horizontal axis.
[0157] Particularly in a second embodiment, the second chamber may
have a sample outlet. The sample outlet may be provided in the
lower portion of the second chamber, for instance the lower 10%,
more preferably lower 5% and ideally the lowest part of the second
chamber. The outlet may be in the bottom wall of the second chamber
or preferably in a corner of the second chamber, ideally the corner
opposing the inlet.
[0158] Particularly in a second embodiment, the second chamber may
be connected to a third chamber, preferably by a channel. The inlet
to the third chamber may be provided in the upper portion of the
third chamber, for instance the upper 20%, preferably upper 10% and
ideally upper 5%. The inlet may be in the top wall of the third
chamber. The inlet may be provided in the corner of the third
chamber.
[0159] Particularly in a second embodiment, the channel connecting
the second chamber to the third chamber may have a plurality of
sections. The channel may have one or more horizontal sections
and/or one or more vertical sections.
[0160] Particularly in a second embodiment, the third chamber may
have a non-linear cross-section. The chamber may have a
cross-section formed by a horizontal top wall, inclined lower wall
and end walls joining the top and lower walls. The transition end
walls may be curved or linear. The cross-section may be relative to
a horizontal axis.
[0161] Particularly in a second embodiment, the third chamber may
have a sample outlet. The sample outlet may be provided in the
lower portion of the third chamber, for instance the lower 10%,
more preferably lower 5% and ideally the lowest part of the third
chamber. The outlet may be in the bottom wall of the third chamber
or preferably in a corner of the third chamber, ideally the corner
opposing the inlet.
[0162] The sample denaturation step or a part thereof may have a
first state in which it is isolated from one of more of the other
steps in the cartridge and/or from one or more other parts of the
sample denaturation step. The one or more other steps may be a
sample receiving step and/or sample extraction step and/or sample
retention step and/or washing step and/or sample amplification step
and/or electrophoresis step and/or analysis step. The sample
denaturation step or part thereof may have the first state during
contacting of the sample with the chamber and/or second chamber
and/or third chamber and/or during denaturation of the sample. The
sample preparation step or a part thereof may be provided with one
or more valves, preferably to provide the isolation. The first
inlet to the amplification step and/or amplification chamber may be
provided with a valve, preferably of the open state to closed state
type. The first outlet from the amplification step and/or
amplification chamber may be provided with a valve, preferably of
the open state to closed state type. The outlet from the
denaturation step to the electrophoresis step and/or the channel
connected to the outlet of the third chamber may be provided with a
valve, preferably of the closed state to open state type.
[0163] The electrophoresis step may be provided on the device. The
electrophoresis step may be provided on the same device as the
sample receiving step and/or sample preparation step and/or sample
amplification step. The electrophoresis step may include a
channel.
[0164] The channel may be connected to the amplification step
and/or denaturing step.
[0165] The channel may extend from the plane of the device to a
location behind the plane of the device.
[0166] The electrophoresis step may be provided on an element. The
element may be a part of or be separate from the device. The
element may be planar. The element may include one or more
channels. The element may include one or more channels in which
electrophoresis is provided. One or more electrodes may be provided
on the element. One or more electrodes may be provided to load the
sample into the element. One or more electrodes may be provided to
perform the electrophoresis step. The electrodes may be provided in
portions which have a greater depth than one or more other parts of
the element. The one or more other parts of the element may include
the part in which the channel is provided. The electrodes may be
provided in portions which adjoin end portions of the element. The
end portions may provide the mounting for the element on a carrier
and/or relative to the device, such as a cartridge.
[0167] The connection between the one or more electrodes and the
operating electronics for the instrument may be provided by one or
more pins mounted on the element. The one or more pins may be
spring loaded. The one or more pins may be partially or fully
recessed into a surface of the element, particularly the greater
depth portion(s) thereof. The connection may be provided or may be
further provided by one or more pins mounted on the instrument. The
one or more pins may be spring loaded. The connection may be made
when the element is put in the use position.
[0168] The element, and particularly a channel therein, may be
connected to the device, such as a cartridge, by a conduit. The
conduit may be flexible. The conduit may be a tube.
[0169] The channel may be connected to a chamber. The chamber may
contain a liquid to matrix interface, preferably a horizontal
interface. A pump, preferably an electrochemical pump, preferably
the fourth pump may convey the sample to the chamber. The pump,
preferably the electrochemical pump, may also convey a buffer
and/or formamide to the chamber. The buffer and/or formamide may
displace the content of the amplification and/or PCR chamber into
the chamber. The buffer and/or formamide may include one or more
components for the electrophoretic separation and/or analysis. The
one or more components may include a size standard.
[0170] The sample may be concentrated before the start of
electrophoresis.
[0171] The sample may be concentrated before the sample enters the
matrix. The sample may be concentrated by the electrophoretic
velocity on one side of the interface exceeding the opposing
electroosmotic velocity and/or by the electrophoretic velocity on
the other side of the interface being less than the opposing
electroosmotic velocity.
[0172] The sample may be collecting and/or concentrated at a first
location. The sample may be further collected and/or concentrated
at a second location.
[0173] The sample may be collecting and/or concentrated at a first
location in the form of an interface. The sample may be further
collected and/or concentrated at a second location in the form of
an interface. The first interface may be planar. The second
interface may be planar. The first interface may be provided by a
series of surfaces. The second interface may be provided by a
series of surfaces. The series of surfaces may be provided by
particles or beads or channels or mixtures thereof.
[0174] The sample may be collecting and/or concentrated at a first
interface. The sample may be further collected and/or concentrated
at a second interface. The sample may be stacked at a first
interface. The sample may be further stacked at a second
interface.
[0175] The first interface may be a liquid to liquid interface or
liquid to solid or gel interface or solid or gel to solid or gel
interface. The first interface may be a membrane. The second
interface may be a liquid to liquid interface or liquid to solid or
gel interface or solid or gel to solid or gel interface. The second
interface may be a membrane. The first and second interfaces may be
of the same or different types.
[0176] The sample or a part thereof may be collected and/or
concentrated by flowing a first fluid past a first side of an
interface. The sample or a part thereof may be collected and/or
concentrated by flowing a second fluid past a second side of an
interface.
[0177] The sample may be fed to one side of the interface, for
instance the first side. A reagent, for instance a buffer may be
fed to the other side of the interface, for instance the second
side. The channel feeding the sample and/or the channel feeding the
reagent to the channel containing the interface may be at least
partially with the channel containing the interface. The channels
may be aligned at an angle of less than 30.degree.. Both channels
may be so aligned. The channel feeding the sample and/or the
channel feeding the reagent to the channel containing the interface
may be curved so as to align their flow with the direction of flow
within the channel containing the interface.
[0178] The channel for electrophoresis may be provided at an angle
to the interface, for instance greater than 75.degree.. The channel
may be perpendicular to the interface, particularly the plane
thereof. The channel for electrophoresis may be provided at an
angle to the first and the second interface, for instance greater
than 75.degree.. The channel may be perpendicular to the first and
the second interface, particularly the plane thereof.
[0179] The sample or a part thereof may be collected and/or
concentrated at the first interface and then at the second
interface. Conditions on one or both sides of the first interface
may be varied to cause collection and/or concentration at the first
interface. Conditions on one or both sides of the second interface
may be varied to cause collection and/or concentration at the first
interface. Conditions on one or both sides of the first interface
may be varied to cause collection and/or concentration at the
second interface. Conditions on one or both sides of the second
interface may be varied to cause collection and/or concentration at
the second interface. The conditions which are varied may be one or
more of reagent or reagents present, the reagent or reagents
concentration, pH, temperature, conductivity of the components
present or electrical potential present. The conditions may vary at
or in proximity with the interface.
[0180] The electrical potential may be applied by a voltage across
a first electrode and a second electrode. The first electrode may
be provided to one side of the interface, particularly the first
and the second interfaces. The second electrode may be provided to
the other side of the interface, particularly the first and second
interfaces. The first electrode may be provided in a channel or
chamber connected to, but spaced from the channel through which the
sample is introduced and/or in which the interface is provided. The
second electrode made be provided in the channel for
electrophoresis. The second electrode may be provided beyond the
channel for electrophoresis, compared with the position of the
first electrode.
[0181] The second interface may lead to the channel for
electrophoresis. The second interface may be in contact with the
matric within the channel for electrophoresis.
[0182] The sample may be introduced to a channel, that channel
being in contact with a first interface. That channel may be in
contact with a second interface. The first and second interfaces
may be provided at opposing ends of the channel. The first and
second interfaces may be provided in opposition to one another,
with a length of channel there between. The length of channel there
between may include the channel in which electrophoresis is
provided.
[0183] An electrode may be provided on the side of the first
interface away from the channel. An electrode may be provided on
the side of the second interface away from the channel. An
electrical potential may be applied to one or both of the
electrodes, preferably across the electrodes.
[0184] At least a part of the sample, such as DNA in the sample,
may be moved towards the first interface by the electrical
potential. The first interface may be downstream of a second
interface relative to the direction in which the sample flows into
the channel. The electrical potential may be applied as the sample
flows through the channel. The flow may be from an inlet to an
outlet. A waste sample chamber may be provided downstream of the
channel.
[0185] The first and/or second interface may be impermeable to one
or more components of the sample, such as DNA. The first and/or
second interface may be impermeable to components of greater than
SkDa, or even greater than 8 kDa.
[0186] A further material may flow into the channel, preferably
after the sample flow and/or after the at least a part of the
sample is at the first interface. The further material may displace
the sample flow from the channel. One or more other materials may
flow through the channel between the sample flow and the further
material.
[0187] The further material may provide a matrix for the
electrophoresis in the channel. The further material may be
introduced into the channel and then altered to provide the matrix
for electrophoresis. The further material may be altered by the
application of light, such as UV light, and/or heating. The further
material may be altered by polymerisation.
[0188] The one or more other materials may include one or more
buffers and/or one or more salt removal agents and/or one or more
DNA purification reagents and/or one or more PCR primer removal
reagents.
[0189] The sample may be introduced to a channel, that channel
being in contact with a first interface. The first interface may be
provided at one end of a channel, with a length of channel there
between. The length of channel there between may include the
channel in which electrophoresis is provided.
[0190] An electrode may be provided on the side of the interface
away from the channel. An electrode may be provided on the other
side of the interface, with the length of the channel provided
between that electrode and the other electrode. An electrical
potential may be applied to one or both of the electrodes,
preferably across the electrodes.
[0191] At least a part of the sample, such as DNA in the sample,
may be moved towards the interface by the electrical potential. The
interface may be provided in a wall of the channel through which
the sample flows and/or may be across a channel extending off the
channel within which the sample flows. The electrical potential may
be applied as the sample flows through the channel. The flow may be
from an inlet to an outlet. A waste sample chamber may be provided
downstream of the channel.
[0192] The first interface may be impermeable to one or more
components of the sample, such as DNA. The first interface may be
impermeable to components of greater than 5 kDa, or even greater
than 8 kDa.
[0193] The electrical potential may be used to transfer the at
least a part of the sample from the interface to the matrix in
which electrophoresis is conducted. The electrical potential may be
reversed to provide this transfer.
[0194] One or more other materials may flow through the channel
after the sample flow. The one or more other materials may include
one or more buffers and/or one or more salt removal agents and/or
one or more DNA purification reagents and/or one or more PCR primer
removal reagents. The channel may be connected to the
electrophoresis channel. The electrophoresis channel may be linear.
The electrophoresis channel may have a side channel, preferably the
side channel is connected to the channel. The electrophoresis
channel may have a second side channel. The second side channel may
be axially aligned with the first side channel or may be offset
relative thereto. The electrophoresis channel may be provided with
an electrode an one end of a separation length and a second
electrode at the other end of a separation length. The first side
channel and/or second side channel may be provided with an
electrode. One or more of the electrodes may have a coating, for
instance a platinum coating, gold coating, carbon coating, nickel
coating. One or more of the electrodes may be of platinum, gold,
carbon or nickel.
[0195] The channel may be provided with a first side channel
through which the sample or at least a part thereof is introduced.
The first side channel may provide flow in the direction of gravity
to the channel. The channel may be provided with a second side
channel, preferably through which the sample or a part thereof
exits the channel. The second side channel ma provide flow in a
direction against gravity away from the channel. The junction
between the first side channel and the channel may be spaced along
the channel when compared with the junction between the second side
channel and the channel.
[0196] A detection location may be provided at a position along the
separation length.
[0197] The sample amplification step may include a split into a
first channel and a second channel. The first channel may be
connected to the amplification step and/or amplification chamber as
described above. The second channel may be connected to a second
amplification step and/or amplification channel.
[0198] The sample amplification step may include a supply of sample
to a first channel and a second channel. The first channel may be
connected to the amplification step and/or amplification chamber as
described above. The second channel may be connected to a second
amplification step and/or amplification channel.
[0199] The first amplification step and/or amplification chamber
may be connected in series with the second amplification step
and/or amplification chamber. The first amplification step and/or
amplification chamber may be connected in parallel with the second
amplification step and/or amplification chamber.
[0200] The second amplification step and/or second amplification
chamber may have any of the features, options and possibilities set
out elsewhere, including those of the amplification step and/or
amplification chamber.
[0201] The second amplification step and/or second amplification
chamber may be provided with a quantification unit, for instance
for the amount of sample therein, ideally the amount of DNA. The
quantification unit may provide the amount of sample at one or more
times before, during or after amplification in the second
amplification step and/or second amplification chamber.
[0202] The quantification unit may include one or more reagents
provided in or introduced to the second amplification step and/or
second amplification chamber.
[0203] The quantification unit may include a device sensitive to a
characteristic of the sample and the amount thereof. The
characteristic may be light, particularly fluorescent light. The
quantification unit may include the optical system and/or light
detector used in the electrophoresis step and/or analysis step.
[0204] The sample preparation step may include one or more
chambers, preferably into which the sample passes.
[0205] The chamber may be provided connected to one or more further
chambers. Each of the chambers may be provided with a one or more
particles. The particles may be beads. One or more of the particles
may be magnetic. The one or more particles may have a magnetic
material within a surface layer or layers. The particles may be
provided with one or more reagents or materials which releasable
bind and/or link and/or combine with a part of the sample, for
instance DNA. The particles, such as beads, may be stored in the
chamber before use. The particles, such as beads, may be introduced
to the chamber to prepare it for use, for instance within 5 hours,
or even within 1 hour, of use occurring.
[0206] A plurality of chamber may be provided, connected in series.
Two or more of the chambers may have an inlet in the upper portion
of the chamber, for instance the upper 20%. The inlet may be in the
top wall of the chamber. Two or more of the chambers may have an
outlet in the lower portion of the chamber, for instance the lower
20%, more preferably lower 10% and ideally the lowest part of the
chamber. The outlet may be in the bottom wall of the chamber.
[0207] The chambers may be connected to each other by one or more
channels. Preferably the channel has a plurality of sections. The
channel may have one or more vertical sections and/or a one or more
horizontal sections.
[0208] Two or more of the chambers may have the same configuration
and/or shape. One or more of the chambers may have a different
configuration and/or shape to one or more of the others.
[0209] One or more of the chambers may be a channel or passageway
which is larger in respect of one or more dimensions than the
channel leading to it and/or from it. The one or more particles may
be provided in the channel or passageway which is larger.
[0210] One or more chambers may be provided having a particulate
collection and/or holding location. Flow into the chamber
preferably passes through the particulate collection and/or holding
location, preferably preferentially to flow through other locations
in the chamber. The particulate collection and/or holding location
may be a recess in the bottom of the chamber.
[0211] One or more chambers, channels or passageways may be
provided in which the one or more particles are provided in a
channel connected to the one or more chambers, channels or
passageways. The one or more particles may be displaceable from the
channel into the one or more chambers, channels or passageways. A
material may be provided in the channel to displace the one or more
particles.
[0212] Any of the aspects of the invention may include any of the
following options, features or possibilities.
[0213] The sample may be received from one or more of: a swab, a
buccal swab, a cotton swab, a soft swab, a solution, a suspension,
an item of clothing, an item placed in the mouth, a cigarette or
piece thereof, chewing gum or saliva.
[0214] The sample may be a skin sample, blood sample, cell sample,
bodily fluid sample, hair sample, saliva sample or sample
containing one or more of these.
[0215] The sample may be a forensic sample. The sample may be a
medical sample.
[0216] The analysis may be for diagnostic purposes. The analysis
may be for forensic purposes.
[0217] The analysis may be for use in the consideration of marker
targets, diagnostic assays, disease markers, biobanking
applications, STR based targets in transplants, identification of
drug resistant microorganisms, blood testing, mutation detection,
DNA sequencing, food analysis, pharmogenetics and pharmogenomics,
medical fields, biotech fields, in determining familial
relationships, paternity testing and pedigree testing in
animals.
[0218] The analysis may be for use in border control, security or
customs situations and/or uses.
[0219] The device may be a microfluidic device. The instrument may
incorporate a microfluidic device. The device may be a device
processing a sample of less than 50:1, preferably less than 30:1,
more preferably less than 20:1, potentially less than 10:1 in one
or more steps. The device may be a device processing a fluid,
particularly a liquid, of less than 50:1, preferably less than
30:1, more preferably less than 20:1, potentially less than 10:1 in
one or more steps.
[0220] The device may process and/or contain a fluid, particularly
a liquid, of less than 1 ml, possibly less than 500:1, possibly
less than 250:1, potentially less than 200:1, possibly less than
175:1, possibly less than 50:1, preferably less than 30:1, more
preferably less than 20:1, potentially less than 10:1 in one or
more of the following steps: a sample receiving step and/or sample
preparation step and/or sample extraction step and/or sample
retention step and/or purification step and/or washing step and/or
elution step and/or amplification step and/or PCR step and/or
denaturing step and/or investigation step and/or electrophoresis
step and/or detection step and/or analysis step and/or results
output step.
[0221] The device may incorporate one or more channels or chambers
with a maximum dimension of less than 1000:m, possible less than
750:m and preferably less than 550:m.
[0222] The device may incorporate one or more channels or chambers
with a maximum dimension of less than 500:m, possible less than
250:m and preferably less than 100:m.
[0223] The device may include a chambers provided with one or more
reagents. One or more chambers may be so provided. The reagents may
be different. The reagents may be in liquid form. The reagents may
be provided on and/or in the surface of a solid. The solid may be
one or more beads. The solid may be magnetic.
[0224] One or more reagents may be provided for cell lysis. One of
more reagents may be provided for a selective extraction of DNA
containing material from other material. One or more reagents may
be provided for washing. One or more reagents may be provided for
elution, particularly from the surface of a solid. One or more
reagents may be provided for amplification, particularly PCR based
amplification. One or more reagents may be provided for denaturing.
One or more reagents may be provided for electrophoresis.
[0225] Preferably the device has a stored form and a use form. In
the use form, the sample to be processed may be loaded into the
device. Preferably one or more reagents are pre-loaded into the
device and/or are present in the device when in the stored form.
One or more reagents may be loaded into the device in the use
form.
[0226] The device and/or method may include one or more pumps.
Preferably the device only includes pumps of a single type.
Preferably the pumps of the single type are identical with respect
to chamber shape and/or electrode positions and/or electrode
materials and/or orientation and/or chamber volume and/or pump
electrolyte and/or pump electrolyte concentration.
[0227] One or more, preferably all, of the pumps may be
electrochemical pumps.
[0228] The device may have an orientation of use, preferably one
electrode in the pump chamber is provided above the other. The pump
chamber may have a height greater than its width. The pump chamber
may have a width greater than its depth.
[0229] The pump chamber may have an outlet. Preferably the outlet
is provided in the upper section of the pump chamber. The upper
section may be the upper 20%, preferably 10%, and more preferably
5% of the height of the chamber. The outlet may be in the top wall
of the chamber.
[0230] The pump chamber may contain NaCl. The molarity of the
electrolyte in the pump chamber may be between 0.2M and 3M,
preferably 1M+/-15%.
[0231] The electrophoresis step and/or electrophoresis cartridge
section may be provided with a channel, for instance a capillary
for electrophoresis.
[0232] The channel may be provided with a matrix. Preferably the
matrix resists the passage of elements, the resistance being
related to the size of the element. Preferably different size
elements migrate through the matrix at different rates, the larger
migrating slower.
[0233] The channel may be provided with an inert bed of particulate
material to form the matrix.
[0234] The channel may be provided with a gel, particularly a
polymer gel. The channel may be provided with polyhydroacrylamide,
polydimethylacrylamide or mixtures there of. The channel may be
provided with a cross-linked polymer. The cross-linking of the
polymer may be provided in situ.
[0235] One or more surfaces of the channel may be treated, for
instance with a hydrophilic coating, for instance
poly(hydroxyethlacrylamide).
[0236] The channel may be provided with a matrix during
electrophoresis. The channel may be provided without a matrix prior
to electrophoresis, with the matrix being introduced before
electrophoresis commences. The matrix or a material for forming the
matrix may be stored at a location removed from the channel in
which electrophoresis is provided. The matrix or material for
forming the matrix may be stored in a chamber. The chamber may be
connected by a channel to the channel in which electrophoresis is
provided.
[0237] The matrix and/or material for forming the matrix may be
altered before use in the electrophoresis step. The alteration may
be provided before and/or during and/or after the matrix and/or
material for forming the matrix is provided in the channel. The
alteration may be polymerisation. The alteration may be caused
and/or triggered by heating and/or the application of light, such
as UN light. The alteration may be applied to all of the matrix
and/or material for forming the matrix or only a part thereof. One
or more parts of the matrix may be prevented from alteration, for
instance by masking those parts and/or excluding heat and/or
excluding light from them.
[0238] The sample receiving step may include the transfer of a
sample from outside the device and/or instrument, to inside the
device and/or instrument. The sample receiving step may receive the
sample from a collection device or from a storage device. The
sample receiving step may include the transfer of the sample to a
channel or chamber within the device.
[0239] The sample preparation step may include contacting the
sample with one or more reagents and/or one or more other
components. The reagents and/or other component may be used to
prepare the sample for one or more of the subsequent steps.
[0240] The sample extraction step may be part of or separate from
the sample preparation step. The sample extraction step may include
contacting the sample with one or more reagents and/or components
which select the sample component(s) relative to one or more waste
components in the sample. The selected sample component(s) may be
removed from the waste component(s) and/or the waste component(s)
may be removed from the selected sample components. The waste
component(s) may flow away from the extraction step. The waste
component(s) may be washed away from the extraction step using one
or more further reagents and/or components.
[0241] The sample retention step may be a part of or may be
separate from the sample preparation step and/or sample extraction
step. The sample retention step may include contacting the sample
with one or more reagents and/or components which retain the sample
component(s) relative to one or more waste components in the
sample. The sample component(s) may be retained on one or more
beads. The beads may be magnetic. The retained sample component(s)
may be removed from the waste component(s) and/or the waste
component(s) may be removed from the retained sample components.
The waste component(s) may flow away from the retention step. The
waste component(s) may be washed away from the retention step using
one or more further reagents and/or components. The waste
component(s) may flow past the location of retention. The waste
component(s) may be washed away using one or more further reagents
and/or components which flow past the location of retention.
[0242] The retained and/or selected sample may be eluted,
preferably with the eluent conveying the retained and/or selected
sample to the next step.
[0243] The purification step may be a part of or may be separate
from the sample preparation step and/or sample extraction step
and/or sample retention step. The purification step may separate
the selected sample components, for instance DNA, from one or more
waste components of the sample, for instance cellular material, PCR
inhibitors and chemical inhibitors.
[0244] The washing step may be a part of or may be separate from
the sample preparation step and/or sample extraction step and/or
sample retention step and/or purification step. The washing step
may remove one or more components of the sample from the location
of one or more other components of the sample.
[0245] The elution step may be a part of or may be separate from
the sample preparation step and/or sample extraction step and/or
sample retention step and/or purification step and/or washing step.
The elution step may remove one or more components of the sample
from a first form into a second form. The first form may be bound
to a surface or substrate, for instance on a bead. The second form
may be in a liquid, for instance the eluent.
[0246] The amplification step may include contacting the sample
with one or more reagents and/or components to cause amplification.
The amplification step may include contacting the sample with
conditions, preferably of a cyclic nature, to cause amplification.
The amplification may be provided by a PCR step.
[0247] The denaturing step may prepare the sample for
electrophoresis. The denaturing step may include contacting the
sample with one or more reagents and/or components. The denaturing
step may include contacting the sample with conditions, preferably
of a cyclic nature, to cause denaturing.
[0248] The investigation step may provide a characteristic for one
component of the sample which differs from the characteristic for
one or more other components of the sample. The characteristic may
be one or more detectable positions and/or one or more signals
and/or one or more intensities and/or one or more colours and/or
one or more concentrations and/or presence of one or more
characteristics and/or absence of one or more characteristics.
[0249] The electrophoresis step may be part of or may be separate
from the investigation step. The electrophoresis step may include
transferring the sample to a start location for electrophoresis
and/or a mobility based separation and/or a size based separation.
The start location may be in a channel. The electrophoresis step
may include one or more voltage conditions. One or more voltage
conditions may be used to transfer the sample to the start
location. One or more voltage conditions may be used to provide the
separation. The analysis step may establish one or more of the
characteristics of the sample.
[0250] The analysis may interrogate the instrument, particularly
the device, and/or may seek a response from the instrument,
particularly the device. The analysis may subject the instrument,
particularly the device, to an operation, for instance the
application of light. The analysis may consider the response to the
operation, for instance the light returning.
[0251] The analysis step may include one or more operations
involving an interaction with the device. The analysis step may
include one or more operations not involving an interaction with
the device. One or more of the interactions may be electromagnetic
interactions.
[0252] The analysis step may apply light to the device. The
analysis step may receive light from the device. The analysis step
may establish the relative position of the elements having a
characteristic, for instance an allele having a fluorescent dye.
The analysis step may establish the relative size of the elements
having a characteristic, for instance an allele having a
fluorescent dye. The analysis step may generate one or more
results. The light may be of visible and/or non-visible
wavelengths. The results output step may display the one or more
results from the analysis step and/or a processed form thereof.
[0253] The results output step may transmit the one or more results
from the analysis step and/or a processed form thereof to a remote
location. The results output step may compile the one or more
results into a transmission form. The transmission may be via a
telecommunications network. The results may be provided in a format
compatible with one or more software applications.
[0254] The results output step may be followed by a further
processing step. The further processing may interpret the results
to provide further results. The further processing step may analyse
the results to provide a DNA profile for the sample. The further
processing step may provide an indication of a match between the
sample and a database record of a sample. The further processing
step may be provided at a location remote from the instrument. The
further processing step may be provided at a location connected to
the instrument, at least part of the time, by a telecommunications
network. The further processing step may return to the instrument
and/or a computer, preferably within 200 m of the site of the
instrument, the further processed results.
[0255] The results may be processed on the instrument to give
processed results. The processed results may extract from the
results the signals, sections of signals or positions attributable
to a characteristic being analysed for, such as an allele. The
results and/or processed results may be provided to the results
output step.
BRIEF DESCRIPTION OF THE DRAWINGS
[0256] Various embodiments of the present invention will now be
described, by way of example only, and with reference to the
accompanying drawings in which:
[0257] FIG. 1 is a schematic illustration of the stages involved in
the consideration of a sample from collection to results and
illustrates the positioning of the embodiments of the present
invention in that context;
[0258] FIG. 2 is a schematic illustration of the key steps provided
on or by an instrument embodying the present invention;
[0259] FIG. 3a is a front face view of part of a cartridge
embodying the present invention;
[0260] FIG. 3b is a table of dimensions and volumes for a cartridge
according to the present invention, and components thereof;
[0261] FIG. 4 is a front face view of a further part of the
cartridge of FIG. 3a and embodying further features of the present
invention;
[0262] FIG. 5a is a side view of the section of the cartridge of
FIGS. 3a and 4 where in joins the electrophoresis cartridge
section;
[0263] FIG. 5b is a front view of the electrophoresis cartridge
section shown in FIG. 5a, with the section of the cartridge
omitted;
[0264] FIGS. 6a to 6e are schematic illustrations of alternative
arrangements for contacting the fluid and beads;
[0265] FIG. 7 is an illustration of an alternative structure for
providing sample to the PCR chamber;
[0266] FIG. 8 is a front view of the electrophoresis cartridge
section showing an alternative form of injector;
[0267] FIG. 9 is a schematic illustration of the parallel PCR
chamber arrangement used in providing real time PCR and feedback of
the results;
[0268] FIG. 10a is an illustration of a closing valve used in the
present invention;
[0269] FIG. 10b is an illustration of an opening valve used in the
present invention;
[0270] FIG. 11 shows an option for the archiving of a part of the
sample handled;
[0271] FIG. 12 is a schematic front view of one embodiment of the
instrument;
[0272] FIG. 13 is a side view showing the insertion of the
cartridge into the instrument;
[0273] FIG. 14 is a schematic of the light source, optics and
detector setup for the electrophoresis section of the
instrument;
[0274] FIG. 15 is an electropherogram showing the variation in
signal from the detector setup with time;
[0275] FIG. 16 is a schematic of an example of a system for
detecting fluorescence;
[0276] FIG. 17 is a plot of LED spectrum, light reflected, and
residual LED light over a range of wavelengths;
[0277] FIG. 18 is a plot of power of the LED-module over time;
[0278] FIG. 19 is an illustration showing beam shape and size as
measured by the laser camera;
[0279] FIGS. 20a and 20b are plots of CCD signal v/s wavelengths
for static fluorescence measurements; and
[0280] FIG. 21 is a plot of CCD signal v/s time for dynamic
fluorescence measurements;
[0281] FIG. 22 is an illustration of a PCR chamber according to a
further embodiment;
[0282] FIG. 23 is an illustration of the position of stacked
Peltier effect devices;
[0283] FIG. 24 is an illustration of an embodiment for loading a CE
channel
[0284] FIG. 25 is an illustration of a further embodiment for
loading a CE channel;
[0285] FIG. 26 is an illustration of a further embodiment of a PCR
chamber;
[0286] FIG. 27 is a front face view of a cartridge according to an
embodiment;
[0287] FIG. 28a is a front face view of a cartridge according to a
different embodiment;
[0288] FIG. 28b is a table of dimensions and volumes for the FIG.
28a cartridge;
[0289] FIG. 29a is a perspective view of an embodiment of the
instrument;
[0290] FIG. 29b is a front view of the instrument of FIG. 29a;
[0291] FIG. 29c is a side view of the instrument of FIG. 29a;
[0292] FIG. 30 is a perspective view of another instrument
embodiment;
[0293] FIG. 31a is an illustration of a carrier, cartridge and CE
chip embodiment;
[0294] FIG. 31b is an illustration of a detail of the carrier to
cartridge engagement;
[0295] FIG. 32a is an illustration of a carrier to CE chip
engagement;
[0296] FIG. 32b is a cut away illustration of a part of the FIG.
32a engagement;
[0297] FIG. 33a is an illustration of the tube and cartridge
connection;
[0298] FIG. 33b is an illustration of the tube to CE chip
connection;
[0299] FIG. 34a is an illustration of the carrier being inserted
into the instrument;
[0300] FIG. 34b is an illustration of the inserted carrier;
[0301] FIG. 35a is an illustration of the cartridge and carrier in
the insertion form;
[0302] FIG. 35b is an illustration of the cartridge and carrier in
the use form;
[0303] FIG. 35c is an illustration of the cartridge returned to the
carrier;
[0304] FIG. 36a is a perspective view of the position of the pair
of calipers;
[0305] FIG. 36b is a perspective view of the back of the pair of
calipers;
[0306] FIG. 36c is a plan view of the caliper structure in the open
form;
[0307] FIG. 36d is a plan view of the caliper structure in the
closed form;
[0308] FIG. 37a is a perspective view of the second support of the
carrier and CE chip;
[0309] FIG. 37b is a partial cut away illustration of the second
support and CE chip;
[0310] FIG. 38 is a perspective view of the CE chip heater
board;
[0311] FIG. 39 is a perspective view of an embodiment of the
optics;
[0312] FIG. 40a is a perspective view of the alignment
structure;
[0313] FIG. 40b shows the alignment structure of FIG. 40a in the
stowed position;
[0314] FIG. 40c shows the alignment structure of FIG. 40a in the
use position;
[0315] FIG. 41a shows three positions for an alternative PCR
chamber embodiment;
[0316] FIG. 41b shows two positions for a further PCR chamber
embodiment;
[0317] FIG. 41c shows three positions for a still further PCR
chamber embodiment;
[0318] FIG. 42a shows a CE chip embodiment;
[0319] FIG. 42b shows a detail of the CE chip of FIG. 42a;
[0320] FIG. 43 shows an approach to loading sample to the CE
step;
[0321] FIG. 44 shows an alternate approach to loading sample to the
CE step;
[0322] FIG. 45 shows a further alternative for loading sample to
the CE step
[0323] FIG. 46 shows a further embodiment of a PCR chamber.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0324] In a variety of cases it is desirable to be able to analyse
a biological sample to obtain information on the sample and/or one
or more components of the sample. Such cases include medical
diagnostics, for instance to look for disease markers, and forensic
science, for instance to establish a DNA profile.
[0325] At present, such analyses are conducted by highly trained
scientists in a laboratory environment. This means that a
significant amount of effort and experience goes into the handling
of the samples, the use of the analysis equipment and the
formulation of the conclusions reached. However, the need to convey
the sample to a laboratory environment and then receive the results
back from the laboratory environment introduces a potential time
delay between obtaining the sample and obtaining the results
thereon. The need to use a laboratory environment and highly
trained scientists potentially adds to the time required, as the
supply of such people and resources is limited. The need to use a
laboratory environment and highly trained scientists potentially
adds to the cost as there are capital and running costs associated
with such facilities and the scientists.
[0326] If fewer laboratory style environments are to be used for
the analysis or the staff used are less specialised, then there is
the potential for problems with the analysis, unless a proper and
reliable system is provided.
[0327] The present invention has amongst its potential aims to
enable analysis of samples at a greater variety of locations and/or
non-laboratory type locations. The present invention has amongst
its potential aims to enable analysis by personnel having a lower
level of training and/or experience. The present invention has
amongst its potential aims to enable lower cost and/or faster
analysis of samples. The present invention has amongst its
potential aims to enable greater use and/or more successful use of
analysis by law enforcement authorities.
[0328] Many of the concepts and issues to be addressed by the
invention are best understood by way of the following examples. It
should be noted, however, that these examples are by their very
nature detailed and exhaustive, and that benefits from the present
invention arise even when only small sections of the examples are
implemented in other embodiments of the present invention.
[0329] The various embodiments and examples explain the invention
initially in the context of a reference sample; that is a sample
collected from a known individual under controlled conditions. An
example of a reference sample would be a sample collected by a swab
from the buccal cavity of a person who has been arrested, the
sample being collected at a police station. The invention is also
suited to casework samples; that is a sample collected from a
location from an unknown individual under non-controlled
conditions. An example would be a spot of blood collected by a swab
from a crime scene, with the source of the blood unknown. Where the
differences between reference samples and casework samples have an
impact on the preferred forms of the instrument, cartridge and
methods, the casework sample embodiments are separately
described.
[0330] The substitution of one or more components by one or more
different components or different arrangements of components is
also envisaged where particular conditions or issues arise. Again,
after the discussion of the reference sample and casework sample
contexts for the instrument, these alternatives are described.
[0331] As a starting point, it is useful to establish the context
of the instrument, cartridge and methods of use in the overall
context in which they may be used, by way of example. Thus in FIG.
1 there is a schematic of the overall process into which the
present invention fits. This overall process includes a sample 1
which is gathered in a sample collection stage 3. This is followed
by a sample preparation stage 5. In the subsequent sample loading
stage 7, a prepared cartridge 9 is loaded with the collected and
prepared sample 1. The next stage is the cartridge installation
stage 15 in which the cartridge 9 is introduced to the instrument
11. The instrument 11 also receives various inputs 13 at the sample
loading stage 7 and/or at the cartridge installation stage 15
and/or subsequently.
[0332] The structure and processes performed within the instrument
11 and cartridge 9 are described further below in the context of
FIG. 2.
[0333] Once the instrument 11 has completed these stages and
achieved the analysis, the next stage is the results stage 17. This
is followed by one or more output stages 19, and potential further
stages 21 which integrate the analysis into the criminal justice
system of that jurisdiction. A wide range of possible links between
the various output stages 19 and further stages 21 may be possible,
with some being linked to just one stage and others be the result
of multiple such stages and/or combinations thereof.
[0334] An output stage 19 may include the transmission of the
results from the instrument to a remote location for processing.
The processing may be performed using complex software and/or
hardware tools, before the final results are returned to the
instrument 11 or to another computer. Processing the results at a
remote location may be preferably in terms of the size, cost or
complexity of the software/hardware needed to perform the
processing thus only being provided at a limited number of
locations, rather than a part of each instrument.
[0335] The following description of the operation of the instrument
11, in a generally sequential manner, provides full details of the
key instrument stages and their interrelationship.
[0336] Referring to FIG. 2, the instrument has a sample receiving
step 200, sample preparation step 202, sample amplification step
204, electrophoresis step 206 and analysis step 208 and data
communication step 210.
[0337] In the sample receiving step 200, the sample 1 is
transferred from a sample storage and/or processing stage 5, which
is outside of the cartridge 9 and instrument 11, to a location on
the cartridge 9.
[0338] The initial collection device is frequently a swab. The swab
is used to pick up the sample 1 from an article or substrate.
[0339] In the sample preparation step 202, the key components
within the sample are contacted with the reagents and/or components
intended to prepare the sample for the subsequent steps. In this
embodiment, the sample preparation step 202 contacts the sample
with beads to retain the DNA and recover it, whilst the other
components which are not to be recovered flow through and away. The
sample preparation step 202 also includes contact with a wash agent
to improve the separation of the DNA from the other components. The
wash agent flows through the chamber holding the beads and retained
DNA and flows to a further chamber, a waste chamber. The wash agent
is followed by an elution agent to release the DNA from the beads
for the subsequent steps.
[0340] In the sample amplification step 204, the DNA is contacted
with amplification reagents and provided with the conditions
necessary to achieve amplification through PCR.
[0341] In the electrophoresis step 206, the amplified DNA is
conveyed to a start point for a mobility based separation within a
capillary. An electric field is then used to separate the complex
DNA amplicons into different size clusters.
[0342] In the analysis step 208, the channel is inspected to
establish the relative position and hence size of elements detected
in the capillary. This is achieved by an excitation light source,
fluorescent markers associated with the elements to be detected and
suitable optics to detect the fluorescent light resulting.
[0343] In the data communication step 210, the instrument compiles
the necessary data packet for transmission and transmits it to a
remote location for consideration. The data packet includes
information on the electrophoresis results, sample identity and
other information. The analysed results may be received by the
instrument as part of the data communication step 210.
[0344] Some data processing may be performed on the instrument
itself, for instance to deconvolute the analysis results to
indicate the peaks indicative of alleles present.
[0345] The instrument can be provided in a format which considers a
single sample at a time, or can be provided in a format which
considers multiple samples at a time. The multiple samples may each
be run on separate cartridges, but modified cartridges which handle
multiple samples are possible. The handling of multiple cartridges
is beneficial in allowing a single set of controllers, power
supplies, optics and the like to consider multiple samples, with
reduced capital costs.
Cartridge
[0346] Key to the operation of the instrument is a disposable,
single use cartridge 9. This cartridge 9 is intended to only
process and provide the results for analysis on a single occasion.
The disposable nature of the cartridge 9 places a number of
constraints on the cartridge 9 in terms of the materials which can
be used, because of the need to keep manufacturing, assembly or
purchase costs low.
[0347] The detailed layout of the cartridge 9 is now described.
Later, a description of the sequence of operation of the elements
which make up the cartridge is provided.
[0348] FIG. 3a is an illustration of that part of the sample
receiving step 200 provided on the cartridge 9, the whole sample
preparation step 202 and the whole sample amplification step 204.
The subsequent steps and their respective pasts of the cartridge 9
are illustrated separately.
[0349] FIG. 3b provides details of the volumes of the various
chambers used, the depths (into the page in effect) for the various
components and the overall dimensions of this part of the cartridge
9.
[0350] The cartridge 9 is provided with a sample introduction
chamber 302 connected to a channel 304 leading to the outside of
the cartridge 300. This forms those parts of the sample receiving
step 200 provided on the cartridge 9.
[0351] The sample preparation step 204 follows. To provide this,
the sample introduction chamber 302 is connected to a pumping fluid
channel 306 and hence to a first electrochemical pump 308. The
sample introduction chamber 302 has an outlet channel 310 which
passes valve 312 and provides an inlet to purification buffer
chamber 314. Valve 312 is initially open.
[0352] Purification buffer chamber 314 is connected via channel 316
to bead storage chamber 318. The bead storage chamber 318 is
connected via channel 320 to initial mixing chamber 322. The outlet
channel 324 from initial mixing chamber 322 is blocked by closed
valve 326, but a vent channel 328 is open because valve 330 is open
initially.
[0353] The outlet channel 324 leads past valve 326 to a first
further mixing chamber 332 and then through channel 334 to second
further mixing chamber 336. The outlet 338 from the second further
mixing chamber 336 leads past valve 340 to incubation chamber 342,
where bubble mixing assists the DNA to bead binding process.
[0354] The incubation chamber 342 has a vent channel 344 provided
with valve 346 and an outlet channel 348 which is initially closed
by valve 350. The incubation chamber 342 is also provided with a
pumping fluid inlet channel 352 which passes valve 354 and is
connected to second electrochemical pump 356.
[0355] The outlet channel 348 from the incubation chamber 342 leads
to capture chamber 358 where the beads and hence bound DNA are
collected. The capture chamber 358 is provided with a first vent
channel 360 which passes first valve 362 and second valve 364. The
capture chamber 358 is also provided with a second vent channel 366
which passes first valve 368 and second valve 370.
[0356] Also connected to capture chamber 358 is wash buffer channel
372. The wash buffer channel is connected to first valve 374 and
second valve 376 and leads from second electrochemical pump 356
through wash buffer chamber 378 to the capture chamber 358.
[0357] Also connected to capture chamber 358 is an elution liquid
channel 380. The elution liquid channel 380 is connected to first
valve 382, elution liquid storage chamber 384, second valve 386 and
back to third electrochemical pump 388.
[0358] The capture chamber 358 has a wash outlet channel 390 which
splits into a first wash outlet channel section 392 which passes
valve 394, and into a second wash outlet channel section 396 which
passes valve 398. After passing their respective valves 394, 398,
the first wash outlet channel section 392 and second wash outlet
channel section 396 rejoin one another to form further wash channel
400. The further wash channel 400 leads past valve 402 into waste
chamber 404. The waste chamber 404 is vented along vent channel 406
past valve 408. These elements provide the sample preparation step
202.
[0359] To provide the sample amplification step 204, capture
chamber 358 is also provided with elution outlet channel 410 which
leads past valve 412 and past valve 414 and into PCR chamber 416.
The outlet channel 418 from the PCR chamber 416 leads past valve
420 into archive chamber 422. The archive chamber 422 is vented
through vent channel 424. The role of the archive chamber 422 is
described further below.
[0360] Provided within the PCR chamber 416 is a bead loaded with
the reagents, a multimix, needed for the PCR process. The
reagents/multimix include primers dNTPs and PCR reaction mix,
including Tris buffer, MgCl.sub.2, NaCl and BSA. These reagents are
released into the sample once it contacts the bead in the PCR
chamber 416 and the temperature is raised above ambient
temperature.
[0361] The above circuit overall, is sufficient to receive, retain,
wash, elute and perform PCR on the sample, as well as storing the
waste from the process and an archive of the PCR product.
[0362] Subsequently, the arrangement shown in FIG. 4 can be used to
transfer the now amplified DNA from the PCR chamber 416 into the
electrophoresis step 206.
[0363] In FIG. 4, the PCR chamber 416 is the same PCR chamber 416
which was illustrated in FIG. 3 and described above. Other features
were omitted from FIG. 3 to improve the clarity of that Figure.
[0364] Leading from the PCR chamber 416 is a denaturing feed
channel 500 which is connected to an amplified material mixing
chamber 502. The amplified material is pumped from PCR chamber 416
by the action of fourth electrochemical pump 504 which is connected
to channel 506, hence to denaturing reagent storage chamber 508 and
through channel 510 to the PCR chamber 416. Formamide is provided
in the denaturing reagent storage chamber in the preferred
form.
[0365] These components are isolated from the PCR chamber 416
during the sample amplification step 204 by closed valve 512 and
closed valve 514. Both valve 512 and 514 are opened and valves 516
and 518 are closed to convey the amplified material away from the
PCR chamber 416.
[0366] From the denaturing feed channel 500, the amplified material
and denaturing reagents enter the first amplified material mixing
chamber 502, pass through channel 520, into second amplified
material mixing chamber 522, through channel 524 and into third
amplified material mixing chamber 526. Whilst the third amplified
material mixing chamber 526 fills, valve 528 is shut and vent 530
is open. An overall volume of 45:1 is provided, 5:1 from the PCR
chamber and 40:1 from the denaturing reagent storage chamber
508.
[0367] The amplified material is held in the third mixing chamber
526 for the necessary time and at the necessary temperature to
complete the denaturing process. Once this has been achieved, the
valve 528 is opened and further pumping by the fourth
electrochemical pump 504 pumps the denatured material to the
electrophoresis step inlet 532. At the inlet 532, the denatured
material passes out of the plane of the cartridge 9 and to the
electrophoresis cartridge section behind. Once past through the
inlet 532, valve 534 is shut to isolate the cartridge 9 from the
electrophoresis cartridge section 600.
[0368] The overall result of this structure is the pumping of the
amplified DNA to a start point for the electrophoresis step
206.
[0369] The transfer from PCR to CE steps is provided in a way which
allows easy integration of the steps, does not impact upon the
temperature and pressure stability required in PCR and achieves
minimal sample loss during transfer. Automated mixing of the sample
and size standards during transfer and possibilities for
pre-concentrating the sample before CE are also rendered
possible.
[0370] The overall configuration of the electrophoresis step 206
can be seen in the side view of FIG. 5a and front view of FIG.
5b.
[0371] The inlet 532 leads from the plane of the cartridge 9,
through into the plane of the electrophoresis cartridge section
600. Here, the inlet 532 leads into the top section 602 of an
electrophoresis feed reservoir 604. The top section 602 is empty,
but the lower section 606 is provided with the gel 608 which also
fills the capillary 610. The sample is pumped into the
electrophoresis feed reservoir 604 by a fourth electrochemical
pump, not shown.
[0372] Sample flow from the reservoir 604 into the correct position
within the capillary 610 is achieved using electrophoresis as the
transport mechanism.
[0373] In this embodiment, the injector structure provided within
the capillary cartridge section 600 is a double T injector. This
includes a first electrode location 612, second electrode location
614 provided at the other end of the long capillary 616 in which
the size based separation is achieved. A third electrode location
618 and fourth electrode location 620 are provided in side arms 622
and 624 respectively. The side arms are offset relative to one
another, with side arm 624 further towards the second electrode
location 614, than the side arm 622.
[0374] Initially, sample is drawn from the liquid phase in the
reservoir 604 through the interface with the gel provided in the
reservoir 604 and hence into the gel by a voltage applied to the
electrode present at the third electrode location 618. Once the
sample has been drawn past the fourth electrode location 620, a
voltage is also applied to the electrode at the fourth electrode
location. Generally, the electrode at the third electrode location
may be at a voltage of 600V and the electrode at the fourth
electrode location may be at a voltage of 200V. The voltage may be
floating for the electrodes at the first 612 and second 614
electrode locations.
[0375] This situation results in sample being drawn along side arm
624, along the section 626 and into side arm 622, such that sample
is present in the two side arms 622 and 624 and the section 626 of
the capillary 616.
[0376] This gives the plug of sample upon which the
electrophoresis's to act in the section 626.
[0377] To reduce the cost of the electrodes used, consistent with
the cartridge being single use, platinum coated, gold coated,
carbon, nickel and other lower cost electrodes may be used.
[0378] Once positioned, the separation voltages are applied: 1500V
at the electrode at the second electrode location 614; 0V at the
electrode at the first electrode location 612; and 200V at the
electrodes present at the third electrode position 618 and fourth
electrode positions 620.
[0379] The capillary 616 is filled with a gel matrix which
preferentially retards the speed of progress of elements within the
DNA as their size increases. The result is a size based separation
of the elements, with the faster elements reaching the detection
location 626 first and the slowest reaching the detection location
628 last. The different times at which the signals are generated
and form the electropherogram indicate the size of the element
behind that signal.
[0380] It is possible to assist in the interpretation of the
unknown element sizes by using a size standard within the
capillary. This is provided with a different dye colour or
otherwise rendered distinct. The method set out in U.S. patent
application No. 61/096,424, the contents of which are hereby
incorporated by reference, offers approaches for determining the
sizes of the unknowns from the size standard.
[0381] The setup and operation of the light source, optics and
detector is described in detail below.
[0382] Other embodiments of the cartridge have also been
developed.
[0383] As shown in FIG. 27, the cartridge 27-01 has been modified
by providing the electrochemical pumps 27-03, 27-05, 27-07, 27-09
with connections between the wires leading to the electrodes in the
pumps and the power source not shown of the Pogo.TM. pin type. The
pins 27-11 are spring loaded in the recesses of the cartridge 27-01
and in use contact similar spring loaded pins (not shown) on the
other side of the cartridge to instrument interface. A reliable
electrical contact is thus provided and the cartridge is more
robust against damage during storage, installation and use than
designs in which the wires for the electrochemical pumps protruded
from the side of the cartridge.
[0384] The form shown in FIG. 27 also features guide holes 27-13
which are used in the alignment of the cartridge and instrument, as
described in more detail below.
[0385] A preferred embodiment of the cartridge is shown in FIG.
28a. This is an illustration of that part of the sample receiving
step 200 provided on the cartridge 28-09, the whole sample
preparation step 202, the whole sample amplification step 204, the
whole sample denaturation step and the feed to the capillary
electrophoresis step 206.
[0386] FIG. 28b provides details of the volumes of the various
chambers used, the depths (into the page in effect) for the various
components and the overall dimensions of this part of the cartridge
28-09.
[0387] The cartridge 28-09 is provided with a sample introduction
chamber 28-302 connected to a channel 28-304 leading to the outside
of the cartridge 28-09. This forms those parts of the sample
receiving step 200 provided on the cartridge 28-09.
[0388] The sample preparation step 204 follows. To provide this,
the sample introduction chamber 28-302 is connected to a pumping
fluid channel 28-306 and hence to a first electrochemical pump
28-308. The sample introduction chamber 28-302 has an outlet
channel 28-310 which passes valve 28-312 and provides an inlet to
bead storage chamber 28-318. Valve 28-312 is initially open.
[0389] The bead storage chamber 28-318 has an outlet channel 28-316
leading to binding buffer storage chamber 28-314. This sequence of
chambers is reversed compared with the FIG. 3a embodiment. The
binding buffer storage chamber 28-314 has an outlet channel 28-320
which leads to mixing/purification chamber 28-322.
[0390] Mixing/purification chamber 28-322 is connected via channel
28-324 through valve 28-326 and via channel 28-500 to first further
mixing chamber 28-332. The outlet channel 28-324 from
mixing/purification chamber 28-322 is blocked by closed valve
28-326, but a vent channel 28-328 is open because valve 28-330 is
open initially.
[0391] The outlet channel 28-324 leads past valve 28-326 to a first
further mixing chamber 28-332 and then through channel 28-334 to
second further mixing chamber 28-336. The outlet 28-338 from the
second further mixing chamber 28-336 leads past valve 28-340 to
incubation chamber 28-342, where bubble mixing assists the DNA to
bead binding process. The incubation chamber 28-342 may be actively
heated or may simply provide the necessary dwell time and/or other
binding conditions needed.
[0392] The incubation chamber 28-342 has a vent channel 28-344
provided with valve 28-346 and an outlet channel 28-348 which is
initially closed by valve 28-350. The incubation chamber 28-342 is
also provided with a pumping fluid inlet channel 28-352 which
passes valve 28-354 and is connected to second electrochemical pump
28-356.
[0393] The outlet channel 28-348 from the incubation chamber 28-342
leads to capture chamber 28-358 where the beads and hence bound DNA
are collected. The capture chamber 28-358 is provided with a first
vent channel 28-360 which passes first valve 28-362 and second
valve 28-364. The capture chamber 28-358 is also provided with a
second vent channel 28-366 which passes first valve 28-368 and
second valve 28-370.
[0394] Also connected to capture chamber 28-358 is wash buffer
channel 28-372. The wash buffer channel is connected to first valve
28-374 and second valve 28-376 and leads from second
electrochemical pump 28-356 through wash buffer chamber 28-378 to
the capture chamber 28-358.
[0395] Also connected to capture chamber 28-358 is an elution
liquid channel 28-380. The elution liquid channel 28-380 is
connected to first valve 28-382, elution liquid storage chamber
28-384, second valve 28-386 and back to third electrochemical pump
28-388.
[0396] The capture chamber 28-358 has a wash outlet channel 28-390
which splits into a first wash outlet channel section 28-392 which
passes valve 28-394, and into a second wash outlet channel section
28-396 which passes valve 28-398. After passing their respective
valves 28-394, 28-398, the first wash outlet channel section 28-392
and second wash outlet channel section 28-396 rejoin one another to
form further wash channel 28-400. The further wash channel 28-400
leads past valve 28-402 into waste chamber 28-404. The waste
chamber 28-404 is vented along vent channel 28-406 past valve
28-408. These elements provide the sample preparation step 202.
[0397] To provide the sample amplification step 204, capture
chamber 28-358 is also provided with elution outlet channel 28-410
which leads past valve 28-412 and past valve 28-414 and past valve
28-502 and into PCR chamber 28-416. The outlet channel 28-418 from
the PCR chamber 28-416 leads past valve 28-420 and past valve
28-504 and past valve 28-506 into archive chamber 28-422. The
archive chamber 28-422 is vented through vent channel 28-424. The
role of the archive chamber 28-422 is as described further
above.
[0398] Provided within the PCR chamber 28-416 is a bead loaded with
the reagents, a multimix, needed for the PCR process. The
reagents/multimix include primers dNTPs and PCR reaction mix,
including Tris buffer, MgCl.sub.2, NaCl and BSA. These reagents are
released into the sample once it contacts the bead in the PCR
chamber 28-416 and the temperature is raised above ambient
temperature.
[0399] The above circuit overall, is sufficient to receive, retain,
wash, elute and perform PCR on the sample, as well as storing the
waste from the process and an archive of the PCR product.
[0400] The PCR part of the circuit has been moved to the upper
section of the cartridge compared with the previous embodiments so
as to present it physically closer to the CE chip.
[0401] Subsequently, the further arrangement shown in FIG. 28a can
be used to prepare, denaturation step, and transfer the now
amplified DNA from the PCR chamber 28-416 into the electrophoresis
step 206.
[0402] Leading from the PCR chamber 28-416 is outlet channel
28-418. This splits after valves 28-420 and 28-504 into a
denaturing feed channel 28-550 and the channel leading to the
archive chamber 28-422. The denaturing feed channel 28-550 is
connected to a denaturation chamber 28-552. The amplified material
is pumped from PCR chamber 28-416 by the action of fourth
electrochemical pump 28-554 which is connected to channel 28-556,
hence to denaturing reagent storage chamber 28-558 and through
valve 28-560 and channel 28-562 to the PCR chamber 28-416.
Formamide is provided in the denaturing reagent storage chamber
28-558 in combination with the size standards to be used in the
capillary electrophoresis step.
[0403] These components are isolated from the PCR chamber 28-416
during the sample amplification step 204 by closed valve 28-502 and
closed valve 28-420. Both valve 28-502 and 28-420 are opened and
valves 28-414 and 28-506 are closed to convey the amplified
material away from the PCR chamber 28-416 to the denaturation
chamber 28-552. This is vented through valve 28-564, with exit
channel 28-566 closed by valve 28-568.
[0404] The amplified material is held in the denaturation chamber
28-552 for the necessary time and at the necessary temperature to
complete the denaturing process. Once this has been achieved, the
valve 28-568 is opened and further pumping by the fourth
electrochemical pump 28-554 pumps the denatured material to the
electrophoresis step inlet 28-570.
[0405] At the inlet 28-570, the denatured material passes out of
the plane of the cartridge 9 and through a tube to the
electrophoresis cartridge section behind. The overall result of
this structure is the pumping of the amplified DNA to a start point
for the electrophoresis step 206.
[0406] Details of the connection of the inlet 28-570 to the CE chip
are provided below.
[0407] Throughout the operations described above and in the
sections that follow, various checks are made on operating
conditions, component performance and successful operation so as to
ensure the processing is correctly provided from start to finish.
Errors or problems are indicated to the operator.
[0408] Cartridge Sequence of Operation
[0409] The sequence of operation, purely by way of example, applied
to the cartridge shown in and described in relation to FIGS. 3a and
b is as follows, with sample timings also given.
TABLE-US-00001 Time Purpose and since start (sec) Change notes 0.0
Incubation chamber 358 - adjust temperature to 25.degree. C. 0.9
Valve 312 - opening valve - heat on 31.5 First electrochemical pump
308 - on 73.3 Valve 330 - closing valve - heat off 121.1 Valve 312
- opening valve - heat off 138.7 First electrochemical pump 308 -
off 187.8 Valve 326 - opening valve - heat on 212.3 Valve 312 -
opening valve - heat on 233.9 Valve 330 - closing valve - heat off
236.0 First electrochemical pump 308 - on 324.3 Valve 312 - opening
valve - heat off 368.6 Valve 326 - opening valve - heat off 370.4
Valve 346 - closing valve - heat on 401.0 First electrochemical
pump 308 - off 461.4 Valve 346 - closing valve - heat off 653.4
Valve 350 - opening valve - heat on 655.1 Magnet - field applied to
chamber 656.4 Valve 326 - opening valve - heat on 684.5 First
electrochemical pump 308 - on 783.4 Valve 326 - opening valve -
heat off 804.1 Valve 394 - closing valve - heat on 815.4 Valve 340
- closing valve - heat on 829.6 Valve 350 - opening valve - heat
off 840.8 Magnet - field removed from chamber 867.5 First
electrochemical pump 308 - off 894.2 Valve 394 - closing valve -
heat off 944.5 Valve 368 - opening valve - heat on 975.5 Valve 340
- closing valve - heat off 977.2 Second electrochemical pump 356 -
on 1025.8 Valve 354 - closing valve - heat on 1036.2 Valve 368 -
opening valve - heat off 1050.8 Second electrochemical pump 356 -
off 1079.7 Valve 324 - opening valve - heat on 1080.6 Valve 368 -
opening valve - heat on 1116.3 Valve 354 - closing valve - heat off
1118.0 Second electrochemical pump 356 - on 1181.3 Valve 370 -
closing valve - heat on 1196.4 Valve 368 - opening valve - heat off
1228.3 Valve 324 - opening valve - heat off 1233.9 Second
electrochemical pump 356 - off 1244.2 Valve 398 - opening valve -
heat on 1249.4 Valve 324 - opening valve - heat on 1271.8 Valve 370
- closing valve - heat off 1273.1 Magnet - field applied to chamber
1284.7 Second electrochemical pump 356 - on 1328.6 Valve 324 -
opening valve - heat off 1333.8 Valve 402 - closing valve - heat on
1334.7 Valve 408 - closing valve - heat on 1379.9 Valve 398 -
opening valve - heat off 1383.8 Magnet - field removed from chamber
1393.9 Second electrochemical pump 356 - off 1419.5 Valve 362 -
opening valve - heat on 1435.4 Valve 402 - closing valve - heat off
1465.1 Valve 408 - closing valve - heat off 1466.0 Second
electrochemical pump 356 - on 1474.6 Valve 374 - closing valve -
heat on 1493.6 Valve 362 - opening valve - heat off 1501.8 Valve
382 - opening valve - heat on 1504.8 Valve 362 - opening valve -
heat on 1508.7 Second electrochemical pump 356 - off 1531.9 Third
electrochemical pump 388 - on 1578.8 Incubation chamber 358 -
adjust temperature to 60.degree. C. 1585.0 Valve 374 - closing
valve - heat off 1586.6 Valve 362 - opening valve - heat off 1588.5
Valve 364 - closing valve - heat on 1633.3 Valve 382 - opening
valve - heat off 1640.4 Third electrochemical pump 388 - off 1679.0
Valve 364 - closing valve - heat off 1881.0 Valve 412 - opening
valve - heat on 1882.9 Valve 382 - opening valve - heat on 1906.2
Magnet - field applied to chamber 1914.9 Third electrochemical pump
388 - on 1952.3 Incubation chamber 358 - adjust t to 25.degree. C.
2010.0 Third electrochemical pump 388 - off Magnet - field removed
from chamber Valve 382 - opening valve - heat off Valve 412 -
opening valve - heat off 2017.3 Valve 420 - closing valve - heat on
Isolate PCR Valve 414 - closing valve - heat on chamber 2173.3
Valve 420 - closing valve - heat off Valve 414 - closing valve -
heat off 2185.0 Incubation chamber temperature control - off
[0410] Cartridge Alternatives
[0411] There are a variety of alternatives for the various
components within the cartridge and/or their operation. Some of
these are now described, by way of example only.
[0412] 1) Bead Handling
[0413] As described above, the cartridge makes use of a bead
storage chamber 318 from which the beads are washed in operation.
This washing action provides contact between the sample, reagents
and the beads. Mixing results in the beads taking up the DNA in the
sample and retaining it. Subsequent retention of the beads allows
the DNA to be separated from the rest of the sample and allows
washing stages to improve further this separation.
[0414] It is important to ensure that the beads are displaced from
their storage location, such that the beads are available, in
contact with the relevant liquids, to perform their task.
Modifications to the manner in which the beads are stored and/or
dispensed can assist in this. The beads may be stored away from the
cartridge. They may be introduced to the cartridge to prepare it
for use.
[0415] Firstly, it is possible to provide a dispersant together
with the beads so as to keep them dispersed and hence more easily
collected and carried by the fluid flow. This can help prevent
blockages and/or agglomerations of beads. Different dispersants
and/or variations in the amount provided can be used to tailor
this.
[0416] Secondly, it is possible to provide the beads in a series of
bead storage chambers, rather than in a single chamber. FIG. 6a
illustrates one such arrangement, where the beads are split into
three groups, each in its own chamber 700. In this way, the contact
between the fluid and the beads is staggered and a compacted mass
of beads is avoided on the lead edge of the fluid. A variation on
this is provided in FIG. 6b, where a first bead storage chamber
700a is separated from the second bead storage chamber 700b by a
mixing chamber 702.
[0417] Thirdly, the contact can be provided with a thin chamber 704
whereby the transition of the fluid from the thing channel 706 into
the chamber causes non-laminar flow and hence improved mixing. The
provision of the beads spread along the length of the chamber 704
also means that they do not contact the fluid all at the same
time.
[0418] Fourthly, the flow direction and/or chamber design can be
modified to encourage displacement of the beads from their storage
position into a mixed form with the fluid. Thus in the FIG. 6d
form, the fluid enters the chamber 700 in one bottom corner 708 and
displaces, arrows, the beads resting in that part. A swirling flow
within the chamber 700 gives mixing, before the fluid and bead
mixture exits the chamber 700 through the other bottom corner
710.
[0419] Fifthly, the beads can be stored in a side arm 712 or other
form of passage. As the flow of fluid passes through thin chamber
714 and past the junction 716 with the arm 712, a force is applied
behind the mass of stored beads in the side arm 712. This forces
the mass of stored beads towards and into the junction 716 where
they gradually contact and are swept away by the fluid flow.
Gradual dispersal of the beads into the fluid is provided. The
motive force behind the beads can be provided by a similar
structure to that used to move material in the context of the
closing valves described herein.
[0420] 2) PCR Chamber Filling
[0421] In the above system, the amount of the processed sample
which is made available to the PCR stage is controlled by the
relative height of the outlet from the PCR chamber to the archive
chamber leading to overflow of excess sample into the archive
chamber. This results in a PCR chamber which is not completely full
of sample during PCR. As PCR involves heating of the sample,
evaporation and/or condensation of part of the sample may occur at
a location outside of the PCR chamber. This can reduce the reagents
present in the PCR chamber and hence reduce the efficiency of the
PCR stage.
[0422] In an alternative form, the PCR chamber is entirely filled
with the sample before PCR is started. This is achieved using the
arrangement of FIG. 7 where the majority of the components have the
same structure and function as shown in the FIG. 3 and FIG. 4
description. The differences are in the section around the PCR
chamber 416.
[0423] In this alternate form, the PCR chamber 1416 is fed material
along channel 1413. Initially, the path of least resistance to this
fluid flow is through the PCR chamber 1416, along channel 1500,
past opened valve 1502 and onto vent 1504. The vent 1504 is
hydrophobic and so allows the passage of the air displaced from the
PCR chamber 1416 and channel 1500 by the material's advance. Once
the fluid reaches the vent 1504, however, the path of least
resistance changes and further flow occurs along channel 1418 past
valve 1428 and into archive chamber 1422, which is provided with
vent 1424. By this time, the PCR chamber 1416 is completely full of
liquid and hence the volume of the liquid subjected to PCR is
guaranteed.
[0424] As before, the valves around the PCR chamber 1416 are closed
during the amplification itself, so as to isolate the PCR chamber
1416.
[0425] In a third alternative, the configuration shown in FIG. 22,
the PCR chamber 22-01 is along channel 22-03. Initially, the path
of least resistance to this fluid flow is through the inlet 22-05
to the PCR chamber 22-01. Once the PCR chamber 22-01 has filled,
the liquid overflows through exit 22-07 into channel 22-09 which is
a continuation of channel 22-03. Further fluid flow simply
by-passes the PCR chamber 22-01 and flows through channel 22-03 and
then channel 22-09. To control the flow correctly, the dimension A
of the inlet 22-05 is greater than the dimension B of the outlet
22-07. The dimension is preferably greater in terms of the
cross-sectional area, perpendicular to the direction of flow. The
complete filling of the PCR chamber 22-01 ensure the volume of the
liquid subjected to PCR is guaranteed.
[0426] Various shapes are possible for the PCR chamber. FIG. 26
provides an example in which the PCR chamber 26-01 is formed as
smooth as possible. This assists with full fluid contact with the
surfaces and hence complete and accurate filling of the PCR chamber
26-01. The sample flows along channel 26-03 and enters the PCR
chamber 26-01 via inlet 26-05 provided towards the bottom of the
PCR chamber 26-01. The sample fills the PCR chamber 26-01 before
overflowing through outlet 26-07 provided towards the top of the
PCR chamber 26-01 and into channel 26-09.
[0427] In the embodiment of FIG. 46, a variation on the above
principle is provided. The flow to the PCR chamber 46-100 passes
along channel 46-102 and past valve 46-104. The channel 46-102
turns as it approaches the chamber 46-100 and provides inlet
channel 46-106. The natural flow is along this route. As the flow
progresses, the PCR chamber 46-100 fills, with the gas exiting
through outlet channel 46-108. The outlet channel 46-108 has a
similar configuration to inlet channel 46-106, but the
cross-sectional area of the outlet channel 46-108 is much smaller
than that of the inlet channel 46-106. As a result, when the liquid
reaches the outlet channel 46-108, the flow resistance increases
greatly and flow is redirected along the by-pass channel 46-110 in
preference. Both the outlet channel 46-108 and the by-pass channel
46-110 lead past valve 46-112 to exit channel 46-114. The Peltier
effect device heats the area within the dotted lines and so ensures
that as much of the space between the two valves, 46-104 and 46-112
is heated so as to minimise any condensation within that space.
3) Sample Concentration Before Capillary Electrophoresis
[0428] In some instances, it may be helpful to increase the
concentration of the sample prior to its use in the electrophoresis
step and/or to reduce the size of the sample as it is injected.
[0429] Once suitable approach for doing so is set out in European
patent publication no 1514100, the contents of which are
incorporated herein by reference. This technique uses careful
balancing of the electrophoretic velocity of the DNA and the
opposing electroosmotic velocity to concentrate the DNA at the
liquid to gel interface. A change in conditions can then be used to
drawn the concentrated DNA into the electrophoresis step as a
concentrated and small sample.
[0430] Another option is hydrodynamic stacking. This is based upon
the variation in the flow velocity between sample and the location
from which the size based separation starts, for instance through
the use of adjustments to conductivity, buffer components, pH and
the like. An example of such an approach is field amplified sample
stacking, FASS. This provides higher electric fields in the lower
conductivity zones than in the higher conductivity zones. The
sudden potential drop at the interface between the two zones causes
sample stacking there.
[0431] Mechanical pre-concentration is also a possibility. Packed
beds, nanochannels, immobilised polymers and membranes all offer
the possibility of trapping and concentrating the sample.
Electro-elution, where by the release of the sample is caused by
the application of an electric potential to a membrane, is one
possibility.
[0432] A combined technique approach to pre-concentration may be
particularly beneficial. Such an approach is shown in FIG. 24, in
the case of CE channel being in the same plane as the rest of the
cartridge, and FIG. 25, in the case of the CE channel not being in
the same plane as the rest of the cartridge.
[0433] As illustrated, the combined flow 24-01, 25-01 of DNA
containing sample and formamide pass valve 24-03, 25-03 and then
reach a junction 24-05, 25-05. The Y-shaped junction brings the
combined flow 24-01, 25-01 into proximity with the running buffer
flow 24-07, 25-07 in channel 24-08, 25-08. These flows cross the CE
channel 24-09, 25-09 and any excess passes to chamber 24-11, 25-11.
The left-hand detail shows the construction present at the
intersection of the CE channel 24-09, 25-09 and the channel 24-08,
25-08.
[0434] In the FIG. 24 form, the stacking interface 24-11 is
provided between the combined flow 24-01 and buffer flow 24-07. The
electric potential is provided by electrode 24-13. The second
stacking function is provided by the membrane 24-15 provided
between the buffer flow 24-07 and the CE channel 24-09.
[0435] In the FIG. 25 form, the stacking interface is similarly
provided.
4) Alternative Electrophoresis Channel Configuration
[0436] In the embodiment described above, the injector is of the
double T type. As an alternative, it is possible to use a
cross-channel injector, as shown in FIG. 8.
[0437] In this case, the reservoir 604, channel 610 and other parts
leading to the fourth electrode location 620 are the same. The arm
624 provided with the fourth electrode location 620 and the arm 622
provided with the third electrode location 618 are aligned on a
common axis and at 90.degree. to the main capillary 616.
[0438] The sample is drawn towards the electrode at the third
electrode position 618 by the application of a voltage. To prevent
dispersion of the sample into the main capillary, towards the first
612 and/or second 614 electrode locations, a voltage is applied to
the electrode at the first electrode location 612 and to the
electrode at the second electrode location 614. This has the effect
of pinching the part of the sample at the intersection of the main
capillary 616 and the arms 622, 624, and maintaining the minimal
size of the plug which is then used in the capillary
electrophoresis.
[0439] A further electrophoresis channel configuration is shown in
FIG. 43. In this case, the sample flows along channel 43-100 from
inlet 43-102 to outlet 43-104. A potential difference is applied
between locations A and B. This draws the DNA in the sample towards
the membrane 43-106. The membrane is sized, 10-14 kDa cutoff, to
retain the DNA. The separation matrix is then flowed into the
channel 43-100; UV activation may be provided, as discussed
elsewhere. The same buffers at location A, B and in the matrix are
then provided for the electrophoretic separation to be provided
through the application of a potential difference between A and
B.
[0440] The polarity may be provided in the reverse direction before
the CE run, for instance to ensure the buffer extends from A to B.
DNA is not lost as the flow will maintain it on the membrane
43-106.
[0441] Between loading to the membrane 43-106 and the CE
separation, it is possible to introduce a variety of
reagents/buffers into locations A and/or B and/or the channel
43-100 to assist in purifying the DNA and/or to optimise CE
conditions, for instance through removal of excess salts and/or
unincorporated PCR primers. Both locations A and B have their own
inlets and outlets for this purpose.
[0442] A still further configuration is shown in FIG. 44. In this
case, again the sample flows through channel 44-100 from inlet
44-102 to outlet 44-104. A potential difference between A and B is
used to attract and retain the DNA on a membrane 44-106. By
swapping to an electrolyte flow through channel 44-100 and changing
the potential difference it is possible to load the DNA to the
matrix in main channel 44-108. The CE can then be performed.
[0443] Again one or more cleaning or condition controlling steps
may be provided before CE is conducted.
[0444] A yet further configuration is shown in FIG. 45. In this
case, the arm 45-100 leading the sample into the main channel
45-102 where CE is performed extends downwards, at least partially
aligned with gravity. The arm 45-104 leading away from the main
channel 45-102 extends upward, at least partially aligned with
gravity. In this way gravitation effects promote retention within
the main channel 45-102, rather than encouraging flow away from it
and into another arm.
5) Cartridge Variant for Real Time PCR Performance
[0445] In the cartridge 9 described above, the cartridge 9 is being
used to consider a reference sample. In this alternative
embodiment, the changes to the cartridge 5009 beneficial to the
consideration of a casework sample are considered.
[0446] A major difference between a casework sample and a reference
sample is that whilst the amount of DNA recovered in a reference
sample has a degree of consistency, and is of a high level, this is
not the case for a casework sample. The manner in which the sample
is left, the passage of time, the collection process and other
factors can all result in the amount of DNA in a casework sample
being unpredictable, and often lower, than desired.
[0447] To counteract this, the casework sample processing seeks to
ensure that the amount of DNA arising from the amplification
process is within certain bounds.
[0448] To do this, the casework sample provides for parallel
processing of the sample, particularly in terms of the sample
amplification step 204.
[0449] The sample receiving step 200 and sample preparation step
202 are basically the same as previously described. The difference
comes in the sample amplification step 206.
[0450] The channel 5410 containing the eluted DNA from the beads
held in the incubation chamber 5358 leads to a junction 5700 where
the flow is split into two separate streams 5702, 5704.
[0451] The first stream 5702 passes into a PCR chamber 416 of the
type previously described (and is not illustrated further). The
subsequent handling of this by the cartridge 9 is as described
above, save for the possible changes in the sample amplification
conditions/duration described shortly.
[0452] The second stream 5704 passes into a second separate PCR
chamber 5706. This second PCR chamber 5706 contains a bead provided
with a coating containing the necessary regents for PCR and for a
quantification analysis.
[0453] During processing, PCR is advanced in the PCR chamber 416
and in the second PCR chamber 5706, in parallel. After a given
number of PCR cycles for the second PCR chamber 5706, the contents
of the second PCR chamber 5706 are considered to establish the
quantity of DNA which has been generated by the PCR cycles up to
that point. This can be equated to the amount of DNA present within
the original sample and hence the amount of DNA the PCR chamber 416
is working on. As a result of the quantification, the PCR
conditions and/or cycle number for the PCR chamber 416 can be
varied to optimise the quality of amplification product.
[0454] Further details on the operation of such a system and the
use of this feed back are to be found in 61/026,869, the contents
of which are incorporated herein by reference, particularly as they
relate to the parallel conduct of PCR and the use of the results
from one PCR to control and/or modify the conduct of the other
PCR.
[0455] Suitable reagents include the Plexor HY kit available from
Promega Inc, 2800 Woods Hollow Road, Madson, Wis. 53711, USA and
Quantifiler.RTM. Duo DNA quantification kit available from Applied
Biosystems, Foster City, Calif., 944404, USA.
[0456] To establish the quantity of DNA present, it is necessary to
interrogate the sample using an excitation light source and then
quantify the amount of light arising. To do this, light from a
light source is conveyed to the second PCR chamber 5706 and
focussed thereon using a lens system. The excitation light
interacts with the dye(s) associated with the sample. The
fluorescent light generated is detected and is proportional to the
quantity of DNA present.
[0457] The light source used could be the same light source as is
used for the electrophoresis step 206, and described in detail
below. The light would be conveyed to the second PCR chamber 5706
by an optical fibre. Because the Peltier heater/coolers are
positioned in front of and behind the second PCR chamber 5706, the
light for the detection is introduced from the side of the
cartridge 9. The light source may be a laser, for instance of the
type and/or with the set up discussed further below in the
electrophoresis step 206. As an alternative, however, it is
possible to use a light emitting diode based light source, as
described below.
[0458] Depending upon the quantity, the number of cycles used in
the PCR chamber 416 may be increased, decreased or kept at the
normal level, so as to provide a quantity of DNA within the desired
range after PCR has been completed in PCR chamber 416.
[0459] In the context of real time quantification and/or the
handling of samples from crime scenes (rather than those taken
under controlled conditions from individuals), differences in the
implementation of the invention may be provided. These may
include:
[0460] 1) The parallel processing of the sample so as to allow the
results from a first processing of the sample to inform on the
optimum conditions etc to be used in the main processing of the
sample. Further details of such an approach are to be found in
WO2009/098485, the contents of which are incorporated herein by
reference with respect to the parallel processing and consideration
of samples and the feedback of information from one processing to
the other.
[0461] 2) The efficiency of the extraction should be as high as
possible, for instance through optimised sample recovery, lysis and
amplification. The use of various processes and/or reagents to
separate the DNA of interest from problematic components, such as
PCR inhibitors, is beneficial in this respect.
[0462] 3) The cartridge used will feature many of the steps and
components exemplified above, but with the incorporation of the
parallel PCR circuit and the ability to analyse the results
therefrom, for instance using a laser or LED to apply light to the
liquid, with the return light being detected to inform on the PCR
process. Photo diodes and/or cameras can be used in the light
detection. A control material may be provided within the sample to
provide a reference value with respect to the light detected.
[0463] 4) The instrument would benefit from being able to run
positive and/or negative controls. These could be run in the same
cartridge as the sample. The controls may be handled by the
operator in the same manner as the sample of interest so as to
inform on contamination risks. The controls may just be run
periodically so as to check on the instrument, for instance in the
form of a calibration check.
Cartridge Components
[0464] Within the cartridge are a significant number of components,
with each being optimised with respect to its role and its role in
combination with the other components.
1) Valves
[0465] To minimise manufacturing costs and give consistent
operation, all of the valves in the cartridge are one of two types.
The two types are a closing valve 2000; FIG. 10a; and an opening
valve 2002; FIG. 10b.
[0466] The closing valve 2000 is shown schematically in FIG. 10a.
The closing valve 2000 is positioned above, relative to the
direction of gravity, the channel 2004 to be closed. The closing
valve 2000 is formed by a conduit 2006 which is in fluid
communication with the channel 2004 and is in fluid communication
with the bottom of a valve reservoir 2008. The valve reservoir 2008
is filled with paraffin wax and is 3 mm in diameter and is provided
with the conduit 2006. On the top of the valve reservoir 2008, a
gas passage 2010 provides fluid communication with a valve gas
reservoir 2012. The valve gas reservoir 2012 is full of air.
[0467] The dotted line in FIG. 10a shows that part of the location
of the closing valve 2000 which is in contact with a heater
element, not shown, provided on the adjoining printed circuit board
of the instrument.
[0468] When the closing valve 2000 is to be activated, the heater
element is caused to heat up. This both melts the paraffin wax in
the valve reservoir 2008 and causes the air in the valve gas
reservoir 2012 to expand. The expansion of the air provides the
driving force to displace the melted paraffin wax from the valve
reservoir 2008 into the conduit 2006 and then into the channel
2004.
[0469] The volume of paraffin wax displaced is controlled by the
temperature to which the valve gas reservoir 2012 is heated
(variation in pressure) and the duration of the heating applied (as
the paraffin wax soon solidified once the heating is switched
off).
[0470] Continued displacement of the paraffin wax into the channel
2004 causes the paraffin wax to expand in each direction along the
channel 2004.
[0471] In some cases, the fluid in the channel will not compress or
move in one direction (or is limited in the extent possible) and so
the flow of the paraffin wax within the channel 2004 occurs
preferentially in the other direction. Normally, the paraffin wax
is displaced into the channel 2004 until a 2 mm to 10 mm length of
the channel 2004 is filled. With the heat removed, the paraffin wax
sets in this new position and the channel 2004 is reliably
sealed.
[0472] The section where the channel 2004 is to be shut, is
deliberately chosen to be horizontal, relative to the direction of
gravity, as this assists the retention of the paraffin wax at the
location to be sealed.
[0473] To assist further in the formation of the seal, it is
beneficial to arrange the closing valve so that it is between one
or two upward, relative to the direction of gravity, bends. As
shown in FIG. 10a the bend 2014 provides assistance in the accurate
formation of the seal within the channel 2004.
[0474] The opening valve 2002 is shown schematically in FIG. 10b.
The opening valve 2002 is positioned as a part of the channel 2004
the fluid flows through. The opening valve 2004 is formed by a
valve chamber 2020 which has an inlet 2022 from the channel 2004 in
a first side wall 2024 and an outlet 2026 leading to the
continuation of the channel 2004 in the opposing side wall
2028.
[0475] The paraffin wax is positioned in the initial section 2030
of the valve chamber 2020. Downstream of this initial section 2030,
is a trap section 2032. The dotted line in FIG. 10b shows that part
of the opening valve 2002 which is in contact with a heater
element, not shown, provided on the adjoining printed circuit board
of the instrument.
[0476] When the opening valve 2002 is to be activated, the heater
element is caused to heat up. This melts the paraffin wax in the
initial section 2030. By the time the paraffin is melted, or
shortly thereafter, an electrochemical pump upstream of the opening
valve 2002 has been activated for sufficient time to cause a
pressure build up, upstream of the opening valve 2002. This
pressure causes the driving force to displace the melted paraffin
wax from the initial section 2030 and downstream into the trap
section 2032. Once in the trap section 2032, the passage 2034 above
the paraffin wax is clear allowing fluid communication through the
opening valve.
[0477] With the heat removed, the paraffin wax sets in this new
position and the channel 2004 and passageway 2034 is reliably
opened.
[0478] The section where the channel 2004 is to be opened is
deliberately chosen to be horizontal, relative to the direction of
gravity, as this assists the retention of the paraffin wax in the
trap section 2032.
[0479] In some applications, particularly those close to the high
temperatures used in the PCR chamber, the valves benefit from using
a high melting point wax. This melts at greater than 95.degree. C.
and so does not melt under PCR conditions. In some cases, the valve
performance can be improved further by using a high melting point
and lower melting point mixture; with the lower melting point wax
tending to fill any cracks which form in the higher melting point
wax.
[0480] A further valve embodiment is shown in FIG. 47. The channel
47-100 is connected to the valve by a side channel 47-102 as usual.
The side channel 47-102 leads to a first chamber 47-104. This is
connected via a short channel 47-106 to a larger second chamber
47-108.
2) Chambers
[0481] Within the cartridge, a variety of chambers are provided for
a variety of purposes.
[0482] To achieve those purposes efficiently and effectively, the
chamber designs are optimised in various ways.
[0483] With respect to the incubation chamber 358, this is provided
with a broad base which is generally horizontal. In operation, the
offset magnet (not shown) is used to restrain the magnetic beads in
position during washing and during elution. The broad base provides
a suitable location to which the beads can be drawn and secured,
whilst exposing them to the wash flow or to the elution flow.
[0484] The sloping walls within the incubation chamber 358 and the
bubble mixing chamber 342 are provided to promote the flow of
eluent, introduced into the chambers at the top, to the outlet at
the bottom of the chamber.
[0485] The angular corners are used to generate improved pressure
gradients from the inlet for a part of the process to the outlet in
that respective part of the process.
[0486] The first further mixing chamber 332 and second further
mixing chamber 336 are provided to encourage non-laminar flow
within the flow route. As the fluid transitions from the channel,
with its cross-section, to the chambers, with their increased
cross-section, non-laminar flow arises. This gives good mixing for
the different density fluids and particles which are all to be
mixed. Such mixing forms are significantly better in this respect
than bubble mixing alone or piezoelectric based mixing.
[0487] The PCR chamber 416 has two principle embodiments; as
described above. In each, the PCR regents are provided within the
degradable shell of a bead located within the PCR chamber 416. To
ensure proper flow of the liquids around and past the bead, the
bead is provided with a bead seat. This provides a defined rest
position for the bead, but as the bead is only contacted at
discrete locations when in the seat, fluid is still able to flow
past the bead. The seat ensures that the bead does not block at
inlet to and/or outlet from the PCR chamber 416. The seat ensures
that there are no large areas of the bead surface, and hence of the
reagents, which are isolated for fluid contact.
[0488] In the second of the PCR chamber 416 embodiments, described
in the alternatives for the cartridge section, the PCR chamber 416
is completely filled with fluid. This gives a reproducible volume
of fluid in the PCR process. The same position arises with the
third embodiment, FIG. 22.
[0489] In the first of the PCR chamber 416 embodiments, the maximum
level of fluid within the PCR chamber 416 is controlled by the
relative height of the outlet within the chamber. The outlet in
effect acts as an overflow for the fluid, once the PCR chamber 416
has filled to this level. A head space remains above the fluid,
within the PCR chamber 416.
3) Vents
[0490] To allow fluid flow, air or sample, around the cartridge 9,
various vents need to be provided for various chambers.
[0491] To prevent any risk or suggestion that material can enter
the cartridge 9 through such vents, each of the vents is provided
with a filter element to exclude particulate material. In addition,
when a vent is part of the active processing on the cartridge 9,
the vent is under positive pressure and so air is flowing out
through the vent. This too assists in preventing any risk of
particulate material entering the cartridge 9.
[0492] In some situations, it is desirable to be able to allow air
to pass through the vent freely, but for the vent to resist the
passage of any subsequent liquid. An example is to be found in the
alternative PCR chamber 416 filling embodiment. To provide this,
those vents are hydrophobic. The vent may be hydrophobic because of
the base material forming the vent and/or because of a treatment
applied to the material of the vent. Such a treatment can be
provided, for instance, by using polypropylene material and/or by
providing a polysulphone coating.
4) Archive
[0493] As described above, the fluid not needed in the PCR chamber
416, is pumped onward to an archive chamber 422.
[0494] The purpose of the archive chamber 422 is to provide a
storable record of the sample supplied to the sample amplification
stage 204, and the PCR chamber 416 in particular.
[0495] If needed, the sample in the archive chamber 422 can be
accessed at a later date to enable a further amplification and
analysis to be performed. Further processing in this way is useful
where it is necessary to repeat the analysis, for instance by way
of verification. Alternatively, further processing enables a
different amplification and analysis protocol to be applied, for
instance, a protocol suitable for low levels of DNA within the
sample.
[0496] In the form shown in FIG. 3, the archive chamber 422 is an
integral part of the overall cartridge 9.
[0497] In an alternative, form shown in FIG. 11, the archive
chamber 2422 is still fed the surplus sample through a channel 2418
leading away from the PCR chamber, not shown.
[0498] The archive chamber 2422 is positioned on a stub 2750 which
extends from the side of the cartridge 9. The stub 2750 is
connected to the cartridge 9 during normal use, but a line of
weakness 2752 is provided. This allows the stub to be snapped off
the cartridge 9 after the completion of the processing. This means
the archive function can be provided by only storing the stub 2750,
rather than have to store the far larger overall cartridge 9. Given
the number of samples which may be considered, and the time for
which they have to be stored, saving of storage space is a
significant issue.
[0499] To seal the archive chamber 2422, once it has been loaded, a
closing valve 2754 is provided on the cartridge 9 side of the line
of weakness 2752 and a further closing valve 2756 is provided on
the stub 2750 side of the line of weakness 2752. These valves are
activated to place paraffin wax in the channel 2418 on either side
of the line of weakness 2752. To provide for long term storage, a
further closing valve 2758 is provided on the channel leading from
the archive chamber 2422 to the vent 2424.
[0500] Just as the cartridge 9 is provided with an identifier,
which is used to link it in the records to the sample loaded upon
it, then the stub 2750 is also provided with a common identifier so
as to maintain the link after the stub 2750 is broken off the
cartridge 9.
5) Reagents
[0501] Various options exist for the provision of the reagents
needed in the various steps of the processing. As far as possible,
so as to keep the processing as simple as possible for the user,
the cartridge 9 is provided with pre-loaded reagents. Examples of
such pre-loaded reagents would include the bead provided in the PCR
chamber 416; with the bead carrying the PCR regents inside. Other
pre-loaded regents include the various wash liquids and elution
liquids described in the methodology above.
[0502] If necessary, one or more reagents can be provided separate
from the cartridge 9, and be loaded onto the cartridge at or close
to the time of use. This may be necessary where the reagent is
unable to withstand prolonged storage under the conditions to which
the cartridge 9 is exposed. These may be conditions of temperature
and/or mechanical conditions such as vibration or orientation.
[0503] A preferred form of reagent provision is provided where the
reagent(s) are provided as part of a solid phase reagent or solid
phase reagent storage component, with release of the reagent being
triggered by an increased temperature. Gel forms of reagent and/or
reagent storage component, preferably triggered to release by the
application of higher temperatures are also a useful option.
6) Electrochemical Pumps
[0504] To simplify the construction and costs of the cartridge, a
common approach is used to providing the motive power to the
various operations on the cartridge; electrochemical pumps. Each of
the electrochemical pumps consists of a pair of electrodes immersed
in the electrolyte. The flow of a current results in off gassing.
The off gas collects in the top of the electrochemical pump,
increases in pressure and leaves the pump via the outlet in the top
of the pump. This off gas pushes ahead of itself other fluids
encountered in the channels and chambers. The off gas contributes
to bubble mixing in some of the stages.
[0505] To give a desired extent of pumping, the volume of the
electrochemical pump can be varied. The extent of pumping can be
delivered in one, two or more goes, as turning off the current
stops the pumping action.
[0506] The rate of pumping and/or pressure delivered can be varied
by varying the molarity of the electrolyte. Sodium chloride is the
preferred electrolyte; used at 1M; and used in conjunction with
aluminium electrodes.
7) Electrophoresis Matrix
[0507] The material provided within the capillary of the
electrophoresis stage is important to the reliability and
resolution of the analysis obtained.
[0508] Various possible materials can be used in the capillary.
These include the use of polymer matrix, for instance a
polyhydroacrylamide, a polydimethylacrylamide or mixtures there of.
The polymers may be cross-linked to give the desired properties
and/or formed into their state of use within the capillary, after
loading. It is also possible to use an inert bed of particulate
material to form the matrix in which the size based separation is
achieved.
[0509] As well as optimising the performance through the properties
of the gel, it is also possible to treat the capillary walls to
improve properties. For instance it is possible to apply
hydrophilic coatings, such as poly(hydroxyethlacrylamide).
[0510] A potential methodology for the electrophoresis matrix is to
store that material in a chamber which is a part of the CE chip,
but not use that chamber for the CE separation. Instead, when
required for use, the stored matrix is moved from the chamber into
the capillary so as to fill it to the desired degree. As a result
of loading just before use, the matrix is no subject to
sedimentation effects; these can have a detrimental effect on the
analysis. Pressure loading can be used for this purpose.
[0511] Another potential methodology is to fill the main channel
and arms of the CE chip with the matrix. Those parts of the CE chip
where the matrix is not needed, for instance aside from the main
channel, may be masked. In this way, when UV light is applied the
parts where the matrix is not needed retain the matrix unaltered.
The unaltered matrix can be washed away. Where the matrix is
exposed to UV light it is altered and resists washing away.
8) CE Chip Design
[0512] A preferred configuration for the CE chip is shown in FIG.
42a and the detailed partial view of FIG. 42b.
[0513] The end portions 42-100 cooperate with the carrier when the
chip is mounted within it. The external profile of the base of the
CE chip is designed to match with that defined by the raised
surface around the CE chip heater board, described elsewhere in
this document.
[0514] As described below, a number of electrodes are required in
different parts of the channels provided within the CE chip so as
to load the sample and then perform the necessary separation to
give the analysis. These electrodes within the channels are
connected to pins 42-102 which extend above the plane of the CE
chip. These pins 42-102 are positioned so that they are within the
cut away portion of the second support and so are exposed. This
allows suitable connections to be made to these pins 42-102 so as
to apply the necessary voltages to them and to the electrodes
connected to them.
[0515] The CE chip is shown with a single channel in which CE is
performed, but channels suitable to perform separations on multiple
samples could be provided.
9) PCR Chamber Sealing
[0516] In the embodiments described elsewhere, the chambers and the
valves which are used to seal the channels leading to and from them
are separate. In the following embodiment, the chambers and the
valves are integrated as a single component.
[0517] As shown in FIG. 41a, the PCR chamber 41-100 is provided in
the cartridge. However, the walls defining the circumference, at
least, of the chamber 41-100 are rotatable within the body of
material forming the cartridge. In the lefthand form, the rotatable
wall is positioned such that the holes therein are aligned with the
inlet channel 41-102 and the loading outlet channel 41-104. As a
result, liquid can enter and gas leaves the chamber 41-100 until
the chamber is full, centre form. The rotatable wall can then be
rotated to align the holes therein with the inlet channel 41-102
and the dispense outlet 41-106, right hand form, to allow the
contents to be emptied.
[0518] A variant of this approach is shown in FIG. 41b, where inlet
channel 41-100 is connected to outlet channel 41-108. Rotation
aligns the holes with dispense inlet 41-110 and dispense outlet
41-106.
[0519] The variant in FIG. 41c uses the arrangement to seal the
chamber during PCR. In the left hand form, the inlet channel 41-102
is connected to and fills the chamber up to the level of the outlet
channel 41-108. Partial rotation offsets the holes in the rotating
wall from alignment with any of the inlets/outlets, centre form.
After PCR, further rotation aligns the holes with the dispense
inlet 41-110 and dispense outlet 41-106.
[0520] The extent of rotation may be limited by abutment surfaces
provide in the cartridge wall which abut surfaces on the rotating
walls or vice versa. Partially circular forms for the hole in the
cartridge which receives the rotating walls and/or vice versa may
also be used to control or limit rotation in one or both
directions.
[0521] Rotation may be provided by cooperation between an actuator
and a slot in the circular wall.
[0522] Rotation may cause pads or other pliable material to be
compressed or otherwise deformed to give sealing.
[0523] One or more of the channels may serve as a light path,
rather than or in addition to being a fluid flowpath, so as to
allow an investigatory instrument to shine light into the liquid
contained within the chamber. Such an embodiment is useful in the
context of the cartridge variant for real time PCR discussed
above.
Instrument Configuration and Appearance
[0524] The instrument 11 is illustrated in FIG. 12 and is provided
within a casing 8000. The mid section 8002 of the instrument 11 is
provided with a door 8004 provided with a latch 8006. Behind the
door 8004 is the location at which the cartridge 9 is mounted in
use. This location is a position in which the plane of the
cartridge 9 is parallel to the plane of a printed circuit board
8008. At the location, the cartridge 9 and components on the
printed circuit board 8008 contact one another.
[0525] Behind the printed circuit board 8008 are the electronics
for operating and controlling the components provided on the
printed circuit board 8008. These include the power supplies,
voltage controllers, temperature controllers and the like.
[0526] The upper section 8010 of the instrument 11 provides the
display 8012 by means of which the user inputs information into the
instrument 11 and receives visual information from the instrument.
The software and hardware for operation of the display 8012 are
provided on a computer positioned behind the display screen 8012 in
the upper section 8010.
[0527] The lower section 8014 of the instrument 11 contains the
high voltage power supply and controller for the laser used in the
inspection of the capillary electrophoresis. Also in this lower
section 8014 are the charge couple device used to sensor the
fluorescence and the optics for conveying the light to and from the
capillary.
[0528] Another embodiment of the instrument is shown in FIGS. 29a,
29b and 29c. The instrument 29-11 is provided within a casing
29-8000. The upper section 29-8002 of the instrument 11 is provided
with a door 29-8004. The door 29-8004 is a combination of a top
section 29-8006 and front section 29-8008 of the casing
29-8000.
[0529] The lower section 29-8010 of the instrument 11 provides the
display 29-8012 by means of which the user inputs information into
the instrument 11 and receives visual information from the
instrument 11.
[0530] The window 29-8014 allows for visual inspection of the
cartridge used. A series of light bars 29-8016 are used to indicate
the extent of progress through the steps involved; the more of the
bar which is lit the greater the extent of the step performed.
[0531] A stylus 29-8018 is used by the operator to interact with
the display 29-8012.
[0532] Various control buttons 29-8020 are provided below the
screen 29-8012.
[0533] The overall dimensions of the instrument are width, W, 419
mm, overall height, OH, 621 mm, depth, D, 405 mm.
[0534] The side panel 29-8022 is removable for maintenance
purposes.
[0535] The embodiment of FIG. 30 shows the door 30-8004 structure
more clearly, together with the workspace 30-8024 that is accessed
through it. The workspace 30-8024 includes the slot into which the
cartridge carrier 30-8026 is inserted. The cartridge carrier
30-8026 is as described elsewhere in this document. The workspace
30-8024 also includes the lane finding apparatus 30-8028.
[0536] The cover 30-8030 in the side panel 30-8032 is opened by
rotation to allow access to the optics for maintenance
purposes.
Cartridge to Instrument Interface
[0537] As described above, once the cartridge 9 is loaded with the
sample, the cartridge 9 is loaded into the instrument 11 for the
processing to be conducted.
[0538] As a first step, the latch 8004 is released and the door
8002 is opened.
[0539] To insert the cartridge 9, FIG. 13, the section of the
cartridge 9 which bears the PCR chamber 416 is inserted into a slot
8023 between the components which will control the PCR process.
These components include the thermoelectric heaters/coolers,
Peltier devices 8025, and fans 8027 there for. These components are
free to travel to a limited extent to help with the locating of the
cartridge 9 within the slot 8023, whilst being forcibly returned to
the optimum position after insertion so as to give effective
heating/cooling.
[0540] The cartridge 9 is provided with a series of recesses which
cooperate with dowels extending through the printed circuit board
8008 to accurately register the cartridge 9 relative to the printed
circuit board 8008. The dowel arrangement is such that the
cartridge 9 cannot be fitted the wrong way round.
[0541] Once positioned, the cartridge 9 is provided in a plane
which is parallel to the plane of the printed circuit board 8008.
Both components have flat surfaces facing one another so as to
assist with the good contact needed between them.
[0542] The closing of the door 8002 and operation of the latch 8004
applies a compressive force to the cartridge 9 by way of a series
of spring loaded pins mounted on the inside surface of the door
8002. This helps hold the cartridge 9 in firm contact with the
printed circuit board 8008.
[0543] The printed circuit board 8008 is important to the
successful operation of the invention. It provides the energy
sources for the various components to be driven on the cartridge 9.
In effect, the drivers are all provided in the cartridge 9, but the
energy sources are provided on the printed circuit board 8008. In
this way, the precision operation needed is ensured by the
expensive and bespoke electronics and arrangement of the printed
circuit board 8008; a reusable component of the instrument. In this
way, the cartridge 9 is simple and self-contained. This reduces the
complexity of the interface between the two and also removes the
risk of contamination of the contents of the cartridge 9. The only
transfer between the printed circuit board 8008 and the cartridge 9
is conducted and radiated heat from the heaters and the magnetic
field provided by the magnet.
[0544] The components provided on the printed circuit board
include: [0545] a) The electrical contacts 9000 which connect to
the pins of the electrochemical pump electrodes on the cartridge 9.
These provide the electrical power, when needed, to operate the
electrochemical pumps. [0546] b) The electrical heaters 9002 which
are used to apply heat to the valves on the cartridge so as to open
or close the valves depending upon their type. These are square
areas of resistance heating material which is applied by printing a
paste to the desired location. The heating effect is improved if
the square block is rotated through 45.degree. relative to the axis
of the channel subject to the valve. [0547] c) The magnet 9004
which is advanced into proximity with the cartridge 9 when it is
desired to retain the beads and prevent them from moving. The
magnet 9004 is retracted away from the cartridge 9 when it is
desired to release the beads within the chamber 358. [0548] d) The
sensors 9006 are providing feed back and/or verification of the
conditions induced by the heaters etc.
Alternatives for Cartridge to Instrument Interface
[0549] If it is necessary to alter or improve the contact between
the cartridge and the printed circuit board, there are various
options for doing so, including the following: [0550] a) The
loading provided by the sprung pins mounted on the door 8002 can be
increased. This applies a force to the cartridge 9 and pushes it
against the printed circuit board 8008. [0551] b) The cartridge 9
can be mechanically clipped to the printed circuit board 8008, with
the clip(s) applying a compressive force. [0552] c) The cartridge 9
can be provided with a compressible substrate mounted on the
surface which is intended to contact the printed circuit board. In
this way, when then cartridge 9 and printed circuit board 8008 are
pushed together, the substrate will provide good all over contact.
The substrate can be a solid material, paste or even a liquid. The
materials of the substrate, or parts there of, are selected so as
to provide maximum thermal conductivity, for instance. Particles,
nanoparticles or other materials may be added to alter the
properties. The substrate may be protected, prior to use, by a
peelable backing. [0553] d) As described above, the components
(such as heaters etc) are provided in a fixed position on the
printed circuit board 8008. This means they move with the printed
circuit board 8008. It is possible to provide one or more, and even
each of these components with a degree of independent movement. For
instance, they may be provided with a sprung mounting on the
printed circuit board. In this way, each is able to independently
adjust its position, forward and backwards, relative to the
cartridge. [0554] e) As shown in FIG. 23, it is possible to provide
the section of the cartridge 9 which bears the PCR chamber 416 in
opposition to stacked components which will control the PCR
process. In this example, the stack includes a first Peltier device
23-01 in contact with the cartridge 9 and in contact with and
aligned with a second Peltier device 23-03. The stacking of the
devices allows high temperatures, for instance greater than
150.degree. C. to be obtained within the PCR chamber. Such
temperatures are beneficial in terms of melting the high melting
point wax seals described elsewhere within this document. [0555] f)
Alternative forms of heater may be used instead of Peltier effect
device. For instance infra red heating devices may be used. The
material around the PCR chamber, or a part of that material, may be
capable of resistance heating to give the necessary heating for the
chamber. Resistance heaters positioned against the cartridge may be
used. Microwave heating may be used.
Alternative Cartridge to Instrument Interface
[0556] In the alternative embodiments of the instrument described
above in relation to FIGS. 29a, b, c and FIG. 30, the cartridge is
not loaded directly into the instrument. Instead, once loaded with
the sample, the cartridge 31-01 is loaded into a cartridge carrier
31-03.
[0557] The use of the carrier 31-03 means that the cartridge 31-01
and the CE chip can be constructed separately. This allows
different material and/or different production tolerances to be
used for the different components; a beneficial effect on cost
and/or performance and/or the balance between those can thus be
provided.
[0558] The carrier 31-03 also allows for easy assembly of the
required components and their insertion into the instrument in a
unitary form. At the same time, the carrier is designed so as to
allow separate alignment checking and adjustment for the cartridge
and the CE chip so that both are in their correct, optimised
position within the instrument.
[0559] If desired, the cartridge position can be checked and any
alignment adjustment necessary can be made. Before CE starts, a
separate check can be made on the alignment of the CE chip, within
any adjustments it needs being made before CE starts.
[0560] The cartridge carrier 31-03 is illustrated in FIG. 31a. The
cartridge carrier 31-03 includes a first support 31-05 and a second
support 31-07 which is perpendicular to the first support
31-05.
[0561] The first support 31-05 is used to carry the cartridge
31-01. The second support 31-07 is used to carry the capillary
electrophoresis, CE, chip; this interaction is described further
below.
[0562] The prepared cartridge 31-01 is presented with its face
31-09 to the face 31-11 defined by the first support 31-05. An
externally threaded screw 31-13 provided at each corner of the
first support 31-05 is received into an opposing aperture 31-15
provided at each corner of the cartridge 31-01. Rotation of the
screws 31-13 causes them to engage with and enter an internal screw
thread provided in the apertures 31-15. Further tightening mounts
the cartridge 31-01 on the first support 31-05 and hence the
carrier 31-03 in a secure and known position.
[0563] The interaction between the cartridge 31-01 and the carrier
31-03 is shown in more detail in FIG. 31b in relation to one of the
screws 31-13.
[0564] The screw 31-13 is provided with a knurled head 31-17. The
threaded engagement occurs between the end 31-19 of the screw 31-13
and the aperture 31-21 in the cartridge 31-01. A jam nut 31-23 in
cooperation with a washer 31-25 serves to hold the screw 31-13 on
the carrier when not engaged with a cartridge 31-01 The jam nut
31-23. washer 31-25 and sleeve 31-27 serve to prevent over
tightening between the carrier 31-03 and the cartridge 31-01.
[0565] Rotation of the screw 31-13 pulls the knurled head 31-17 and
the cartridge 31-01 closer together. This causes compression of the
conical spring 31-29 between the knurled head 31-17 and an abutment
surface 31-31 on the first support 31-05. The spring 31-29 assists
in ensuring correct alignment during tightening. Once rotation is
finished, the first support 31-05 and hence carrier 31-03 is in a
known position relative to the cartridge 31-01.
[0566] The CE chip 32-31 is inserted into the carrier 32-03 as
shown in FIG. 32a. The CE chip 32-31 is slid into a slot. As shown
in FIG. 32b, the second support 32-07 provides such a slot 32-33 at
either end for receiving the end portions 32-35 of the CE chip
32-31. An incline 32-37 on the lead edge 32-39 of the CE chip 32-31
engages with the end 32-41 of a spring loaded plunger 32-43 and
causes it to displace outward, arrow A. Once the recess 32-43 is
presented to the end 32-41 of the plunger 32-43, the plunger 32-43
returns, arrow B, and so prevents onward movement of the CE chip
32-31 past the desired position.
[0567] Once the cartridge 31-01 and the CE chip 32-31 are inserted
into the carrier 31-03, 32-03, the fluid connection between the two
is provided by a tube 33-45. The insertion of the cartridge 31-01
into the carrier 31-01 causes the electrophoresis step inlet 28-570
on the cartridge 31-03 (see FIG. 28a) to become connected to the
tube 33-45. As shown in FIG. 33a, the tube 33-45 extends upward,
parallel to the plane of the cartridge 31-01 and the first support
31-05 through an opening 33-47 in the carrier 31-03. As shown in
FIG. 33b, once through the opening 33-47, the tube 33-45 makes a
90.degree. turn into the plane of the second support 31-07 and the
CE chip 32-31. The tube 33-45 is accommodated within the second
support 31-07 above the CE chip 32-31. A further 90.degree. turn
leads the tube 33-45 into the CE chip 32-31. The remaining fluid
transport is handled within the CE chip 32-31 itself, as described
elsewhere in this document.
[0568] After insertion of the cartridge 31-01 and the CE chip 32-31
into the carrier 31-03, as described above, the carrier 31-03 is
ready for insertion.
[0569] As a first step, the door 34-8004 is opened, FIG. 34a, to
expose the workspace 34-8024. The work space 34-8024 includes the
slot 34-47 that the carrier 34-03 is inserted into.
[0570] The carrier 34-03 is inserted into the slot 34-47 until the
second support 34-07 comes to rest on the surface 34-49 of the
workspace 34-8024. The cooperation of the carrier 34-03 with the
slot 34-47 ensures the correct general positioning of the cartridge
34-01 with respect to the instrument, both in terms of lateral and
vertical positioning; FIG. 34b.
[0571] Insertion in this way provides the section of the cartridge
which bears the PCR chamber between the components which will
control the PCR process; as described further below.
[0572] Once inserted, the door 34-8004 is closed. The closing of
the door 34-8004 triggers various actions based upon contact
between the closed door 34-8004 and casing. The clamping of the
cartridge to the PCB, the positioning of the CE chip on the CE chip
heater board, the introduction of the electrical contacts to the
pins provided on the CE chip, the introduction of the electrical
contacts to the pins providing the conduction path to the
electrodes in the electrochemical pumps are all triggered in this
way. The closure of the door 34-8004 is also used to turnoff the
interlock for various safety systems within the instrument. The
interlock prevents, for instance, the laser being active with the
door or any other opening in the instrument's casing being open. a
similar principle applies to the power supplies within the
instrument.
[0573] As with other embodiments, it is important to provide
effective and accurate contact between the cartridge and the
instrument interface. In FIGS. 35a, b and c the provision of the
contact is illustrated.
[0574] FIG. 35a shows the carrier 35-03 in position in the slot
35-47. In the insertion position, as shown, the arrangement
provides for a gap 35-51 between the face 35-53 of the cartridge
35-01 which opposes the face 35-55 of the printed circuit board
35-57 of the instrument.
[0575] In the next step, FIG. 35b, the cartridge 35-01 is moved
into the use position. A platen 35-59 is moved, direction of
arrows, by an actuator, not shown. This causes the cartridge 35-01
to be brought into full contact with the PCB 35-57. The movement is
such that the conical spring 35-29 is further compressed. During
this movement, a series of rods which extend through the PCB 35-37
enter various holes (27-13 in FIG. 27) and so ensure that the
alignment between the cartridge and the PCB is correct in that
orientation too.
[0576] When the use of the cartridge 35-01 has finished, then the
force applied to the platen 35-59 by the actuator is released. As a
result, the carrier 35-03 is returned to the insertion position by
return springs, not shown. The release causes the conical springs
35-29 to pull the cartridge 35-01 back into position inside the
carrier 35-03, FIG. 35c. The carrier 35-03 can then be removed by
lifting it out of the slot 35-47, taking with it the cartridge
35-01.
[0577] The face to face contact between the cartridge and the PCB
provides the majority of the interactions between the cartridge and
the instrument, for instance, heating for valve control, sensor
etc. The contact between the PCR chamber and its temperature
cyclers are provided through further components, however; see FIGS.
36a, b, c and d.
[0578] In FIG. 36a, the cartridge 36-01 is shown inserted into the
slot provided in the instrument. Once inserted, the section of the
cartridge 36-01 bearing the PCR chamber is positioned between a
pair of calipers 36-100. The PCB is cut away at this location so as
to not be in the way of the Peltier effect devices 36-102, 36-108
and pair of calipers 36-100. The calipers 36-100 are floating such
that they do no interfere with the contact sought between the
cartridge 36-01 and the PCB during the movement from the insertion
position to the use position.
[0579] The front caliper 36-100a is provided with a Peltier effect
device 36-102 mounted on a support 36-104 which is capable of
reciprocating movement, arrow C, under the control of actuator
36-106. The actuator 36-106 is also mounted on the pair of calipers
36-100.
[0580] The back caliper 36-100b is provided with a second Peltier
effect device 36-108 mounted fixedly on the caliper 36-100b. The
second Peltier effect device 36-108 is provided in opposition to
the Peltier effect device 36-102.
[0581] In the open position shown in FIG. 36c, such as is provided
with the cartridge in the insertion position, the distance between
the opposing faces 36-110, 36-112 of the Peltier effect device
36-102 and the second Peltier effect device 36-108 is more than the
thickness of that section of the cartridge 36-01 and more than the
thickness of the carrier 36-03 which passes between the pair of
calipers 36-100 during insertion of the carrier 36-03.
[0582] In the closed position shown in FIG. 36d, such as is
provided during the amplification step, the distance is reduced.
This is achieved by the actuator 36-106 moving the Peltier effect
device 36-102 on the front caliper 36-100a towards the cartridge
36-01 and towards the opposing second Peltier effect device
36-100b. This actuation, combined with the floating nature of the
pair of calipers 36-100 brings both of the Peltier effect devices
into firm contact with the cartridge 36-01 on opposing sides
thereof. They are now in position to provide the necessary heating
and/or cooling for the PCR step.
[0583] Thermocouples to sense the temperatures applied, and
potentially to be used to control the temperatures applied, are
provided in close proximity with the Peltier effect devices,
embedded in copper shims, bonded to the Peltier effect devices.
[0584] Before the carrier 36-03 is removed, the actuator 36-106
returns the Peltier effect devices 36-100 to the open position.
[0585] In addition to the carrier allowing for relative movement of
the cartridge to ensure correct positioning with respect to the
PCB, the carrier also allows for totally independent relative
movement of the CE chip. This is importing in ensuring correct
positioning of the CE chip for the CE step. This is achieved by the
structure and operation shown in FIGS. 37a and b.
[0586] As the carrier 37-03 with the CE chip 37-31 in it is
inserted into the slot in the instrument, the second support 37-07
approaches the work surface 37-49. The work surface 37-49 carries a
CE chip board heater 37-100 in the form of a planar surface. this
is surrounded by a raised surface 37-102 which provides a nest for
the CE chip 37-31 once positioned.
[0587] Projecting pins 37-104 on the work surface 37-49 enter
apertures 37-106 provided in the second support 37-07 of the
carrier 37-03; FIG. 37a. In FIG. 37b, the top part of the second
support 37-07 is shown cut away so that the full extent of the CE
chip 37-31 can be seen. The apertures 37-106 in the second support
37-07 align with the slot 37-108 which receives the end portions
37-108, 37-110 of the CE chip 37-31. As a result, the end portions
37-108, 37-110 are also provided with through apertures 37-112a,
37-112b. The projecting pins 37-104 thus pass through these
apertures 36-112a, 36-112b too as the carrier 37-03 approaches the
work surface 37-49.
[0588] The conical ends of the pins 37-104 mean that they enter the
apertures 37-106, 37-112a, b, even where there is potential
misalignment. The fuller diameter parts of the pins 37-104
encourage the CE chip 37-31 into the correct position. The CE chip
37-31 is centred to the CE chip board heater 37-100 as a result.
The CE chip heater board 37-100 and raised surface 37-102 can be
seen clearly in FIG. 38.
Electrophoresis Components
1) Optics
[0589] In the electrophoresis step 206, at the detection location
628, light from a laser 800 is focussed to be incident upon the
fluorescent dye associated with a DNA element to make it
detectable.
[0590] A different dye is used for each different DNA element type;
a type is generally associated with a given locus.
[0591] To get good sensitivity, it is important for the incident
light to be of sufficient intensity for the detectors to receive
sufficient light to be sensitive to the emitted fluorescent light,
but for the intensity not to be so high as to give rise to
photobleaching of the dyes. To provide for this, the following
arrangement is used; FIG. 14.
[0592] The light source is a compact laser 900 which is mounted on
a heat sink 902. The laser 900 is a Cobolt Calypso laser (from
Cobolt AB, Kraftriken 8, SE-104 05, Stockholm, Sweden) and emits at
491 nm with a maximum power of 50 mW. The light emitted by the
laser 900 is fed to a fibre coupler 904 (09 LFC 001, f=3.5 mm from
Melles Griot, 2051 Palomar Airport Road, 200, Carlsbad, Calif.
92011, USA) and hence into an patch cable assembly (M31L01, from
Thorlabs, 435 Route 206 North, Newton, N.J., 07860, USA) and
optical fibre 906 (GIF625, dia 62.5:m, NA=0.275 from Thorlabs, 435
Route 206 North, Newton, N.J., 07860, USA).
[0593] The use of the optical fibre 906 is beneficial as it safely
controls the laser light direction, enables the laser light to be
easily conveyed to the position of use and enables mechanical
stability to be provided within the overall system. At the end of
the optical fibre 906 a power of up to 45.32 mW is still
observed.
[0594] The laser light then passes through a collimator 908
(F230FC-A, F=4.5 mm, NA=0.55, from Thorlabs) and a logpass filter
with a sharp cut-off wavelength, EM filter (Omega Optical XF3093,
T50=515 nm) before reaching the spot mirror 910.
[0595] The spot mirror 910 is used to both direct the laser light
to the detection location 628 of the capillary and to transmit,
anisotropically and without filtering, the fluorescent light
received there from to the detector unit. It is angled at
45.degree. to the beam of laser light. To do this, the reflector
910 consists of a 25 mm round glass disc which transmits all light
from <80 above 380 nm. An ellipse, 2 mm long by 1 mm wide, is
provided at the centre of the reflector 910 (so as to present an
effective 1 mm circular mirror), formed of a highly reflective
mirror layer deposited there (reflectivity of 99.99%).
[0596] Before reaching the detection location 628, the laser light
passes through a focussing lens 912. This can be a microscope optic
or other such adjustable focussing lens. Such optics are useful as
they introduce no optical aberrations to the light, shape the beam
for application to the detection location 628 and don't give any
selective loss of light colours. The power reaching the detection
location 628 is over 27.40 mW.
[0597] The fluorescent light is effectively scattered from the dye
in the capillary 616 in all directions. For the fluorescence light
to reach the detector unit, that light needs to hit the spot mirror
910 at a location outside of the glass spot. If it does so, the
light is transmitted into the detector unit 914.
[0598] The detector unit 914 includes a slit in front of a
spectrometer to obtain diffraction-limited incident light, the
spectrometer provided with a diffraction grating and a lens 918
(LA1608A plano convex, f=50 mm, D=25 mm, with anti-reflective
coating within 350-650 nm, made of BK7 glass, Thorlabs Inc), to
direct the light to the charge coupled device 916. The CCD 916 has
spectroscopic abilities.
[0599] The CCD 916 generates the signals which are then used to
generate the electropherogram, an example of which is shown in FIG.
15
[0600] Using such an approach, a sensitivity approaching that of
laboratory style electrophoresis instruments can be reached. The
instrument is able to detect down to the presence of 2.5 .mu.M of
fluorescein dye at pH 7.
[0601] In an alternative approach, certain problems with the
stability of the fibre optics can be avoided by providing an open
beam approach to delivering the light from the laser to the
channel.
[0602] An alternative embodiment of the optics is shown in the cut
away perspective view of FIG. 39. The instrument casing 39-01
provides various mounts for the optics. The light is generated by
the laser head 39-03 operated under control by the laser controller
39-05. The light enters the optics 39-07 and is directed at the
channel in the CE chip, not shown, mounted in the CE chip heater
board 39-09.
[0603] The return light enters the optics 39-07 and is directed
back to the spectrometer 39-11 and CCD camera 39-13. Above the CE
chip heater board 39-09 is the chip alignment structure 39-15 which
is described further below.
2) Calibration and Verification for Optics
[0604] When first using the optics for detecting the
electrophoresis results, and periodically thereafter, it is
beneficial to ensure that the optics are properly calibrated to the
capillary 616 at the detection location 628 in the electrophoresis
cartridge section. This ensures best transmission of the excitation
light into the detection location 628, best recovery of the
fluorescence light from the dyes encountered at the detection
location 628 and the performance of the detection at the detection
location 628 (and hence at the correct distance from the point at
which the sample is injected).
[0605] To achieve these aims, the electrophoresis cartridge section
is provided with various aids. These are intended to allow
automated verification and calibration of the position by the
instrument 11.
[0606] Firstly, a fixed marker is provided on the electrophoresis
cartridge section, a known distance along the capillary 616 and a
known distance perpendicular to the capillary 616, from the
detection location 628. When the laser light is incident upon the
fixed marker, a response is detected by the CCD 916. The position
of the incident laser light is thus known. The incident position of
the laser light along the capillary is thus correct. The known
distance of the fixed marker from the detection location 628,
perpendicular to the capillary 616 can then be used to adjust the
position at which the laser light is incident so as to correspond
with the detection location 628. X and Y axis verification of the
incident laser light position corresponding with the detection
location 628 is thus provided. The marker could be a physical mark
(for instance etched) on the cartridge and/or a coloured mark (for
instance a dye) and/or a quantum dot.
[0607] To provide for the verification on the Z axis, the working
distance between the lens and the capillary 616, a known source,
with a known characteristic is provided on the electrophoresis
cartridge section at a known Z axis distance relative to the
correct Z axis distance of the capillary 616. By adjusting the
focus of the lens so as to maximise the response by the CCD 916,
the correct working distance for the known source is established.
An adjustment can then be made to reflect the relative working
distance for the known source relative to the capillary 616.
Ideally, these are in the same plane at the same working distance
so as to allow the known to provide direct verification for the Z
axis position relative to the capillary 616.
[0608] As an alternative means of verification on the position, it
is possible to use the marker for the X axis and then use variation
in transmission to check the Y axis position. Thus a marker is used
to determine the correction position along the axis of the
capillary 616. The adjustment can then scan in the Y axial
direction are use the CCD (or another detector) to consider the
variation with position. The reflected signal will be constant at a
level when the laser light is incident on the cartridge away from
the capillary. When incident light traverses the capillary 616,
then the signal will vary in a predictable manner, so allowing the
position to be set subsequently at the position corresponding to
the middle of the capillary 616 in the signal. To assist in this,
it is possible to introduce a polariser insert for the calibration
part of the process so as to increase the observed variation in the
signal. The polariser is removed before the actual electrophoresis
results collection starts. The effect whose variation is detected
can arise from the capillary 616 itself, a marker at a known
distance from the capillary 616 or a material present in the
capillary 616 (for instance, a dye labelled component provided as
part of a sizing standard, whose mobility is higher than the other
elements of the size standard or unknown elements).
[0609] The FIG. 39 and FIGS. 40a, b and c embodiment shows the
alignment structure 39-15 and its operation.
[0610] The alignment structure 39-15 is in the form of a swing arm
40-100 which can be pivoted relative to the casing 40-102 under the
power of an actuator contained within the swing arm 40-100. The
other end of the swing arm 40-100 is provided with a camera
40-104.
[0611] In the stowed position, FIG. 40b, the swing arm is
positioned in contact with a hard stop 40-106 mounted on the casing
40-102 too. In the check position, FIG. 40c, the actuator has
caused the swing arm 40-100 to swing away from the casing 40-102
and so position the camera 40-106 over the channel 40-108 in the CE
chip 40-31.
[0612] In the use position, triggered by the operator, a laser is
activated and this creates a diffraction pattern which can be seen
on the camera display. The adjustment for the CE chip position is
used to move the CE chip until the diffraction pattern indicates
that the middle of the channel has been located. The alignment of
the channel with the optics used in the analysis is thus provided.
The camera can also be used to achieve focussing of the system in
the Z axis adjustment.
3) Electrophoresis Environment Control
[0613] For the necessary resolution to be obtained in the
electrophoresis step 206, the temperature of the capillary 616 and
its contents need to be carefully controlled at the optimum
temperature. In the present embodiment, the electrophoresis
cartridge section is in contact with a thermally conductive block,
with a series of resistance heaters provided on the opposing side
of the block. These are provided with controllers and are capable
of maintaining the temperature of the electrophoresis cartridge
section at the optimum temperature +/-0.3.degree. C.
[0614] In addition, the cavity that the electrophoresis cartridge
section is provided in is thermostatically controlled at the
optimum temperature. This reduces still further temperature
variation before, during and after use.
[0615] The use of a CE chip heating bed, and raised surface around
it, is beneficial in controlling the temperature within the CE
chip. The nest so formed ensures consistent positioning and good
contact.
4) Use of LED's as Light Source
[0616] FIG. 16 depicts a schematic of an example of a system for
detecting fluorescence. The system includes light emitting diodes
(LEDs), e.g., high power cyan LEDs, to provide excitation
wavelength light to detect dyes combined with biological samples.
The system also includes a bifurcated optical fibre assembly made,
e.g., from high transmission fused-silica cores with high numerical
apertures (NAs), e.g., NA=0.22. The LED excitation system described
herein can be applied for DNA detection in capillary
electrophoresis systems in mobile analytical units. The compactness
and light weight of the LED system enables automating assays for
nucleic acid studies. Using the compact and light weight system
allows creating bench-top analysis systems that can be used both in
the laboratory and in the field.
[0617] In some implementations, two LEDs are assembled in parallel
and supplied with a stabilized DC voltage of 3.6 V. The current
passing through the LED assembly is 1.8 A. The junction is
maintained at 15.+-.1.degree. C. by a
Proportional-Integrative-Derivative (PID) control loop (Model
TE-36-25 from T.E. Technology, Inc.) acting on two 13.times.13 mm
thermoelectric modules. To save power, and space, two Peltiers
modules are controlled in parallel and the thermocouple sensor is
placed on only one of them assuming that, by construction symmetry,
they both behave similarly. An aluminum heat sink and a fan (12 V
DC) complete the cooling module. This module extends the lifetime
of the LEDs by two orders of magnitude. Without cooling the
junction, the supplied current is 2.7 A.
[0618] The first step of collimation is the use of an
acrylic-molded lens from Lumiled, which collimates the emitted
light to a 15.degree. cone half-angle (NA.about.n
sin(2.sub.1/2).about.0.26). The light is then focused onto a
plano-convex lens (f=35 mm, D=25 mm; NA.about.D/2f.about.0.36).
NA.sub.LED<NA.sub.lens, or the numerical apertures are matched.
The distance between the apex of the lens and the plane of the
collimator, L.sub.max, is adjusted by a micrometer screw to
maximize the power read by a calibrated silicon photodiode sensor.
The value obtained (25 mm) is only close to the focal length f
since the collimated LED is not a point source. The light beam is
then refocused onto a collimation package assembled around an
aspheric lens (f=10 mm, D=5 mm; NA.about.D/2f.about.0.25, Ocean
Optics Ltd) within an anodized aluminum lens tube of length l=30
mm. Each LED is thus coupled into one arm of a 2 m-long bifurcated
silica core (O=600 .mu.m, NA=0.22) optical fibre assembly
(attenuation: 0.013 dB/m at 505 nm--relative transmission: 82% (arm
1) and 87% (arm 2)).
[0619] Table 1 illustrates a power optimization of the system
depicted in FIG. 16. The power at 505 nm, P505, is read by the
silicon photodiode while the distance between the LED collimator
and the lens surface (L.sub.max), the lens geometry, and the lens
tube length (l) are changed. Only one arm of the bifurcated fibre
is used.
TABLE-US-00002 TABLE 1 Lens I Lmax Psos Hemispherical 3 cm 20 mm
225.2 .mu.W Hemispherical 5 cm 18 mm 200.4 .mu.W Hemispherical 8 cm
19 mm 222.8 .mu.W Cylindrical 3 cm 9 mm 170.9 .mu.W Cylindrical 5
cm 9 mm 164.1 .mu.W Plano-convex 3 cm 16 mm 220.9 .mu.W
Plano-convex 5 cm 15 mm 204.1 .mu.W Plano-convex 8 cm 15 mm 173.7
.mu.W None No'ne 12 mm 187.4 .mu.W
[0620] For the bias values described above, when both arms of the
fibre are used, the power at 505 nm read by the photodiode is 820
.mu.W.
[0621] FIG. 17 is a plot of LED spectrum, light reflected, and
residual LED light over a range of wavelengths (nm). FIG. 17
illustrates an LED spectrum obtained in the cooled CCD (diodes:
Ug=2.0 V; I=0.3 A; T=15.degree. C.), calculated light reflected by
the dichroic mirror, and residual LED light after the emitter. The
insert shows the transmission curves of the dichroic and emitter.
The plot indicates that there is a loss of power when the incident
light is reflected onto the sample. Additionally, light is
red-shifted by 20 nm, which causes some of the LED light to
interfere with the carboxyfluorescein dyes. The choice of available
emitters and dichroic mirrors is limited by the dyes chosen to
label the migrating DNA strands.
[0622] FIG. 18 is a plot of power of the LED-module over time.
During a CE experiment, it is crucial to reduce the fluctuations of
the power of the light source within less than 1%. FIG. 18 shows an
example of the power recorded by the silicon photodiode (Probe
S130A, Thorlabs) using the internal calibration function to record
the power emitted by the fiber-LED assembly at 505 nm over time.
The diodes are supplied with a 3.4 V DC voltage corresponding to a
current of 1.4 A while the junction is maintained at
15.+-.1.degree. C. The room is maintained at a temperature of
22.degree. C. (R.H.=24%). The plot illustrates a temporal power
evolution of the LED-module. The lines mark regimes where the power
drops, e.g., by 4.8 nW/s, 11.6 nW/s, and 5.0 nW/s. Overall, the
power drops by about 1.95 .mu.W over 5 min, i.e. 0.48%.
[0623] FIG. 19 is an illustration showing beam shape and size after
the sample objective as measured by the laser camera. The asymmetry
observed is due to imperfections occurring when the two fibre arms
are fused because of the large core diameter of the fibre,
mismatches between the LED-to-LED and the fiber-to-fibre distances,
and tilt in the optical elements. In the results reported in the
next section, the situation corresponding to the single-spot will
be used. One method includes adjusting all the optics to obtain the
maximum power at the merged end of the bifurcated fibre. This can
yield a misshapen light beam as the core size of each arm is large
(multimode fibre). To characterize the beam shape and size after
the microscope objective, i.e. at the entrance of the microchip, a
Coherent Lasercam II 1/2 camera was placed on an {x,y,z}
translation stage equipped with micrometer precision positioners
and equipped with a Leica HCX PL FLUOTAR (40X, NA=0.75, WD=0.40 mm)
and adjustable filters. The objective was brought within .about.8
mm of the Olympus LUCPLFLN (20X, NA=0.45, WD=6.6-7.8 mm) mounted on
the CE setup. This allowed directly imaging the beam coming out of
the fiber-LED assembly via the CE setup. The micrometer positioners
allowed measuring the dimension of the beam with a precision of 10
.mu.m by moving the camera from one spot of the obtained beam
profile image to another and reporting the traveled distance. The
power can be maximized by adjusting each optical collimation
element (P=1.6 mW at 505 nm) (A) or the collimation elements can be
adjusted to give one single spot (P=1.0 mW at 505 nm) (B).
[0624] The system was employed for both static and dynamic
fluorescence measurements. For the static fluorescence
measurements, a 1 .mu.M fluorescein, 6-FAM or rhodamine B solution
is loaded into the microchannel by using a standard laboratory
vacuum line (13 PSI (0.88 atm) depression) to pull the solution
through the channel via 2-mm-diameter access holes. The glass
microchannel is anisotropically etched with fluorhydric acid (HF)
in Schott Borofloat.RTM. low-fluorescence glass (CE chip X8050,
Micronit, B.V., The Netherlands). It is semi-elliptic with a width
of 50 .mu.m, a depth of 20 .mu.m and a length of 85 mm. The plastic
microchannels are hot-embossed into a 1.1-mm-thick cyclic olefin
copolymer (COC) sheet at .about.160.degree. C. from a reactive-ion
etched Si(100) master. The channel section is tapered with a
25.degree. taper angle and has a width of 60 .mu.m (top) and 39
.mu.m (bottom), a depth of 20.mu. and a length of 85 mm. Glass
capillaries that are 1-cm-long (inner diameter 4 mm) borosilicate
are epoxy-glued onto the access holes to act as reservoirs (or
wells). All solutions are filtered with a nylon membrane (pore
diameter: 0.2-.mu.m) to remove small particles that will clog the
channel.
[0625] The loaded chip is placed on the CE setup and the focus of
the 63X sample objective is aligned with the bottom of the channel.
The emitted fluorescent light is gathered onto the 26.6
mm.times.6.7 mm (1024.times.255 pixels) array of the
thermoelectrically cooled Andor CCD. The processed signal is
vertically binned from the software-restricted central rows
irradiated by the light focused onto the spectrometer entrance
slit. The CCD is cooled down to -50.degree. C. to reduce the binned
dark counts to 270 while the exposure time is 0.05 s.
[0626] FIGS. 21A and 21B are plots of CCD signal v/s wavelengths.
The plots indicate the vertically-binned signal from a 1 .mu.M
6-FAM solution loaded into a glass microchannel (A) and a 1 .mu.M
fluorescein solution loaded into a plastic COC channel (B). The
counts from the same microchannel filled with water are subtracted
to take into account the autofluorescence of the glass or plastic
microdevice. The power emitted from the system is 0.98 mW and 1.03
mW at 505 nm for glass and COC, respectively. This is obtained by
supplying the two LEDs (placed in series) with a constant current
of 0.74 A, which corresponds to a voltage of 7.0 V. Due to the
choice of filters (emitter cut-on: T50 at 535 nm), only the tail of
the fluorophore emission is observed (fluorescein:
8..sup.em.sub.max=513 at pH=13, 6-FAM: 8.sup.em.sub.max=517 at
pH=9. The signal-to-noise ratio is 87 for 1 .mu.M 6-FAM in glass
and 36 for 1 .mu.M fluorescein in COC. The SNR is lower in glass
because 6-FAM is known to photobleach faster than fluorescein. The
detection limit parameters for glass and plastic CE microdevices
are summarized in Table 2.
TABLE-US-00003 TABLE 2 Device Power at Maximum signal-to- material
Fluorophore 505 nm counts noise ratio Glass 1 uM 6-FAM 0.98 mW 720
36 COC 1 uM fluorescein 1.03 mW 1750 87
For dynamic fluorescence measurements, glass microchannels are
loaded with reagents similar to the reagents for the static
measurement testing, but a first sequence of reagents are flushed
through the microdevice to reduce the effect of the electroosmotic
flow (EOF) that opposes the electrophoretic flow and results in
peak distortion from a Gaussian shape and therefore loss of
resolution. EOF arises from the re-equilibration of the electrical
double layer arising from the surface charge of the microchannel
walls after the perturbation caused by the migrating charges under
the electric field. The EOF can be efficiently controlled by using
a coating polymer matrix such-as poly-N-hydroxyethylacrylamide
(pHEA) dissolved in water at 0.1% w/v.
[0627] The DNA fragments are separated by electrophoretically
migrating within a sieving polymer matrix such as POP-5.TM.
(Applied Biosystems, Inc.), a mixture of polyacrylamides in an
appropriate buffer, according to their size and interactions with
the polymer network. After the pHEA coating has been applied, IX
A.C.E..TM. buffer (Amresco, Inc.) is flushed into the channel by
vacuum followed by POP-5.TM.. A 1 .mu.M solution of a poly-adenine
oligonucleotide labeled with 6-FAM is placed in the sample well and
will be electrokinetically injected in the separation channel via a
cross-injection geometry. 1X A.C.E..TM. buffer is placed in the
sample waste, buffer waste, and waste wells to ensure ionic
conductivity in the whole device.
[0628] FIG. 21 is a plot of CCD signal v/s time for dynamic
fluorescence measurements. The plot indicates fully binned CCD
signal showing the peak corresponding to the elution of the 1 .mu.M
oligonucleotide (elution time, t.sub.el=77 s) detected by the
optical module. The nature of the peak is confirmed by the spectrum
obtained in the CCD at t=77 s. It is similar to the peak shown in
FIG. 20a. The signal-to-noise ratio of 10 can be improved by
uniformly heating the chip to 50.degree. C. The plot shows the
result of the migration of the oligonucleotide while the LED-fibre
assembly delivers about 980 .mu.W at 505 nm. The two LEDs, placed
in parallel, are supplied with 3.9 V (I=1.9 A) while the junction
is kept at 15.degree. C. The migration field in the separation
channel is 110 V/cm.
[0629] In this manner, an optical excitation module capable of
visualizing a 1 .mu.M oligonucleotide migrating in a glass
microchannel loaded with a sieving matrix is assembled and tested.
The output fibre beam size and divergence, the power distribution
in the beam exiting the fibre assembly as well as the output power
stability over time approach the specifications of existing LIF
setups. A modified epifluorescence microscope arrangement is used
in conjunction with a lightweight compact fixed spectrograph built
around ion-etched grating and aligned with a cooled Charge-Coupled
Device (CCD) camera for added sensitivity. Fluorescent dyes such as
fluorescein, 6-carboxyfluorescein (6-FAM) and rhodamine B can be
detected in conventional plastic (cyclic olefin copolymer) and
glass microchannels at submicromolar levels. A migrating
single-stranded oligonucleotide DNA fragment (10-mer) labeled with
6-FAM can also be detected with high signal-to-noise ratio when
electrophoretically migrated in the microchannels at 100 V/cm. LEDs
operated in conjunction with Peltier elements controlled by a
Proportional Integrative Derivative (PID) module can be used to
replace bulky, expensive and power-consuming Argon ion lasers
conventionally used in Laser Induced Fluorescence (LIF) Capillary
Electrophoresis (CE) experiments. The LEDs in the system can be
HP803-CN obtained from Roithner LaserTechnik GmBH or Luxeon Star
series from Philips Lumiled Lighting Company that offer LEDs
emitting at 505.+-.15 nm with a full-width at half maximum of 20
nm. The LEDs are available with a Lambertian profile with a
half-cone angle of 75.degree., which is not suited for microchip
applications. However, these are high power LEDs with a nominal
radiometric output power of 45 or 80 mW. When properly collimated,
the available power becomes relevant to applications of DNA
detection by CE.
[0630] While this specification contains many specifics, these
should not be construed as limitations on the scope of the
disclosure or of what may be claimed, but rather as descriptions of
features specific to particular implementations of the disclosure.
Certain features that are described in this specification in the
context of separate implementations can also be implemented in
combination in a single implementation. Conversely, various
features that are described in the context of a single
implementation can also be implemented in multiple implementations
separately or in any suitable subcombination. Moreover, although
features may be described above as acting in certain combinations
and even initially claimed as such, one or more features from a
claimed combination can in some cases be excised from the
combination, and the claimed combination may be directed to a
subcombination or variation of a subcombination.
[0631] Similarly, while operations are depicted in the drawings in
a particular order, this should not be understood as requiring that
such operations be performed in the particular order shown or in
sequential order, or that all illustrated operations be performed,
to achieve desirable results. In certain circumstances,
multitasking and parallel processing may be advantageous. Moreover,
the separation of various system components in the implementations
described above should not be understood as requiring such
separation in all implementations, and it should be understood that
the described program components and systems can generally be
integrated together in a single software product or packaged into
multiple software products.
[0632] Thus, particular implementations of the disclosure have been
described. Other implementations are within the scope of the
following claims. For example, the actions recited in the claims
can be performed in a different order and still achieve desirable
results. In some implementations, the sharpness of the cut-on edge
of the dichroic mirror can be improved and the lower wavelength
T.sub.50 can be shifted to a lower wavelength to improve the
signal-to-noise ratio. In some implementations, the diodes can be
operated in a pulsed AC mode where the "on" time is synchronized
with the frame acquisition of the CCD camera, thereby also
extending the lifetime of the LEDs. In some implementation, a
customized LED array can be used that does not have the mold that
yields divergent light. In some implementations, the collimation
parts can be embedded in a rigid casing made, e.g., from black
anodized aluminum.
[0633] In some implementations, the LED-based detection system
described in this disclosure can be used as the microfluidic
electrophoresis system that is described in the attachment, which
is enclosed as part of the present disclosure.
5) Size Standards
[0634] The size standards used in the invention are beneficially
stored within the formamide pump liquid.
[0635] The size standards may be provided according to the form
detailed in International Patent Application no PCT/GB2009/002186,
the contents of which are incorporated herein by reference,
particularly with respect to the provision of and use of size
standards which operate within a single CE channel, together with
the sample being considered.
Instrument Performance
[0636] The result of the above embodiment is the provision of an
instrument, cartridge and operating method which provides quick,
reliable sample analysis, whilst doing so at a wide variety of
locations and when operated by a wide variety of people.
[0637] By way of abilities are performance, the invention provides
a fully integrated instrument capable of performing extraction,
PCR, electrophoresis and analysis, whilst requiring minimal
training and/or intervention by the user. In its optimum form, a
fully automated system from start to finish is provided, the user
simply needing to load the cartridge into the instrument and start
it.
[0638] The modular nature of the instrument allows for upgrading of
one or more modules without impact on the other modules. The data
output format has been carefully selected to allow the analysis of
the data outputted by a variety of existing analysis software
applications, such is I.sup.3 of Forensic Science Service Limited,
and future software applications.
[0639] The end result of the analysis may be a profile for the
sample and/or an indication of a match between the sample and a
database recorded sample and/or other interpretation based
data.
[0640] The use of a single cartridge type to handle a wide variety
of sample from a wide variety of sources is beneficial. The
methodology is able to handle samples originating from buccal
swabs, cotton and other soft swabs, aqueous samples, clothing
samples, cigarette butts, chewing gum and the like.
[0641] The methodology is also able to separate the useful DNA from
residual cellular material, PCR inhibitors (such as ethanol, indigo
etc) and chemical inhibitors.
[0642] The instrument is fully portable and so can be used in a
wide variety of locations. The fully sealed and protected nature of
the cartridge means that contamination is not a risk, even where
the instrument is used outside of laboratory standard conditions.
The instrument operates off a standard mains power supply,
110-240V, 50 Hz, using a conventional electric plug.
[0643] With respect to the overall time, from the sample receiving
step 202, to the transmission away from the instrument in the data
communication step 210, the embodiment described provides this
process in a time period of 141 minutes. That time period can be
reduced, including by the options and variables set out in the
following paragraphs.
[0644] With respect to the sample receiving step 2002, the
embodiment described provides this step in a time period of 2
minutes. Time periods of between 20 seconds and 5 minutes are
easily achievable, depending upon the loading methodology used and
the number of reagents or samples that need to be loaded.
[0645] With respect to the sample preparation step 202, the
embodiment described provides this step in a time period of 24
minutes. That time period can be reduced by shortening the
residence in one or more of the chambers, for instance the
incubation chamber 358, and/or by reducing the time separation
between a valve being activated and reliance on the outcome of the
activation and/or by reducing the washing and/or elution volumes
used. Time periods of between 15 to 30 minutes are easily
achievable.
[0646] With respect to the sample amplification step 204, the
embodiment described provides this step in a time period of 80
minutes. That time period can be reduced by shortening the number
of cycles used, the duration of one or more parts of a cycle and
the time period after introduction to the chamber and before PCR
starts and/or after PCR finishes and before the sample is removed
to the next stage. Again, the time separation between a valve being
activated and reliance on the outcome of the activation is of
significance. Time periods of between 60 to 120 minutes are easily
achievable.
[0647] With respect to the electrophoresis step 206, the embodiment
described provides this step in a time period of 15 minutes. That
time period can be reduced by the use of higher voltages and/or
faster migration media in the capillary and/or reductions in the
sample introduction time. Time periods of between 1 to 60 minutes
are easily achievable. This functionality is achieved in an
instrument weighing less than 10 kg and occupying a footprint of
less than 0.1 m.sup.2.
Instrument Fields of Use
[0648] The structures and method discussed above are useful in the
consideration of a wide variety of samples, over and above forensic
samples. For instance, they can be used: the consideration of
marker targets, diagnostic assays, disease markers, biobanking
applications, STR based targets in transplants, identification of
drug resistant microorganisms, blood testing, mutation detection,
DNA sequencing and the like. Food analysis, pharmogenetics and
pharmogenomics are also areas of use. A wide variety of uses in the
medical and/or biotech field can make use of the invention.
[0649] The invention is also applicable in situations where
familial relationships need to be determined from DNA, for instance
paternity testing. Pedigree testing in animals is a further
example.
[0650] The use of the invention in border control, security,
customs situations and other governmental type uses is
beneficial.
* * * * *