U.S. patent application number 16/958520 was filed with the patent office on 2021-02-25 for improved cartridge for use in in-vitro diagnostics and method of use thereof.
This patent application is currently assigned to ADOR DIAGNOSTICS S.R.L.. The applicant listed for this patent is ADOR DIAGNOSTICS S.R.L.. Invention is credited to Dalibor HODKO, Vladimir HURGIN, Asaf LIPSHITZ, Mario SCHMITT, Amit SCHNELL, Paul D. SWANSON, Ari TADMOR, Lutz WEBER, Eran ZAHAVI.
Application Number | 20210053051 16/958520 |
Document ID | / |
Family ID | 1000005247700 |
Filed Date | 2021-02-25 |
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United States Patent
Application |
20210053051 |
Kind Code |
A1 |
HODKO; Dalibor ; et
al. |
February 25, 2021 |
IMPROVED CARTRIDGE FOR USE IN IN-VITRO DIAGNOSTICS AND METHOD OF
USE THEREOF
Abstract
A cartridge for use in in-vitro diagnostics, the cartridge
including a cartridge housing, a cartridge element, disposed within
the cartridge housing and defining a plurality of operational
volumes, at least some of the plurality of operational volumes
being mutually linearly aligned, a fluid solution transporter
operative to transfer fluid solutions from at least one of the
plurality of operational volumes to at least another of the
plurality of operational volumes, the fluid solution transporter
including a linearly displaceable transport element operative to
sequentially communicate with interiors of the at least some of the
plurality of operational volumes and a venter, including a linearly
displaceable venting element, operative in coordination with the
fluid solution transporter to vent at least one of the plurality of
operational volumes.
Inventors: |
HODKO; Dalibor; (San Diego,
CA) ; SWANSON; Paul D.; (Santee, CA) ; HURGIN;
Vladimir; (Gan Yavne, IL) ; SCHMITT; Mario;
(Karlsruhe, DE) ; WEBER; Lutz; (Muenchweiler,
DE) ; LIPSHITZ; Asaf; (Modiin, IL) ; ZAHAVI;
Eran; (Rehovot, IL) ; TADMOR; Ari; (Herzlia,
IL) ; SCHNELL; Amit; (Kiryat Tivon, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ADOR DIAGNOSTICS S.R.L. |
Rome |
|
IT |
|
|
Assignee: |
ADOR DIAGNOSTICS S.R.L.
Rome
IT
|
Family ID: |
1000005247700 |
Appl. No.: |
16/958520 |
Filed: |
July 4, 2018 |
PCT Filed: |
July 4, 2018 |
PCT NO: |
PCT/IL18/50726 |
371 Date: |
June 26, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2200/0652 20130101;
B01L 2300/0672 20130101; B01L 2300/044 20130101; B01L 2300/043
20130101; C12N 15/1013 20130101; B01L 3/50273 20130101; B01L
3/502753 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; C12N 15/10 20060101 C12N015/10 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2017 |
IL |
PCT/IL2017/051398 |
Claims
1. (canceled)
2. A cartridge for use in in-vitro diagnostics, the cartridge
comprising: a cartridge housing; a cartridge element disposed
within said cartridge housing and defining a plurality of
operational volumes; a fluid solution transporter operative to
transfer fluid solutions from at least one of said plurality of
operational volumes to at least another of said plurality of
operational volumes; and at least one septum which sealingly
communicates with at least some of said plurality of operational
volumes.
3. A cartridge for use in in-vitro diagnostics according to claim 2
and wherein said at least one septum includes a plurality of
septa.
4. A cartridge for use in in-vitro diagnostics according to claim 2
and wherein said at least one septum is penetrable by a penetrating
element.
5. A cartridge for use in in-vitro diagnostics according to claim 2
and wherein at least one of said plurality of operational volumes
is configured such that the interior thereof may be in magnetic
communication with at least one magnet located exteriorly
thereof.
6. (canceled)
7. A cartridge for use in in-vitro diagnostics according to claim 2
and wherein said fluid solution transporter comprises: a linearly
displaceable transport element operative to sequentially
communicate with interiors of said at least some of said plurality
of operational volumes; and a fluid flow driving assembly
communicating with said linearly displaceable transport
element.
8-9. (canceled)
10. A cartridge for use in in-vitro diagnostics according to claim
2 and wherein said cartridge housing comprises first and second
outer housing portions which are hinged together and at least
partially enclose said cartridge element.
11-12. (canceled)
13. A cartridge for use in in-vitro diagnostics according to claim
2 and wherein said plurality of operational volumes includes a
multiplicity of operational volumes, at least some of which are
configured to allow injection of fluid solutions thereinto.
14. A cartridge for use in in-vitro diagnostics according to claim
2 and also comprising a microfluidic PCR array mounted within said
cartridge housing.
15. A cartridge for use in in-vitro diagnostics according to claim
14 and wherein at least one of said plurality of operational
volumes defines an internal passageway to a port of said
microfluidic PCR array.
16. A cartridge for use in in-vitro diagnostics according to claim
2 and also comprising a sensor array mounted within said cartridge
housing.
17-20. (canceled)
21. A method for use in in-vitro diagnostics, the method
comprising: providing a cartridge having a plurality of operational
volumes, at least some of said plurality of operational volumes
being mutually linearly aligned; transferring fluid solutions from
at least one of said plurality of operational volumes to at least
another of said plurality of operational volumes, said transferring
fluid solutions including linearly displacing a transport element
to sequentially communicate with interiors of said at least some of
said plurality of operational volumes; and venting said at least
one of said plurality of operational volumes.
22. A method for use in in-vitro diagnostics according to claim 21
and wherein said transferring also includes driving said fluid
solutions through said transport element between ones of said
plurality of operational volumes.
23. A method for use in in-vitro diagnostics according to claim 21
and wherein said transferring fluid solutions includes transferring
fluid solutions containing cellular material to a microfluidic PCR
array mounted within said cartridge.
24. A method for use in in-vitro diagnostics according to claim 23
and wherein said transferring fluid solutions also comprises
transferring fluid solutions containing cellular material from said
microfluidic PCR array to a sensor array associated with said
cartridge.
25. A method for use in in-vitro diagnostics according to claim 21
and also comprising injecting material into some of said plurality
of operational volumes prior to supplying cellular material
thereto.
26. A method for use in in-vitro diagnostics according to claim 21
and wherein said transferring fluid solutions comprises: locating a
cell membrane breakdown material in a first operational volume;
locating an open end of a hollow needle into communication with
said first operational volume; drawing at least a portion of said
cell membrane breakdown material into said hollow needle; linearly
displacing said open end of said hollow needle into communication
with a second operational volume having a sample located therein;
and repeatedly drawing said sample and at least some of said cell
membrane breakdown material into said hollow needle and expelling
said sample and said cell membrane breakdown material from said
hollow needle into said second operational volume, thereby mixing
said sample and said cell membrane breakdown material.
27. A method for use in in-vitro diagnostics according to claim 26
and wherein said transferring fluid solutions further comprises:
linearly displacing said open end of said hollow needle into
communication with a third operational volume containing a cell
lysis solution and magnetic beads; drawing at least a portion of
said cell lysis solution and magnetic beads into said hollow needle
into engagement with said sample and said cell membrane breakdown
material; and repeatedly drawing said sample, at least some of said
cell membrane breakdown material, said cell lysis solution and
magnetic beads into said hollow needle and expelling said sample,
said at least some of said cell membrane breakdown material, said
cell lysis solution and said magnetic beads, from said hollow
needle into said third operational volume, thereby releasing
nucleic acids from said sample and binding said nucleic acids to
said magnetic beads.
28. A method for use in in-vitro diagnostics according to claim 27
and wherein said transferring fluid solutions further comprises:
linearly displacing said open end of said hollow needle into
communication with a fourth operational volume containing a wash
buffer; drawing at least a portion of said wash buffer into said
hollow needle into engagement with said magnetic beads together
with said nucleic acids bound thereto; and repeatedly drawing said
wash buffer and said magnetic beads, together with said nucleic
acids bound thereto, into said hollow needle, thereby washing away
cell debris and unbound nucleic acids from said magnetic beads.
29. A method for use in in-vitro diagnostics according to claim 28
and wherein said transferring fluid solutions further comprises:
linearly displacing said open end of said hollow needle into
communication with a fifth operational volume containing an elution
buffer; drawing at least a portion of said elution buffer into said
hollow needle into engagement with said magnetic beads, together
with said nucleic acids bound thereto; and repeatedly drawing said
elution buffer and said magnetic beads, together with said nucleic
acids bound thereto, into said hollow needle, thereby disengaging
said nucleic acids from said magnetic beads.
30. A method for use in in-vitro diagnostics according to claim 29
and wherein said transferring fluid solutions further comprises:
linearly displacing said open end of said hollow needle into
communication with a sixth operational volume having at least one
magnet juxtaposed thereto; transferring said elution buffer and
said magnetic beads, together with said nucleic acids disengaged
therefrom, into said sixth operational volume, said at least one
magnet attracting said magnetic beads; and drawing said elution
buffer, together with said nucleic acids, into said hollow
needle.
31-35. (canceled)
Description
REFERENCE TO RELATED APPLICATIONS
[0001] The following patent applications, the disclosures of which
are hereby incorporated by reference, are believed to be related to
the subject matter of the present application:
[0002] Israel Patent Application No. 249856, filed Dec. 29, 2016
and entitled AN ELECTROPHERETIC CHIP FOR ELECTROPHORETIC
APPLICATIONS, and
[0003] Israel Patent Application No. 249857, filed Dec. 29, 2016
and entitled AN ELECTROPHERETIC CHIP FOR ELECTROPHORETIC
APPLICATIONS.
[0004] The following patent application, the disclosure of which is
hereby incorporated by reference and priority from which is hereby
claimed, is also related to the subject matter of the present
application:
[0005] PCT Patent Application PCT/IL2017/051398, filed Dec. 28,
2017 and entitled CARTRIDGE FOR USE IN IN-VITRO DIAGNOSTICS AND
METHOD OF USE THEREOF.
FIELD OF THE INVENTION
[0006] The present invention relates to in-vitro diagnostics
generally.
BACKGROUND OF THE INVENTION
[0007] Various apparatus and methods for in-vitro diagnostics are
known in the art.
SUMMARY OF THE INVENTION
[0008] The present invention seeks to provide a cartridge and an
improved method for in-vitro diagnostics.
[0009] There is thus provided in accordance with a preferred
embodiment of the present invention a cartridge for use in in-vitro
diagnostics, the cartridge including a cartridge housing, a
cartridge element, disposed within the cartridge housing and
defining a plurality of operational volumes, at least some of the
plurality of operational volumes being mutually linearly aligned, a
fluid solution transporter operative to transfer fluid solutions
from at least one of the plurality of operational volumes to at
least another of the plurality of operational volumes, the fluid
solution transporter including a linearly displaceable transport
element operative to sequentially communicate with interiors of the
at least some of the plurality of operational volumes and a venter,
including a linearly displaceable venting element, operative in
coordination with the fluid solution transporter to vent at least
one of the plurality of operational volumes.
[0010] There is also provided in accordance with another preferred
embodiment of the present invention a cartridge for use in in-vitro
diagnostics, the cartridge including a cartridge housing, a
cartridge element disposed within the cartridge housing and
defining a plurality of operational volumes, a fluid solution
transporter operative to transfer fluid solutions from at least one
of the plurality of operational volumes to at least another of the
plurality of operational volumes and at least one septum which
sealingly communicates with at least some of the plurality of
operational volumes.
[0011] Preferably, the at least one septum includes a plurality of
septa. Additionally or alternatively, the at least one septum is
penetrable by a penetrating element.
[0012] There is further provided in accordance with yet another
preferred embodiment of the present invention a cartridge for use
in in-vitro diagnostics, the cartridge including a cartridge
housing defining a plurality of operational volumes and a fluid
solution transporter operative to transfer fluid solutions from at
least one of the plurality of operational volumes to at least
another of the plurality of operational volumes, at least one of
the plurality of operational volumes being configured such that the
interior thereof may be in magnetic communication with at least one
magnet located exteriorly thereof.
[0013] In accordance with a preferred embodiment of the present
invention the fluid solution transporter includes a linearly
displaceable transport element operative to sequentially
communicate with interiors of the at least some of the plurality of
operational volumes. Additionally or alternatively, the fluid
solution transporter includes a fluid flow driving assembly
communicating with the linearly displaceable transport element.
[0014] Preferably, the linearly displaceable transport element
includes a hollow needle. Additionally or alternatively, the
cartridge for use in in-vitro diagnostics also includes a flexible
tube interconnecting the fluid flow driving assembly with the
linearly displaceable transport element.
[0015] In accordance with a preferred embodiment of the present
invention the cartridge housing includes first and second outer
housing portions which are hinged together and at least partially
enclose the cartridge element.
[0016] Preferably, the venter includes a needle assembly, which
cooperates with the plurality of operational volumes.
[0017] In accordance with a preferred embodiment of the present
invention the cartridge for use in in-vitro diagnostics also
includes a sample insertion subassembly communicating with at least
one of the plurality of operational volumes. Additionally or
alternatively, the plurality of operational volumes include a
multiplicity of operational volumes, at least some of which are
configured to allow injection of fluid solutions thereinto.
[0018] In accordance with a preferred embodiment of the present
invention the cartridge for use in in-vitro diagnostics also
includes a microfluidic PCR array mounted within the cartridge
housing. Additionally, at least one of the plurality of operational
volumes defines an internal passageway to a port of the
microfluidic PCR array.
[0019] Preferably, the cartridge for use in in-vitro diagnostics
also includes a sensor array mounted within the cartridge housing.
Additionally, at least one of the plurality of operational volumes
defines an internal passageway to a port of the sensor array.
Additionally or alternatively, the sensor array communicates with
at least one of the plurality of operational volumes operating as a
waste collection volume.
[0020] In accordance with a preferred embodiment of the present
invention the cartridge for use in in-vitro diagnostics also
includes a first plurality of fluid solution transporter locations
respectively communicating with at least some of the plurality of
operational volumes. Additionally, the cartridge for use in
in-vitro diagnostics also includes a second plurality of venting
element locations respectively communicating with the at least some
of the plurality of operational volumes.
[0021] There is yet further provided in accordance with a still
another preferred embodiment of the present invention a method for
use in in-vitro diagnostics, the method including providing a
cartridge having a plurality of operational volumes, at least some
of the plurality of operational volumes being mutually linearly
aligned, transferring fluid solutions from at least one of the
plurality of operational volumes to at least another of the
plurality of operational volumes, the transferring fluid solutions
including linearly displacing a transport element to sequentially
communicate with interiors of the at least some of the plurality of
operational volumes and venting the at least one of the plurality
of operational volumes.
[0022] In accordance with a preferred embodiment of the present
invention the transferring also includes driving the fluid
solutions through the transport element between ones of the
plurality of operational volumes.
[0023] Preferably, the transferring fluid solutions includes
transferring fluid solutions containing cellular material to a
microfluidic PCR array mounted within the cartridge. Additionally,
the transferring fluid solutions also includes transferring fluid
solutions containing cellular material from the microfluidic PCR
array to a sensor array associated with the cartridge.
[0024] Preferably, the method also includes injecting material into
some of the plurality of operational volumes prior to supplying
cellular material thereto.
[0025] In accordance with a preferred embodiment of the present
invention the transferring fluid solutions includes locating a cell
membrane breakdown material in a first operational volume, locating
an open end of a hollow needle into communication with the first
operational volume, drawing at least a portion of the cell membrane
breakdown material into the hollow needle, linearly displacing the
open end of the hollow needle into communication with a second
operational volume having a sample located therein and repeatedly
drawing the sample and at least some of the cell membrane breakdown
material into the hollow needle and expelling the sample and the
cell membrane breakdown material from the hollow needle into the
second operational volume, thereby mixing the sample and the cell
membrane breakdown material.
[0026] Preferably, the transferring fluid solutions further
includes linearly displacing the open end of the hollow needle into
communication with a third operational volume containing a cell
lysis solution and magnetic beads, drawing at least a portion of
the cell lysis solution and magnetic beads into the hollow needle
into engagement with the sample and the cell membrane breakdown
material and repeatedly drawing the sample, at least some of the
cell membrane breakdown material, the cell lysis solution and
magnetic beads into the hollow needle and expelling the sample, the
at least some of the cell membrane breakdown material, the cell
lysis solution and the magnetic beads, from the hollow needle into
the third operational volume, thereby releasing nucleic acids from
the sample and binding the nucleic acids to the magnetic beads.
[0027] In accordance with a preferred embodiment of the present
invention the transferring fluid solutions further includes
linearly displacing the open end of the hollow needle into
communication with a fourth operational volume containing a wash
buffer, drawing at least a portion of the wash buffer into the
hollow needle into engagement with the magnetic beads together with
the nucleic acids bound thereto and repeatedly drawing the wash
buffer and the magnetic beads, together with the nucleic acids
bound thereto, into the hollow needle, thereby washing away cell
debris and unbound nucleic acids from the magnetic beads.
[0028] In accordance with a preferred embodiment of the present
invention the transferring fluid solutions further includes
linearly displacing the open end of the hollow needle into
communication with a fifth operational volume containing an elution
buffer, drawing at least a portion of the elution buffer into the
hollow needle into engagement with the magnetic beads, together
with the nucleic acids bound thereto and repeatedly drawing the
elution buffer and the magnetic beads, together with the nucleic
acids bound thereto, into the hollow needle, thereby disengaging
the nucleic acids from the magnetic beads.
[0029] Preferably, the transferring fluid solutions further
includes linearly displacing the open end of the hollow needle into
communication with a sixth operational volume having at least one
magnet juxtaposed thereto, transferring the elution buffer and the
magnetic beads, together with the nucleic acids disengaged
therefrom, into the sixth operational volume, the at least one
magnet attracting the magnetic beads and drawing the elution
buffer, together with the nucleic acids, into the hollow
needle.
[0030] In accordance with a preferred embodiment of the present
invention the transferring fluid solutions further includes
linearly displacing the open end of the hollow needle into
communication with a seventh operational volume which communicates
with a microfluidic PCR array, transferring the elution buffer and
the nucleic acids into the microfluidic PCR array, the microfluidic
PCR array generating amplified nucleic acids by amplifying the
nucleic acids, drawing the amplified nucleic acids into the hollow
needle, linearly displacing the open end of the hollow needle into
communication with an eighth operational volume containing a
dilution buffer, drawing the dilution buffer into the hollow
needle, into engagement with the amplified nucleic acids and
repeatedly drawing the dilution buffer and the amplified nucleic
acids into the hollow needle, thereby generating diluted nucleic
acids.
[0031] Preferably, the transferring fluid solutions further
includes linearly displacing the open end of the hollow needle into
communication with a ninth operational volume which communicates
with a sensor array, transferring at least a first portion of the
diluted nucleic acids into the sensor array, thereby washing the
sensor array and thereafter transferring a second portion of the
diluted nucleic acids into operative engagement with the sensor
array.
[0032] Preferably, the transferring fluid solutions further
includes linearly displacing the open end of the hollow needle into
communication with a tenth operational volume which contains a
concentrated discriminator, drawing the concentrated discriminator
into the hollow needle, linearly displacing the open end of the
hollow needle into communication with an eleventh operational
volume which contains a discriminator buffer, drawing the
discriminator buffer into the hollow needle, into engagement with
the concentrated discriminator, repeatedly drawing the
discriminator buffer and the concentrated discriminator into the
hollow needle and expelling the discriminator buffer and the
concentrated discriminator from the hollow needle into the eleventh
operational volume, thereby generating a diluted discriminator,
drawing the diluted discriminator into the hollow needle, linearly
displacing the open end of the hollow needle into communication
with the ninth operational volume which communicates with the
sensor array and transferring the diluted discriminator into
operative engagement with the sensor array.
[0033] In accordance with a preferred embodiment of the present
invention the transferring fluid solutions further includes
linearly displacing the open end of the hollow needle into
communication with a twelfth operational volume which contains a
reporter reconstitution buffer, drawing the reporter reconstitution
buffer into the hollow needle, repeatedly drawing the reporter
reconstitution buffer into the hollow needle and expelling the
reporter reconstitution buffer from the hollow needle into the
twelfth operational volume via a thirteenth operational volume
containing a dried reporter, thereby generating a reconstituted
reporter, drawing the reconstituted reporter into the hollow
needle, linearly displacing the open end of the hollow needle into
communication with the ninth operational volume which communicates
with the sensor array and transferring the reconstituted reporter
into operative engagement with the sensor array.
[0034] In accordance with a preferred embodiment of the present
invention the transferring fluid solutions further includes
linearly displacing the open end of the hollow needle into
communication with a fourteenth operational volume which contains
an array wash buffer, drawing the array wash buffer into the hollow
needle, linearly displacing the open end of the hollow needle into
communication with the ninth operational volume which communicates
with the sensor array and transferring the array wash buffer into
operative engagement with the sensor array.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The present invention will be understood and appreciated
more fully from the following detailed description in which:
[0036] FIGS. 1A-1H are simplified respective front, back, top,
bottom, first side and second side planar views and front and rear
perspective views of a cartridge constructed and operative in
accordance with a preferred embodiment of the present
invention;
[0037] FIG. 2 is a simplified pictorial illustration of the
cartridge of FIG. 1 in an open orientation, prior to sealing
thereof;
[0038] FIGS. 3A, 3B and 3C are simplified respective illustrations
of a core assembly useful in the embodiment of FIG. 2, wherein FIG.
3A is a pictorial illustration of the core assembly, and FIGS. 3B
and 3C are planar view illustrations of respective opposite sides
of a base portion thereof;
[0039] FIGS. 4A-4H are simplified respective front, back, top,
bottom, first side and second side planar views and front and rear
perspective views of a functionally enhanced core assembly, which
can be used in the cartridge of FIGS. 1A-2 with suitable
modifications to the cartridge housing;
[0040] FIG. 5 is a simplified exploded view illustration of the
core assembly of FIGS. 4A-4H;
[0041] FIG. 6A-6H are simplified respective front, back, top,
bottom, first side and second side planar views and front and rear
perspective views of a microfluidic base portion of the core
assembly of FIG. 4A-5;
[0042] FIG. 7 is a simplified exploded view illustration of a top
cover assembly forming part of the core assembly of FIGS. 4A-5;
[0043] FIGS. 8A-8H are simplified respective front, back, top,
bottom, first side and second side planar views and front and rear
perspective views of a main portion of the top cover assembly of
FIG. 7, forming part of the core assembly of FIGS. 4A-5;
[0044] FIGS. 9A-9E are simplified respective front, back and
top/bottom planar views and front and rear perspective views of a
first overmolded septum of the top cover assembly of FIG. 7;
[0045] FIG. 10A-10H are simplified respective front, back, top,
bottom, first side and second side planar views and front and rear
perspective views of a second overmolded septum of the top cover
assembly of FIG. 7;
[0046] FIGS. 11A-11F are simplified respective front, back,
top/bottom and side planar views and front and rear perspective
views of a sample port sealing closure of the top cover assembly of
FIG. 7;
[0047] FIGS. 12A-12D are simplified illustrations showing four
typical stages of insertion of a sample into operative engagement
with the core assembly of FIGS. 4A-11F;
[0048] FIGS. 13A-13G are simplified illustrations of typical
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 13A shows an operational state corresponding to that of FIG.
12D, FIGS. 13A-130 show the microfluidic base portion of FIGS.
6A-6H, and FIGS. 13B-13G show operative engagement with chamber B1
thereof and with the sample receiving chamber thereof;
[0049] FIGS. 14A-14E are simplified illustrations of typical
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 14A shows an operational state subsequent to that of FIG. 13G,
FIGS. 14A-14E showing the microfluidic base portion of FIGS. 6A-6H
and operative engagement with chamber B2 thereof;
[0050] FIGS. 15A-15E are simplified illustrations of typical still
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 15A shows an operational state subsequent to that of FIG. 14E,
FIGS. 15A-15E showing the microfluidic base portion of FIGS. 6A-6H
and operative engagement with chamber B3 thereof;
[0051] FIGS. 16A-16E are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 16A shows an operational state subsequent to that of FIG. 15E,
FIGS. 16A-16E showing the microfluidic base portion of FIGS. 6A-6H
and operative engagement with chamber B4 thereof;
[0052] FIGS. 17A-17E are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 17A shows an operational state subsequent to that of FIG. 16E,
FIGS. 17A-17E showing the microfluidic base portion of FIGS. 6A-6H
and operative engagement with chamber B5 thereof;
[0053] FIGS. 18A-18D are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 18A shows an operational state subsequent to that of FIG. 17E,
FIGS. 18A-18D showing the microfluidic base portion of FIGS. 6A-6H
and operative engagement with chamber B6 thereof;
[0054] FIGS. 19A-19D are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 19A shows an operational state subsequent to that of FIG. 18D,
FIGS. 19A-19D showing the microfluidic base portion of FIGS. 6A-6I
and operative engagement with chamber A3 thereof;
[0055] FIGS. 20A-20D are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 20A shows an operational state subsequent to that of FIG. 19D,
FIGS. 20A-20D showing the microfluidic base portion of FIGS. 6A-6H
and operative engagement with chamber B7 thereof;
[0056] FIGS. 21A-21E are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 21A shows an operational state subsequent to that of FIG.
200.
[0057] FIGS. 21A-21E showing the microfluidic base portion of FIGS.
6A-6H and operative engagement with a PCR amplification subsystem
thereof;
[0058] FIGS. 22A-22G are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 22A shows an operational state subsequent to that of FIG. 21E,
FIGS. 22A 22G showing the microfluidic base portion of FIGS. 6A 6H
and operative engagement with chamber B8 thereof;
[0059] FIGS. 23A and 23B are simplified illustrations of typical
yet further steps in the operation of a cartridge such as that
shown in FIGS. 1A-2 including the core assembly of FIGS. 4A-11F,
wherein FIG. 23A shows an operational state subsequent to that of
FIG. 22G, FIGS. 23A and 23B showing the microfluidic base portion
of FIGS. 6A-6H and operative engagement with chamber A4
thereof;
[0060] FIGS. 24A-24F are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 24A shows an operational state subsequent to that of FIG.
23B.
[0061] FIGS. 24A-24F showing the microfluidic base portion of FIGS.
6A-6H and operative engagement with chamber B10 thereof and with a
sensor array thereof;
[0062] FIGS. 25A and 25B are simplified illustrations of typical
yet further steps in the operation of a cartridge such as that
shown in FIGS. 1A-2 including the core assembly of FIGS. 4A-11F,
wherein FIG. 25A shows an operational state subsequent to that of
FIG. 24F, FIGS. 25A and 25B showing the microfluidic base portion
of FIGS. 6A-6H and operative engagement with chamber A5
thereof;
[0063] FIGS. 26A-26F are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 26A shows an operational state subsequent to that of FIG.
25B.
[0064] FIGS. 26A-26F showing the microfluidic base portion of FIGS.
6A-6H and operative engagement with chamber B10 thereof and with a
sensor array thereof;
[0065] FIGS. 27A and 27B are simplified illustrations of typical
yet further steps in the operation of a cartridge such as that
shown in FIGS. 1A 2 including the core assembly of FIGS. 4A-11F,
wherein FIG. 27A shows an operational state subsequent to that of
FIG. 26F, FIGS. 27A and 27B showing the microfluidic base portion
of FIGS. 6A-6H and operative engagement with chamber A6
thereof;
[0066] FIGS. 28A-28F are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 28A shows an operational state subsequent to that of FIG. 27B,
FIGS. 28A-28F showing the microfluidic base portion of FIGS. 6A-6H
and operative engagement with chamber B10 thereof and with a sensor
array thereof;
[0067] FIGS. 29A and 29B are simplified illustrations of typical
yet further steps in the operation of a cartridge such as that
shown in FIGS. 1A-2 including the core assembly of FIGS. 4A-11F,
wherein FIG. 29A shows an operational state subsequent to that of
FIG. 28F, FIGS. 29A and 29B showing the microfluidic base portion
of FIGS. 6A-6H and operative engagement with chamber A7
thereof;
[0068] FIGS. 30A-30F are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 30A shows an operational state subsequent to that of FIG. 29B,
FIGS. 30A-30F showing the microfluidic base portion of FIGS. 6A-6H
and operative engagement with chamber B10 thereof and with a sensor
army thereof;
[0069] FIGS. 31A-31F are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 31A shows an operational state subsequent to that of FIG. 30F,
FIGS. 31A-31F showing the microfluidic base portion of FIGS. 6A-6H
and operative engagement with chamber B9 thereof and with a sensor
array thereof; and
[0070] FIGS. 32A-32D are simplified illustrations of typical yet
further steps in the operation of a cartridge such as that shown in
FIGS. 1A-2 including the core assembly of FIGS. 4A-11F, wherein
FIG. 32A shows an operational state subsequent to that of FIG. 31F,
FIGS. 32A-32D showing the microfluidic base portion of FIGS. 6A-6H
and operative engagement with chamber B13 thereof and with a sensor
array thereof.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0071] Reference is now made to FIGS. 1A-1H, which are simplified
respective front, back, top, bottom, first side and second side
planar views and front and rear perspective views of a cartridge
constructed and operative in accordance with a preferred embodiment
of the present invention and to FIG. 2, which is a simplified
pictorial illustration of the cartridge of FIG. 1 in an open
orientation, prior to sealing thereof.
[0072] As seen in FIGS. 1A-1H and 2, there is provided a cartridge
100 having first and second planar portions 102 and 104, which are
preferably hinged together by an integrally formed hinge 106.
[0073] First planar portion 102, an outer surface 108 of which is
seen particularly in FIGS. 1A and 1G, preferably is a generally
flat, generally rectangular element and includes first, second and
third cut outs, respectively designated by reference numerals 112,
114 and 116, on a top edge 118 thereof, which cooperate with
similarly spaced cut outs on second planar portion 104, which are
described hereinbelow, to respectively define sample transport
needle, venting needle and syringe piston access locations, as
described hereinbelow. First planar portion 102 also may include a
side cut out 120, for retaining a syringe flange.
[0074] First planar portion 102 also preferably defines a heater
engagement aperture 124, a plurality of frangible seal plunger
access apertures 126 and a magnet engagement aperture 128.
Frangible seal plunger access apertures 126 are preferably
implemented in accordance with the teachings of WO2012019599,
entitled `Device for Transporting Small Volumes of a Fluid, in
particular a Micropump or Microvalve`, the description of which is
hereby incorporated by reference.
[0075] Second planar portion 104, an outer surface 130 of which is
seen particularly in FIGS. 1B and 1H, preferably is a generally
flat, generally rectangular element and includes a cut out 136
which defines, together with cut out 116, the syringe piston access
location, as described hereinbelow. Second planar portion 104 also
preferably includes a sample cover snap fit accommodating cut out
138. Second planar portion 104 also may include a side cut out 140,
for retaining the syringe flange.
[0076] Second planar portion 104 also preferably defines a heater
engagement aperture 144, a plurality of frangible seal plunger
access apertures 146, a plurality of support protrusions 148 and a
generally rectangular carbon array access aperture 150. Frangible
seal plunger access apertures 146 are preferably implemented in
accordance with the teachings of WO2012019599, entitled `Device for
Transporting Small Volumes of a Fluid, in particular a Micropump or
Microvalve`, the description of which is hereby incorporated by
reference.
[0077] Second planar portion 104 preferably also includes first and
second notches. 152 and 154, formed within an upper rim 156
thereof, seen most clearly in FIG. 1C. First and second notches 152
and 154 respectively define, together with first and second cut
outs 112 and 114, sample transport needle and venting needle access
locations.
[0078] Turning now to FIG. 2, it is seen that an inner surface 158
of first planar portion 102 preferably defines a first linear array
160 of venting needle slidable mounting protrusions 162 for
engaging a linearly displaceable venting element, preferably
embodied as a venting needle 164, during shipping and prior to use.
It is also seen that inner surface 158 includes a second linear
array 170 of sample transport needle slidable mounting protrusions
172 for engaging a linearly displaceable transport element,
preferably embodied as a sample transport needle 174, during
shipping and prior to use. It is appreciated that respective
venting needle 164 and sample transport needle 174 are formed with
respective needle griping base portions 176 and 178 and tubing
connectors 180 and 182.
[0079] A sample transport tube 190 is connected to sample transport
needle tubing connector 182 and preferably communicates with a luer
connector 192 of a syringe 194, which is retained in position by a
linear array 196 of syringe supports 198.
[0080] It is also seen that an inner surface 208 of second planar
portion 104 preferably defines a linear array 216 of syringe
supports 218, which cooperate with syringe supports 198 to retain
syringe 194 in position. A core assembly 220 is retained within the
housing preferably at least by first and second linear protrusions
222 and 224.
[0081] It is appreciated that the housing is closed subsequent to
manufacture as by relative rotation of the first and second planar
portions 102 and 104 about hinge 106.
[0082] Reference is now made to FIGS. 3A, 3B and 3C, which are
simplified respective illustrations of core assembly 220 useful in
the embodiment of FIG. 2, wherein FIG. 3A is a pictorial
illustration of the core assembly 220, and FIGS. 3B and 3C are
planar view illustrations of a base portion thereof.
[0083] As seen in FIGS. 3A-3C, core assembly 220 preferably
includes a base portion 230, which is illustrated in FIGS. 3B &
3C, a top cover assembly 240 having first and second septa 242 and
244 overmolded therewith as well as a first array of reagent filing
ports 246 and a second array of reagent venting ports 248, which
are employed during manufacture. Top cover assembly 240 is also
provided with a plurality of alignment apertures 250 for receiving
a corresponding plurality of alignment protrusions 252 on base
portion 230, and a plurality of apertures 260 for accommodating
reagent plugs 270 which are mounted onto base portion 230 and are
preferably implemented in accordance with the teachings of
EP2821138, entitled `Flow Cell with Integrated Dry Substance`, the
description of which is hereby incorporated by reference`. Top
cover assembly 240 is preferably also provided with a plurality of
frangible seal plunger access apertures 276, preferably implemented
in accordance with the teachings of WO2012019599 entitled `Device
for Transporting Small Volumes of a Fluid, in particular a
Micropump or Microvalve` and WO2016000998, entitled `Flow Cell
comprising a Storage Zone and a Duct that can be Opened at a
Predetermined Breaking Point`, the description of which is hereby
incorporated by reference.
[0084] Top cover assembly 240 preferably also includes a flexible
sample insertion port sealing cover 280, which removably and
replaceably covers a sample insertion port 292 of a sample
receiving chamber 293 defined by base portion 230. Flexible sample
insertion port scaling cover 280 is preferably formed with a snap
fit protrusion 294, which engages a corresponding recess 296 formed
in the base portion 230. It is noted that core assembly 220 is
particularly suitable for use in conducting RCA assays.
[0085] Reference is now made to FIGS. 4A-4H, which are simplified
respective front, back, top, bottom, first side and second side
planar views and front and rear perspective views of a functionally
enhanced core assembly 400, useful for both RCA and PCR assays and
which can be used in the cartridge of FIGS. 1A-2 with suitable
dimensional modifications to the cartridge housing, and to FIG. 5,
which is a simplified exploded view illustration of the core
assembly of FIGS. 4A-4H.
[0086] As seen in FIGS. 4A-5, core assembly 400 constitutes a
cartridge element preferably including a microfluidic base portion
410, a cover assembly 420, sealingly engaging the microfluidic base
portion 410 on a first side thereof, a sealing cover film 430,
sealingly engaging the microfluidic base portion 410 on a second
side thereof, a carbon array 440 including a layer of double sided
adhesive and which is mounted by means of the double side adhesive
layer onto the sealing cover film 430, and a transparent carbon
array cover 450.
[0087] As seen in FIG. 5, carbon array 440 includes a carbon array
inlet aperture 460 and a carbon array outlet aperture 462.
Additionally, sealing cover film 430 includes a carbon array inlet
access aperture 470 and a carbon array outlet access aperture 472,
which carbon array inlet access aperture 470 and a carbon array
outlet access aperture 472 are respectively aligned with carbon
array inlet aperture 460 and carbon array outlet aperture 462, when
core assembly 400 is assembled.
[0088] Reference is now made additionally to FIG. 6A-6H, which are
simplified respective front, back, top, bottom, first side and
second side planar views and front and mar perspective views of
microfluidic base portion 410 of the core assembly of FIG.
4A-5.
[0089] As seen in FIGS. 6A-6H, the microfluidic base portion 410 is
a generally planar element preferably injection molded from
polypropylene. A first surface 500 is seen in FIGS. 6A and 6G and a
second, opposite surface 510 is seen in FIGS. 6B and 6H. The
microfluidic base portion 410 is preferably formed with an array
520 of frangible seal access apertures 522, preferably implemented
in accordance with the teachings of WO2012019599A2, entitled
`Device for Transporting Small Volumes of a Fluid, in particular a
Micropump or Microvalve`, the description of which is hereby
incorporated by reference.
[0090] Turning initially principally to FIGS. 6A and 6G, which
illustrate first surface 500, it is seen that there is provided an
array 530 of venting needle guiding protrusions 532 arranged along
a longitudinal axis 534. Each of guiding protrusions 532 defines a
tunnel 536 and all of the tunnels are longitudinally aligned along
axis 534.
[0091] Alongside array 530 is a generally longitudinal array 538 of
reagent venting ports 540. Alongside array 538 is a reagent storage
chamber defining protrusion 550, which preferably defines a
plurality of reagent storage operational volumes or chambers 552,
which are labeled for clarity in FIG. 6A as chambers B1-B14. Each
of the reagent storage chambers is preferably provided with a
throughgoing venting aperture 554 and a throughgoing reagent
transport aperture 556.
[0092] Alongside reagent storage chamber defining protrusion 550 is
a generally longitudinal array 560 of reagent supply ports 562.
[0093] Alongside reagent storage chamber defining protrusion 550 is
a generally longitudinal array 570 of reagent plug receiving ports
572, preferably implemented in accordance with the teachings of
EP2821138, entitled `Flow Cell with Integrated Dry Substance`, the
description of which is hereby incorporated by reference.
[0094] Adjacent array 520 of frangible seal access apertures 522 is
an array 580 of transport needle guiding protrusions 582 arranged
along a longitudinal axis 584, which is preferably parallel to axis
534. Each of guiding protrusions 582 defines a tunnel 586 and all
of the tunnels are longitudinally aligned along axis 584.
[0095] Adjacent array 580 is a microfluidic PCR array amplification
subsystem 600 including a gas spring accommodating protrusion 602,
preferably implemented in accordance with the teachings of
WO2010139295, entitled `Apparatus for Transporting a Fluid within a
Channel Leg of a Microfluidic Element` the description of which is
hereby incorporated by reference, a plurality of PCR reagent plug
receiving ports 604 and a recess 606 which defines a heater
engagement region. Below PCR amplification subsystem 600 there is
provided a sample receiving chamber 610 having a sample insertion
aperture 612.
[0096] Turning now to FIGS. 6B and 6H, which illustrate second
surface 510, it is seen that there is provided an array 630 of
recesses 632, each of which recesses 632 communicates with a
corresponding tunnel 536 of a corresponding venting needle guiding
protrusion 532 arranged along longitudinal axis 534. Recesses 632
in combination with tunnels 536 preferably define a plurality of
venting needle tip locations, which are labeled for clarity in FIG.
6H as venting needle tip locations V11-V23. Some of recesses 632
each communicate with a respective microfluidic channel 634, which
in turn communicates with a venting aperture 554 of a corresponding
one of chambers B1-14. Others of recesses 632 each communicate with
a respective microfluidic channel 636, which in turn communicates
with a corresponding one of reagent storage chambers 640, which am
respectively labeled in FIG. 6B as chambers A1-A7. A further recess
632 communicates with the interior of sample receiving chamber 610
for providing venting thereof. One or more additional recess 632
coupled to a corresponding tunnel may be provided to enable
additional functionality not currently contemplated.
[0097] It is also seen that there is provided an array 730 of
recesses 732, each of which communicates with a corresponding
tunnel 586 of a corresponding transport needle guiding protrusion
582 arranged along a longitudinal axis 584. Recesses 732 in
combination with tunnels 586 preferably define a plurality of
sample transport needle tip locations, which are labeled for
clarity in FIG. 6H as sample transport needle tip locations T1-T23.
Some of recesses 732 each communicate with a respective
microfluidic channel 734, which in turn communicates with a reagent
transport aperture 556 of a corresponding one of chambers B1-14.
Others of recesses 732 each communicate with a respective
microfluidic channel 736, which in turn communicates with a
corresponding one of reagent storage chambers 640, which are
respectively labeled in FIG. 6B as chambers A1-A7.
[0098] A further recess 738 communicates with a respective
microfluidic channel 740, which in turn communicates with the
interior of sample receiving chamber 610 for providing sample
transport. A still further recess 742 communicates with a
respective microfluidic channel 744, which in turn communicates
with chamber A1 via a reagent plug port 572 and a frangible seal
located in an aperture 522 and preferably implemented in accordance
with the teachings of WO2012019599, entitled `Device for
Transporting Small Volumes of a Fluid, in particular a Micropump or
Microvalve` and WO2016000998, entitled `Flow Cell comprising a
Storage Zone and a Duct that can be Opened at a Predetermined
Breaking Point`, the descriptions of which are hereby incorporated
by reference. Additional recesses 746 communicate with respective
microfluidic channels 748, which in turn communicate with a
corresponding reagent transport aperture 556 of a corresponding one
of chambers B1-B14 via a respective reagent plug port 572.
[0099] A yet further recess 750 communicates via a microfluidic
channel 752 with PCR amplification subsystem 600. Microfluidic
channel 752 thus defines an internal passageway to a port of PCR
amplification subsystem 600. PCR amplification subsystem 600
preferably includes a plurality of parallel microfluidic channels
760, each of which communicates with a corresponding PCR
amplification chamber 770. Each chamber 770 communicates via a
corresponding reagent plug 604 with a corresponding gas spring 772
located within protrusion 602 and preferably implemented in
accordance with the teachings of WO2010139295, entitled `Apparatus
for Transporting a Fluid within a Channel Leg of a Microfluidic
Element`, the description of which is hereby incorporated by
reference. Reagent plugs 604 within PCR amplification subsystem 700
are respectively labeled in FIG. 68 as reagent plugs A8-A13.
[0100] A still further recess 780 communicates via a microfluidic
channel 782 with carbon array 440, via an aperture 783 which is
aligned with carbon array inlet access aperture 470 of sealing
cover film 430 and carbon array inlet aperture 460 of carbon array
440. Carbon array 440 communicates with chamber B14 via a venting
aperture 784, which aperture 784 is aligned with carbon array
outlet access aperture 472 of sealing cover film 430 and carbon
array outlet aperture 462 of carbon array 440. Chamber B14
preferably serves as a waste receptacle. It is appreciated that
carbon array 440 is thus vented into chamber B14 by way of venting
aperture 784, which venting aperture 784 interfaces and mutually
connects carbon array 440 and chamber B14.
[0101] It is appreciated that reagent storage chambers B1-B14 and
A1-A7 as well as reagent plugs A8-A13 and sample receiving chamber
610 may also be termed operational volumes, defined by microfluidic
base portion 410 of the cartridge element forming part of core
assembly 400. It is further appreciated that the plurality of
operational volumes including chambers B1-B14, A1-A13 and sample
receiving chamber 610 further includes a multiplicity of
operational volumes formed by the various microfluidic channels,
including channels 634, 636, 734, 736, 740, 744, 748, 752.760 and
782 (FIG. 6B) interconnecting B1-B14. A1-A13 and sample receiving
chamber 610, at least some of which microfluidic channels are
configured to allow injection of fluid thereinto, as described in
greater detail henceforth.
[0102] As appreciated from consideration of FIG. 6A, at least some
of chambers B1-B14 are preferably mutually linearly aligned.
Furthermore, as appreciated from consideration of FIG. 6B, at least
some of chambers A1-A7 and A8-A13 am preferably mutually linearly
aligned.
[0103] Reference is now made additionally to FIG. 7, which is a
simplified exploded view illustration of top cover assembly 420,
forming part of the core assembly of FIGS. 4A-5. Top cover assembly
420 preferably includes a main portion 800, having first and second
septa 802 and 804 overmolded thereonto and a sample inlet scaling
portion 806.
[0104] Reference is now made additionally to FIGS. 8A-8H, which am
simplified respective front, back, top, bottom, first side and
second side planar views and front and rear perspective views of
main portion 800 of the top cover assembly of FIG. 7, forming part
of the core assembly of FIGS. 4A-5.
[0105] As seen in FIGS. 8A-8H, the main portion 800 is a generally
planar element, preferably injection molded from polypropylene. A
first surface 810 is seen in FIGS. 8A and 8G and a second, opposite
surface 820 is seen in FIGS. 8B and 8H. The main portion 800 is
preferably formed with first and second septa receiving apertures
830 and 832 for receiving respective septa 802 and 804 which are
overmolded therein. The main portion 800 also includes an aperture
834 for providing access to array 538 of reagent venting ports 540
of microfluidic base portion 410 and an aperture 836 for providing
access to generally longitudinal array 560 of reagent supply ports
562, the array 570 of reagent plug ports 572 and the array 520 of
frangible seal access apertures 522 of microfluidic base portion
410.
[0106] Turning to FIGS. 8A and 8G, which illustrate first surface
810 and to FIGS. 8B and 8H, which illustrate second surface 820, it
is seen that there is provided an array 840 of venting ports 842,
each of which communicates with the interior of a different one of
chambers B1-B13. It is also seen that there is provided a plurality
of reagent filling ports 850, each of which communicates with the
interior of a different one of chambers B1-B13.
[0107] Main portion 800 of top cover assembly 420 preferably also
includes a flexible sample insertion port sealing cover 860, which
removably and replaceably covers sample insertion port 612 of a
sample receiving chamber 610 defined by base portion 410. Sample
inlet sealing portion 806 is preferably mounted, as by use of an
adhesive, onto an underside surface of flexible sample insertion
port sealing cover 860 for providing sealing of sample insertion
port 612. A preferred embodiment of the sample inlet sealing
portion 806 is illustrated in FIGS. 11A-11F.
[0108] Reference is now made additionally to FIGS. 9A-9E, which ae
simplified respective front, back and top/bottom planar views and
front and rear perspective views of first overmolded septum 802 of
the cover assembly 420 of FIG. 7, preferably implemented in
accordance with the teachings of German Patent Application No. 17
172 994.0, entitled `Multifunctional co-molded housing for
microfluidic card`, the description of which is hereby incorporated
by reference.
[0109] As seen in FIGS. 9A-9E, the first overmolded septum 802 is a
generally rectangular overmolded element having a longitudinal
array 870 of recesses 872 formed therein. Longitudinal array 870 of
recesses 872 sealingly overlies array 530 of venting needle guiding
protrusions 532 arranged along longitudinal axis 534.
[0110] Reference is now made additionally to FIG. 10A-10H, which
are simplified respective front back, top bottom, first side and
second side planar views and front and rear perspective views of a
second overmolded septum of the top cover assembly of FIG. 7,
preferably implemented in accordance with the teachings of German
Patent Application No. 17 172 994.0, entitled `Multifunctional
co-molded housing for microfluidic card`, the description of which
is hereby incorporated by reference.
[0111] As seen in FIGS. 10A-10H, the second overmolded septum 804
is a generally rectangular overmolded element having a side
protrusion 880. A main portion 882 of the second overmolded septum
804 is formed with a longitudinal array 890 of recesses 892.
Longitudinal array 890 of recesses 892 sealingly overlies array 580
of transport needle guiding protrusions 582 arranged along
longitudinal axis 584.
[0112] The side protrusion 880 sealingly overlies the frangible
seals at apertures 522 and is preferably implemented in accordance
with the teachings of WO2012019599 entitled `Device for
Transporting Small Volumes of a Fluid, in particular a Micopump or
Microvalve` and WO2016000998, entitled `Flow Cell comprising a
Storage Zone and a Duct that can be Opened at a Predetermined
Breaking Point`, the descriptions of which are hereby incorporated
by reference.
[0113] It is appreciated that first and second overmolded septa 802
and 804 preferably sealingly communicate, by way of first and
second sets of recesses 872 and 892, with at least some of
plurality of operational volumes or chambers A1-A13 and B1-B14 and
sample receiving chamber 610 when cartridge 100 including core
assembly 400 is in an assembled state.
[0114] It will be appreciated by persons skilled in the art that
cartridge 100 of FIGS. 1A-11F described above is preferably
employed for carrying out a biological process. A preferred
embodiment of this process is summarized in Tables I and II
below.
[0115] Table I sets forth preferred content/function and content
composition of each of chambers B1-B14 and A1-A13 of microfluidic
base portion 410 of functionally enhanced core assembly 400.
[0116] It is understood that, due to core assembly 400 being
suitable for use both with PCR and RCA arrays, selected ones of
chambers B1-B14 and A1-A13 may have dual functionality and contents
thereof may be common to both RCA and PCR arrays, as indicated in
Table I with respect to chambers B2-B5. Additionally, selected ones
of chambers B1-B14 and A1-A13 may have differing contents depending
on whether core assembly 400 is used with a PCR or RCA array, as
indicated in Table I with respect to chambers B6, B7, B9 and
A3.
[0117] It is further understood that selected ones of chambers
B1-B14 and A1-A13 may be useful only in conjunction with one rather
than both of PCR and RCA arrays, as further indicated in Table I
with respect to chambers B1, B8, B10-B13 and A1. A2 and A4-A13. In
the case that a particular chamber in useful only in conjunction
with one rather than both of PCR and RCA arrays, that chamber may
be obviated or may be unfilled and therefore not play a part in the
biological process carried out within cartridge 100 when cartridge
100 is used in conjunction with an array type for which that
chamber is not useful.
TABLE-US-00001 TABLE I Amplification array Chamber
Contents/Function with which used Composition B1 Proteinase K PCR
Proteinase K enzyme in buffer, e.g., TRIS-HCl (ph 7.4). B2 Lysis
solution PCR/RCA Guanidinium Thiocyanate, ionic detergent, buffer
(pH 7.4), Isopropanol, Carrier RNA; modified Sera- Mag .TM.
SpeedBeads magnetic particles. B3 Wash Buffer I PCR/RCA Guanidinium
Thiocyanate, ionic detergent, TRIS-HCl (pH 7.4), isopropanol,
(DEPC)-Water B4 Wash Buffer II PCR/RCA KCl/Tris (pH 7), Ethanol, in
Rnase-free water B5 Wash Buffer III PCR/RCA KCl/Tris (pH 7) in
Rnase- free water B6 Elution Buffer PCR Tris based buffer/DDW
(Ultra-Pure DNase and RNase free water produced by Biological
Industries Beit Haemek, Israel). RCA In RCA, the eluted product is
an elongated and entangled genomic DNA, which must first be cut
into smaller segments using a restriction enzyme and incubation for
5 minutes at 37.degree. C. This will be followed by denaturation
for 2 minutes at 95.degree. C. The elution buffer preferably
comprises SBA. B7 Eluant Dilution PCR/RCA Sample Buffer A (SBA)
Buffer comprising L-Histidine, 1- Thioglycerol in DDW or DDW
(Ultra-Pure DNase and RNase free water produced by Biological
Industries Beit Haemek, Israel). In RCA, elution dilution is done
in SBA. B8 Amplicon Dilution PCR Sample Buffer A (L- Buffer
Histidine, 1-Thioglycerol in DDW). B9 Buffer for Reporter PCR
Either High Salt Buffer reconstitution. (HSB) (NaPO4, NaCl, Triton,
pH 7.4) if reporter is dried in DDW, or DDW if reporter is dried in
HSB. Buffer for Ligation RCA DDW for RCA, reaction plug comprises
dried ligase enzyme B10 Buffer for PCR Buffer for dilution of
Discriminator discriminator mix - dilution, for all preferably High
Salt Buffer discriminators. (HSB) (NaPO4, NaCl, Triton, pH 7.4) B11
Sensor Wash 1 RCA Low Salt Buffer (LSB) (NaPO4, Triton, pH 7.4) B12
Sensor Wash 2 RCA Low Salt Buffer (LSB) (NaPO4, Triton, pH 7.4) B13
Sensor Wash PCR Low Salt Buffer (LSB) (NaPO4, Triton, pH 7.4) B14
Waste Receptacle PCR/RCA A1 Proteinase K RCA Proteinase K enzyme in
buffer, e.g., TRIS-HCl (pH 7.4) A2 Elution Buffer RCA Sample Buffer
A (SBA) (L-Histidine, 1- Thioglycerol in DDW) A3 Bead Removal PCR
Tris based buffer/DDW (Ultra-Pure DNase and RNase free water
produced by Biological Industries Beit Haemek, Israel). RCA In RCA
SBA is used A4 Discriminator Mix 1 PCR Discriminator Mix 1 in TE A5
Discriminator Mix 2 PCR Discriminator Mix 2 in TE A6 Discriminator
Mix 3 PCR Discriminator Mix 3 in TE. A7 Discriminator Mix 4 PCR
Discriminator Mix 4 in TE. A8-A13 Amplification PCR PCR Mix (i.e.
buffer, DNA polymerase and a set of primers. In panels that also
include RNA targets, reverse transcriptase is also included in the
mix). The PCR mix is divided into six channels and dried on reagent
plugs A8-A13. Different types of PCR mix may be mounted on each
reagent plug.
[0118] Table II sets forth a simplified description of a typical
biological process that takes place in chambers B1-B14, A1-A13 and
sample receiving chamber 610, in conjunction with a PCR array such
as PCR amplification subsystem 600, in accordance with a preferred
embodiment of the present invention.
TABLE-US-00002 TABLE II Chamber Biological Process Occurring Within
Chamber B1 Sample transport needle 174 is moved to be in indirect
contact with chamber B1 containing Proteinase K. The piston of
syringe 194 is raised, drawing Proteinase K into syringe 194 via a
fluid path comprising microfluidic channel 734 connected to chamber
B1 at aperture 556, the interior of the sample transport needle 174
and the interior of flexible tube 190. Chamber B1 is vented by
venting needle 164, via microfluidic channel 634 which communicates
with chamber B1 via venting aperture 554. The corresponding
mechanical operation of the relevant elements of cartridge 100 of
FIGS. 1A-2 including functionally enhanced core assembly of FIGS.
4A- 11F is described hereinbelow with reference to FIGS. 13A-13C.
Sample The sample transport needle 174 is moved to be in indirect
contact with sample Receiving receiving chamber 610. The piston of
syringe 194 is lowered, thereby injecting Chamber Proteinase K from
syringe 194 into sample receiving chamber 610. 610 The piston is
moved up and down to mix the Proteinase K and a sample previously
inserted in sample receiving chamber 610. The sample may be any
sample of biological and/or cellular material containing nucleic
acids for analysis. At the end of the mixing step, the piston is
lowered and the mixture returns to sample receiving chamber 610.
Sample incubation of the mixture in sample receiving chamber 610
follows, wherein the sample is heated to 56.degree. C.
(.+-.2.degree. C.) for 8-10 min in the presence of the enzyme
Proteinase K. The enzyme dissolves cell membranes and eliminates
nucleases, which are enzymes that catalyze the hydrolytic cleavage
of phosphodiester linkages in the nucleic acid (NA) backbone, thus
breaking down DNA and/or RNA. At the end of the incubation period
the piston is raised, thereby drawing the Proteinase K and sample
via a fluid path comprising microfluidic channel 740, the interior
of the sample transport needle 174 and the interior of tube 190
into the volume of the cylinder underlying the piston in syringe
194. Sample receiving chamber 610 is vented by venting needle 164,
via microfluidic channel 1349. The corresponding mechanical
operation of the relevant elements of cartridge 100 of FIGS. 1A-2
including functionally enhanced core assembly of FIGS. 4A- 11F is
described hereinbelow with reference to FIGS. 13D-13G. B2 The
sample transport needle 174 is moved to be in indirect contact with
chamber B2, which contains a lysis solution and also contains
magnetic beads. The piston of syringe 194 is lowered and the sample
1204 and proteinase K are mixed with the lysis solution contained
in chamber B2 and the magnetic beads, followed by continuous mixing
through the raising and lowering of the piston of syringe 194
multiple times. The composition of the lysis solution, the physical
transport through the narrow sample transport needle 174 and the
pressure and shear forces of the transport, lyses sample cells.
Guanidinium Thiocyanate is used to lyse cells and as a general
protein denaturant which denatures proteins (including nucleases)
which may otherwise inhibit nucleic acids from binding to the
magnetic beads. Denaturation is through the disruption of hydrogen
bonding and weakening of hydrophobic interactions. Ionic detergent
and isopropanol act as detergents and help solubilize membrane
proteins and lipids, causing the cell to lyse and to release its
contents. Nucleic Acids (NAs) bind to the magnetic beads via a
coating thereof. At the end of the lysis stage, the Nucleic Acids
(NAs) are bound to the magnetic beads. The beads, with the NAs
bound thereto are then attracted by a magnet to the wall of syringe
194. The piston of syringe 194 is lowered and the remaining unbound
material (containing cell debris) is forced back to the chamber B2.
Chamber B2 is vented by venting needle 164, via microfluidic
channel 634 which communicates with chamber B2 via venting aperture
554. The corresponding mechanical operation of the relevant
elements of cartridge 100 of FIGS. 1A-2 including functionally
enhanced core assembly of FIGS. 4A- 11F is described hereinbelow
with reference to FIGS. 14A-14E. B3 The sample transport needle 174
is moved to be in indirect contact with chamber B3, containing wash
buffer I. The magnet is removed and the beads and the nucleic acids
bound thereto are washed. The contents of chamber B3 are drawn into
the syringe 194 by raising the piston of syringe 194. Washing is
done by repeated raising and lowering of the piston of syringe 194,
thereby pumping wash buffer I, beads and the nucleic acids bound
thereto from chamber B3 to the interior volume of syringe 194
underlying the piston of syringe 194 and back multiple times, via
the fluid path formed therebetween by tube 190, the interior of the
sample transport needle 174 and microfluidic channel 734 connected
to chamber B3. At the end of the wash, the magnet is returned to
propinquity with the syringe 194 and attracts the beads, with the
NAs bound thereto, to the wall of syringe 194. The piston of
syringe 194 is lowered and the remaining material is forced back
into chamber B3 via the fluid path. Chamber B3 is vented by venting
needle 164, via microfluidic channel 634 which communicates with
chamber B3 via venting aperture 554. The corresponding mechanical
operation of the relevant elements of cartridge 100 of FIGS. 1A-2
including functionally enhanced core assembly of FIGS. 4A- 11F is
described hereinbelow with reference to FIGS. 15A-15E. B4 The
sample transport needle 174 is moved to be in indirect contact with
chamber B4, containing wash buffer II, and the biological
processing of the sample proceeds as previously described for
chamber B3. At the end of the wash, the magnet is returned to
propinquity with the syringe 194 and attracts the beads, with the
NAs bound thereto, to the wall of syringe 194. The piston of
syringe 194 is lowered and the remaining material is forced back to
chamber B4 via a fluid path, formed between syringe 194 and chamber
B4, by tube 190, the interior of the sample transport needle 174
and microfluidic channel 734 connected to chamber B4. Chamber B4 is
vented by venting needle 164, via microfluidic channel 634 which
communicates with chamber B3 via venting aperture 554. The
corresponding mechanical operation of the relevant elements of
cartridge 100 of FIGS. 1A-2 including functionally enhanced core
assembly of FIGS. 4A- 11F is described hereinbelow with reference
to FIGS. 16A-16E. B5 The sample transport needle 174 is moved to be
in indirect contact with chamber B5, containing wash buffer III,
and the biological processing of the sample proceeds as previously
described for chambers B3 and B4. At the end of the wash, the
magnet is returned to propinquity with the cylinder and attracts
the beads, with the NAs bound thereto, to the wall of syringe 194.
The piston of syringe 194 is lowered and the remaining material is
forced back to chamber B5 via a fluid path, formed between syringe
194 and chamber B5, by tube 190, the interior of the sample
transport needle 174 and microfluidic channel 734 connected to
chamber B5. Chamber B5 is vented by venting needle 164, via
microfluidic channel 634 which communicates with chamber B3 via
venting aperture 554. The corresponding mechanical operation of the
relevant elements of cartridge 100 of FIGS. 1A-2 including
functionally enhanced core assembly of FIGS. 4A- 11F is described
hereinbelow with reference to FIGS. 17A-17E. B6 The sample
transport needle 174 is moved to be in indirect contact with
chamber B6, containing an elution Buffer and the piston of syringe
194 is raised, drawing elution buffer into syringe 194. The magnet
is removed so as to allow the release of beads, with the NAs bound
thereto,- and washing thereof. Washing is done by repeated raising
and lowering of the piston of syringe 194, thereby pumping the
elution buffer, beads and the nucleic acids bound thereto from
chamber B6 to the interior volume of syringe 194, underlying the
piston of syringe 194 and back multiple times, via the fluid path
formed therebetween by tube 190, the interior of sample transport
needle 174 and microfluidic channel 734 connected to chamber B6.
The elution buffer releases the NAs from the beads due to the
change in salt concentration. At the end of the elution the piston
of syringe 194 is raised and all fluids are returned to the syringe
194. Chamber B6 is vented by venting needle 164, via microfluidic
channel 634 which communicates with chamber B3 via venting aperture
554. The corresponding mechanical operation of the relevant
elements of cartridge 100 of FIGS. 1A-2 including functionally
enhanced core assembly of FIGS. 4A- 11F is described hereinbelow
with reference to FIGS. 18A-18D. A3 The sample transport needle 174
is moved to be in indirect contact with chamber A3 for the removal
of magnetic beads. The piston is lowered, and the eluted NAs and
the magnetic beads are injected into chamber A3 via a fluid path
formed between the syringe 194 and chamber A3, by tube 190, the
interior of sample transport needle 174 and microfluidic channel
736 which terminates at an opening of chamber A3. A permanent,
fixed magnet, not forming a part of cartridge 100 but rather
external thereto, is preferably positioned along and generally
parallel to chamber A3 so that it is in propinquity with the wall
of chamber A3. The magnet attracts the beads to the wall of chamber
A3 and the NAs remain in the elution buffer solution. The piston is
subsequently raised to an upper intermediate position, thereby
drawing a desired volume of the eluted and free NAs into the
interior volume of the syringe 194 via the fluid path. Chamber A3
is vented by venting needle 164, via microfluidic channel 636 which
terminates at an opening of chamber A3. The corresponding
mechanical operation of the relevant elements of cartridge 100 of
FIGS. 1A-2 including functionally enhanced core assembly of FIGS.
4A- 11F is described hereinbelow with reference to FIGS. 19A-19E.
B7 The sample transport needle 174 is now moved to be in indirect
contact with the chamber B7, containing an Elution Dilution Buffer.
The piston of syringe 194 is in an upper intermediate position,
with the interior volume of the syringe 194 below the piston of the
syringe 194 and a volume including tube 190 and interior of sample
transport needle 174 containing eluant from chamber A3. Dilution is
performed by mixing the eluant and the elution dilution buffer from
chamber B7 by raising the piston to a fully extended position,
thereby drawing the elution dilution buffer, either Sample Buffer A
or DDW water, from chamber B7 into the interior volume of the
syringe 194, and
by repeated raising and lowering of the piston of syringe 194
multiple times. At the end of the mixing, the piston of syringe 194
is raised and a desired volume of the diluted eluant is drawn into
the interior volume of the syringe 194 via a fluid path formed
between syringe 194 and chamber B7 by tube 190, the interior of the
sample transport needle 174 and microfluidic channel 734 connected
to chamber B7. Chamber B7 is vented by venting needle 164, via
microfluidic channel 634 which communicates with chamber B7 via
venting aperture 554. The corresponding mechanical operation of the
relevant elements of cartridge 100 of FIGS. 1A-2 including
functionally enhanced core assembly of FIGS. 4A- 11F is described
hereinbelow with reference to FIGS. 20A-20D. A8-A13 The sample
transport needle 174 is moved to a transport port, which is in
communication with in the PCR amplification subsystem 600. The
piston is lowered and diluted eluant from chamber B7 is injected
via microfluidic channel 752 into the PCR amplification subsystem
600. PCR amplification subsystem 600 includes a plurality of
parallel microfluidic channels 760, each of which communicates with
a corresponding amplification chamber 770. Each amplification
chamber 770 communicates via a corresponding reagent plug A8-A13
with a corresponding gas spring 772. The gas springs 772 ensure the
equal distribution of the eluant among the various amplification
chambers 770. The piston is raised up and down multiple times so as
to reconstitute and mix a dried PCR mix located on each of reagent
plugs A8-A13, positioned along each microfluidic channel leg of the
subsystem, above each amplification chamber 770. PCR amplification
is performed as described elsewhere herein. At the end of the PCR
amplification, the piston of syringe 194 is raised to an upper
intermediate position drawing a desired volume of amplified NAs,
amplicons, into the interior volume of the syringe 194 via a fluid
path formed between PCR amplification subsystem 600 and syringe 194
by microfluidic channel 752, the interior of sample transport
needle 174 and tube 190. A valve may optionally be included between
PCR amplification subsystem 600 and the needle tip location T11 in
order to enhance venting of PCR amplification subsystem 600. The
corresponding mechanical operation of the relevant elements of
cartridge 100 of FIGS. 1A-2 including functionally enhanced core
assembly of FIGS. 4A- 11F is described hereinbelow with reference
to FIGS. 21A-21E. B8 The sample transport needle 174 is moved to be
in indirect contact with chamber B8, containing an amplicon
dilution buffer, such as Sample Buffer A (SBA). The piston of
syringe 194 is in an upper intermediate position, with the interior
volume of the syringe 194 below the piston of the syringe 194 and a
volume including tube 190 and interior of sample transport needle
174 containing PCR products (amplified NAs, amplicons) from PCR
amplification system 600. Dilution is performed by mixing the
amplicons with the amplicon dilution buffer, by raising the piston
of syringe 194 to a fully extended position, thereby drawing the
Amplicon Dilution Buffer from chamber B8 into the interior volume
of the syringe 194, and by repeated raising and lowering of the
piston of syringe 194 multiple times. At the end of the mixing, the
piston of syringe 194 is raised and diluted amplicons are drawn
into the interior volume of the syringe 194 via a fluid path formed
between syringe 194 and chamber B8 by tube 190, the interior of
transport needle 174 and microfluidic channel 748 connected to
chamber B8. The sample transport needle 174 is next moved to
transport needle tip location T21, which is in fluid communication
with the carbon array 440, and the piston of syringe 194 is lowered
to a lower intermediate position. The diluted amplicons pass over
the carbon array 440 and bind to a trisaccharide (e.g., Raffinose),
which is used to preserve the cartridge during storage. Next, the
piston of syringe 194 is lowered further, thus allowing for more
diluted amplicons to pass over the carbon array 440, and for the
electronic addressing of these amplicons to the array 440 following
its activation. Chamber B8 is vented by venting needle 164, via
microfluidic channel 634 which communicates with chamber B8 via
venting aperture 554. The corresponding mechanical operation of the
relevant elements of cartridge 100 of FIGS. 1A-2 including
functionally enhanced core assembly of FIGS. 4A- 11F is described
hereinbelow with reference to FIGS. 22A-22G. A4 The sample
transport needle 174 is moved to be in indirect contact with the
chamber A4, containing a discriminator mix 1. The piston of syringe
194 is raised to an upper intermediate position drawing a desired
volume of discriminator mix 1 into the interior volume of the
syringe 194 via a fluid path formed between syringe 194 and chamber
A4 by tube 190, the interior of transport needle 174 and
microfluidic channel 736 which terminates at an opening of chamber
A4. Chamber A4 is vented by venting needle 164, via microfluidic
channel 636 which terminates at an opening of chamber A4. The
corresponding mechanical operation of the relevant elements of
cartridge 100 of FIGS. 1A-2 including functionally enhanced core
assembly of FIGS. 4A- 11F is described hereinbelow with reference
to FIGS. 23A and B. B10 The sample transport needle 174 is moved to
be in indirect contact with the chamber B10, containing a Buffer
for Discriminator Dilution. The piston of syringe 194 is in an
upper intermediate position, with the interior volume of the
syringe 194 below the piston of the syringe 194 and a volume
including tube 190 and interior of sample transport needle 174
containing discriminator mix 1. Dilution is performed by mixing
discriminator mix 1 and the discriminator dilution buffer contained
in chamber B10 by repeated raising and lowering of the piston of
syringe 194 multiple times. At the end of the mixing, the piston of
syringe 194 is raised and the diluted discriminator is drawn into
the interior volume of the syringe 194 via a fluid path formed
between syringe 194 and chamber B10 by tube 190, the interior of
transport needle 174 and microfluidic channel 748 connected to
chamber B10. The sample transport needle 174 next moves to the
sample transport needle tip location T21, which is in fluid
communication with the carbon array 440, and the piston of syringe
194 is lowered so that the diluted discriminator passes over the
carbon array 440 and binds through hybridization to specific
targets and locations on the array 440 following incubation.
Chamber B10 is vented by venting needle 164, via microfluidic
channel 634 which communicates with chamber B10 via venting
aperture 554. The corresponding mechanical operation of the
relevant elements of cartridge 100 of FIGS. 1A-2 including
functionally enhanced core assembly of FIGS. 4A- 11F is described
hereinbelow with reference to FIGS. 24A-F. A5 The sample transport
needle 174 is moved to be in indirect contact with the chamber A5,
containing a discriminator mix 2. The piston of syringe 194 is
raised to an upper intermediate position drawing a desired volume
of discriminator mix 2 into the interior volume of the syringe 194
via a fluid path formed between syringe 194 and chamber A5 by tube
190, the interior of transport needle 174 and microfluidic channel
736 which terminates at an opening of chamber A5. The biological
process proceeds as described above for discriminator mix 1.
Chamber A5 is vented by venting needle 164, via microfluidic
channel 636 which terminates at an opening of chamber A5. The
corresponding mechanical operation of the relevant elements of
cartridge 100 of FIGS. 1A-2 including functionally enhanced core
assembly of FIGS. 4A- 11F is described hereinbelow with reference
to FIGS. 25A and B. B10 The sample transport needle 174 is moved to
be in indirect contact with the chamber B10, containing a Buffer
for Discriminator Dilution. The piston of syringe 194 is in an
upper intermediate position, with the interior volume of the
syringe 194 below the piston of the syringe 194 and a volume
including tube 190 and interior of sample transport needle 174
containing discriminator mix 2. Dilution is performed by mixing
discriminator mix 2 and the discriminator dilution buffer contained
in chamber B10 by repeated raising and lowering of the piston of
syringe 194 multiple times. At the end of the mixing, the piston of
syringe 194 is raised and the diluted discriminator is drawn into
the interior volume of the syringe 194 via a fluid path formed
between syringe 194 and chamber B10 by tube 190, the interior of
transport needle 174 and microfluidic channel 748 connected to
chamber B10. The sample transport needle 174 next moves to the
sample transport needle tip location T21, which is in fluid
communication with the carbon array 440, and the piston of syringe
194 is lowered so that the diluted discriminator passes over the
carbon array 440 and binds through hybridization to specific
targets and locations on the array 440 following incubation.
Diluted discriminator mix 2 displaces the previously inserted
diluted discriminator mix 1 and the carbon array 440 is drained of
discriminator mix 1 through aperture 462 leading to chamber B14,
which serves as a waste receptacle. Chamber B10 is vented by
venting needle 164, via microfluidic channel 634 which communicates
with chamber B10 via venting aperture 554. The corresponding
mechanical operation of the relevant elements of cartridge 100 of
FIGS. 1A-2 including functionally enhanced core assembly of FIGS.
4A- 11F is described hereinbelow with reference to FIGS. 26A-F. A6
The sample transport needle 174 is moved to be in indirect contact
with the chamber A6, containing a discriminator mix 3. The piston
of syringe 194 is raised to an upper intermediate position drawing
a desired volume of discriminator mix 3 into the interior volume of
the syringe 194 via a fluid path formed between syringe 194 and
chamber A6 by tube 190, the interior of transport needle 174 and
microfluidic channel 736 which terminates at an opening of chamber
A6. The biological process proceeds as described above for
discriminator mix 1. Chamber A6 is vented by venting needle 164,
via microfluidic channel 636 which terminates at an opening of
chamber A6. The corresponding mechanical operation of the relevant
elements of cartridge 100 of FIGS. 1A-2 including functionally
enhanced core assembly of FIGS. 4A- 11F is described hereinbelow
with reference to FIGS. 27A and B. B10 The sample transport needle
174 is moved to be in indirect contact with the chamber B10,
containing a Buffer for Discriminator Dilution. The piston of
syringe 194 is in an upper intermediate position with the interior
volume of the syringe 194 below the piston of the syringe 194 and a
volume including tube 190 and the interior of sample transport
needle 174 containing discriminator mix 3. Dilution is performed by
mixing discriminator mix 3 and the
discriminator dilution buffer contained in chamber B10 by repeated
raising and lowering of the piston of syringe 194 multiple times.
At the end of the mixing, the piston of syringe 194 is raised and
the diluted discriminator is drawn into the interior volume of the
syringe via a fluid path formed between syringe 194 and chamber B10
by tube 190, the interior of transport needle 174 and microfluidic
channel 748 connected to chamber B10. The sample transport needle
174 next moves to the sample transport needle tip location T21,
which is in fluid communication with the carbon array 440, and the
piston of syringe 194 is lowered so that the diluted discriminator
passes over the carbon array 440 and binds through hybridization to
specific targets and locations on the array 440 following
incubation. Diluted discriminator mix 3 displaces the previously
inserted diluted discriminator mix 2 and the carbon array 440 is
drained of discriminator mix 2 through aperture 462 leading to
chamber B14, which serves as a waste receptacle. Chamber B10 is
vented by venting needle 164, via microfluidic channel 634 which
communicates with chamber B10 via venting aperture 554. The
corresponding mechanical operation of the relevant elements of
cartridge 100 of FIGS. 1A-2 including functionally enhanced core
assembly of FIGS. 4A- 11F is described hereinbelow with reference
to FIGS. 28A-F. A7 The sample transport needle 174 is moved to be
in indirect contact with the chamber A7, containing discriminator
mix 4. The piston of syringe 194 is raised to an upper intermediate
position drawing a desired volume of discriminator mix 4 into the
interior volume of the syringe 194 via a fluid path formed between
syringe 194 and chamber A7 by tube 190, the interior of transport
needle 174 and microfluidic channel 736 which terminates at an
opening of chamber A7. The biological process proceeds as described
above for discriminator mix 1. Chamber A7 is vented by venting
needle 164, via microfluidic channel 636 which terminates at an
opening of chamber A6. The corresponding mechanical operation of
the relevant elements of cartridge 100 of FIGS. 1A-2 including
functionally enhanced core assembly of FIGS. 4A- 11F is described
hereinbelow with reference to FIGS. 29A and B. B10 The sample
transport needle 174 is moved to be in indirect contact with the
chamber B10, containing a Buffer for Discriminator Dilution. The
piston of syringe 194 is in an upper intermediate position with the
interior volume of the syringe 194 below the piston of the syringe
194 and a volume including tube 190 and interior of sample
transport needle 174 containing discriminator mix 4. Dilution is
performed by mixing discriminator mix 4 and the discriminator
dilution buffer contained in chamber B10 by repeated raising and
lowering of the piston of syringe 194 multiple times. At the end of
the mixing, the piston of syringe 194 is raised and the diluted
discriminator is drawn into the interior volume of the syringe 194
via a fluid path formed between syringe 194 and chamber B10 by tube
190, the interior of transport needle 174 and microfluidic channel
748 connected to chamber B10. The sample transport needle 174 next
is moved to the sample transport needle tip location T21, which is
in fluid communication with the carbon array 440, and the piston of
syringe 194 is lowered so that the diluted discriminator passes
over the carbon array 440 and binds through hybridization to
specific targets and locations on the array 440 following
incubation. Diluted discriminator mix 4 displaces the previously
inserted diluted discriminator mix 3 and the carbon array 440 is
drained of discriminator mix 3 through aperture 462 leading to
chamber B14, which serves as a waste receptacle. Chamber B10 is
vented by venting needle 164, via microfluidic channel 634 which
communicates with chamber B10 via venting aperture 554. The
corresponding mechanical operation of the relevant elements of
cartridge 100 of FIGS. 1A-2 including functionally enhanced core
assembly of FIGS. 4A- 11F is described hereinbelow with reference
to FIGS. 30A-F. B9 The sample transport needle 174 is moved to be
in indirect contact with chamber B9, containing a reporter
reconstitution buffer and the piston of syringe 194 is raised,
thereby drawing the reporter reconstitution buffer contained in
chamber B9 into the interior volume of the syringe 194 via a fluid
path formed between syringe 194 and chamber B9 by tube 190, the
interior of transport needle 174 and microfluidic channel 748
connected to chamber B9. Reconstitution is performed by repeated
raising and lowering of the piston of syringe 194 multiple times,
so that the reporter reconstitution buffer flows upon a reagent
plug 572 comprising a dried RED reporter. At the end of the
reconstitution, the piston of syringe 194 is raised and the
reconstituted reporter is drawn into the interior volume of the
syringe 194 via the fluid path formed between syringe 194 and
chamber B9 by tube 190, the interior of transport needle 174 and
microfluidic channel 748 connected to chamber B9. The sample
transport needle 174 is next moved to the transport needle tip
location T21, which is in fluid communication with the carbon array
440, and the piston of syringe 194 is lowered so that the
reconstituted reporter passes over the carbon array 440 and binds
through hybridization to the bound discriminators at specific
locations on the array 440 following incubation. Diluted reporter
displaces the previously inserted diluted discriminator mix 4 and
the carbon array 440 is drained of discriminator mix 4 through
aperture 462 leading to chamber B14, which serves as a waste
receptacle. Chamber B9 is vented by venting needle 164, via
microfluidic channel 634 which communicates with chamber B9 via
venting aperture 554. The corresponding mechanical operation of the
relevant elements of cartridge 100 of FIGS. 1A-2 including
functionally enhanced core assembly of FIGS. 4A- 11F is described
hereinbelow with reference to FIGS. 31A-F. B13 The sample transport
needle 174 is moved to be in indirect contact with chamber B13,
containing a sensor wash. The piston of syringe 194 is raised
drawings Low Salt Buffer (LSB) sensor wash from chamber B13 into
the interior volume of the syringe 194 via a fluid path formed
between syringe 194 and chamber B13 by tube 190, the interior of
transport needle 174 and microfluidic channel 734 connected by
chamber B13. Washing is performed by lowering of the piston of
syringe 194 down fully, either in a single step or in multiple
stages, allowing for multiple washes, and by passing the LSB wash
over the carbon array 440. The wash buffer displaces the previously
inserted diluted reporter and the carbon array 440 is drained of
diluted reporter through aperture 462 leading to chamber B14, which
serves as a waste receptacle. Imaging of the carbon array 440
follows. Chamber B13 is vented by venting needle 164, via
microfluidic channel 634 which communicates with chamber B13 via
venting aperture 554. The corresponding mechanical operation of the
relevant elements of cartridge 100 of FIGS. 1A-2 including
functionally enhanced core assembly of FIGS. 4A- 11F is described
hereinbelow with reference to FIGS. 32A-D.
[0119] Based on the biological process set forth in Table II and
the corresponding mechanical operation of elements of cartridge 100
described therein and henceforth, it is understood that venting
needle 164 is a particularly preferred embodiment of a linearly
displaceable venting element, operative as a venter for venting at
least one of plurality of operational volumes A1-A13. B1-B14 and
sample receiving chamber 610. Furthermore, it is understood that
sample transport needle 174 is a particularly preferred embodiment
of a linearly displaceable transport element operative, in
coordination with venting needle 164, to transfer fluid solutions
from at least one of the plurality of operational volumes A1-A13.
B1-B14 and sample receiving chamber 610 to at least another of the
plurality of operational volumes A1-A13, B1-B14 and sample
receiving chamber 610, by sequentially communicating with interiors
of at least some of the plurality of operational volumes A1-A13,
B1-B14 and sample receiving chamber 610.
[0120] It is further understood that sample transport needle 174 in
cooperation with a fluid flow driving assembly, preferably embodied
as syringe 194 communicating with sample transport needle 174 by
way of flexible tube 190, forms a part of a fluid solution
transporter assembly for transporting fluid between ones of
operational volumes A1-A13. B1-B14 and sample receiving chamber
610. Particularly preferably, the fluid flow driving assembly
formed by syringe 194 is operative to drive fluid solutions through
the transport element formed by sample transport needle 174 and
between ones of plurality of operational volumes A1-A13, B1-B14 and
sample receiving chamber 610.
[0121] It is appreciated that the composition, concentration and
functioning of the various solutions, such as reagents or buffers,
described hereinabove are typically known in the art and are
provided as examples. The role that each chemical plays in the
overall process may change from one sample to another. Similarly,
different samples may require different chemicals, thus changing
the exact contents of each chamber.
[0122] Reference is now made to FIGS. 12A, 12B, 12C and 12D, which
are simplified illustrations of typical initial steps in the
operation of core assembly 400 of FIGS. 4A-11F.
[0123] As seen in FIG. 12A, preferably initially sample insertion
port sealing cover 860 is disengaged from sample receiving chamber
610, by way of raising of sample insertion port sealing cover 860
in a direction indicated by an arrow 1200. Sample insertion port
612 of sample receiving chamber 610 is thereby unsealed, thus
allowing access thereto.
[0124] As seen in FIG. 12B, a sample carrying pipette 1202 is
preferably inserted into sample receiving chamber 610 via sample
insertion port 612, as indicated by an arrow 1203, and a sample
1204 delivered thereto.
[0125] As seen in FIG. 12C, following the delivery of sample 1204
to sample receiving chamber 610 by pipette 1202, sample carrying
pipette 1202 is preferably removed from sample receiving chamber
610, as indicated by an arrow 1205.
[0126] As seen in FIG. 12D, sample insertion port sealing cover 860
is preferably lowered in a direction indicated by an additional
arrow 1206, thus sealing sample 1204 within sample receiving
chamber 610 by way of the sealing of sample insertion port 612 by
sample inlet sealing portion 806.
[0127] Following the insertion of sample 1204 within cartridge 100
and prior to the commencement of the biological processing thereof,
all frangible seals within functionally enhanced core assembly 400
are preferably opened. The opening of the frangible seals may be
carried out using a set of spring loaded plungers external to
cartridge 100. The spring loaded plungers may access the frangible
seals via plurality of frangible seal plunger access apertures 126
and 146 shown in FIGS. 1A and 1B preferably in accordance with the
teachings of WO2012019599, entitled `Device for Transporting Small
Volumes of a Fluid, in particular a Micropump or Microvalve`, the
description of which is hereby incorporated by reference.
[0128] Following the insertion of sample 1204 into sample receiving
chamber 610 and the breaking of the frangible seals within
cartridge 100, cartridge 100 is ready for biological processing of
sample 1204, as described hereinabove with reference to Tables I-II
and hereinbelow with reference to FIGS. 13A-32D. It is appreciated
that although functionally enhanced core assembly 400 is
illustrated in FIGS. 13A-32D as including PCR amplification
subsystem 600, and biological processing of sample 1204
correspondingly including PCR amplification steps, functionally
enhanced core assembly 400 may alternatively be implemented with an
RCA amplification subsystem and the biological processing of sample
1204 therein may be suitably modified.
[0129] Reference is now made to FIGS. 13A-13G, which are simplified
illustrations of typical further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 13A shows an operational
state corresponding to that of FIG. 12D, FIGS. 13A-13G show the
microfluidic base portion of FIGS. 6A-6H and FIGS. 13B-13G show
operative engagement with chamber B1 thereof.
[0130] FIG. 13A shows sample 1204 located in sample receiving
chamber 610 and venting needle 164 and sample transport needle 174
poised above array 630 of recesses 632 and array 730 of recesses
732, respectively. It is understood that venting needle 164 and
sample transport needle 174 are preferably respectively removed
from venting needle slidable mounting protrusions 162 (FIG. 2) and
sample transport needle slidable mounting protrusions 172 (FIG. 2)
and positioned as illustrated in FIG. 13A by way of at least one
needle motion motor, external to cartridge 100. A piston 1300 of
syringe 194 is preferably in a fully lowered position within an
interior volume 1302 of syringe 194, such that syringe 194 is
empty.
[0131] FIG. 13B shows the lowering of venting needle 164, as
indicated by an arrow 1304, and of sample transport needle 174, as
indicated by an additional arrow 1306. A hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location V1
and a hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T2.
[0132] It is appreciated that in order for venting needle 164 to
enter any one of venting needle tip locations V1-V23, venting
needle 164 preferably penetrates corresponding ones of recesses 872
(FIG. 9B) sealingly overlying array 530 of venting needle guiding
protrusions 532 (FIG. 5). Similarly, it is appreciated that in
order for sample transport needle 174 to enter any one of sample
transport needle tip locations T1-T23, sample transport needle 174
preferably penetrates corresponding ones of recesses 892 (FIG. 10B)
sealingly overlying array 580 of transport needle guiding
protrusions 582 (FIG. 5). It is appreciated that septa 802 and 804
including recesses 872 (FIG. 9B) and 892 (FIG. 10B) thus are each
preferably penetrable by a penetrating element, which penetrating
element is preferably embodied as venting needle 164 and sample
transport needle 174, respectively.
[0133] FIG. 13C shows the raising of piston 1300, as indicated by
an arrow 1320, thereby drawing a reaction liquid 1322 held in
chamber B1 at least partially into the interior volume 1302 of
syringe 194 below piston 1300. Piston 1300 is preferably operated
by a syringe motor, external to cartridge 100. Reaction liquid 1322
is preferably a buffer containing Proteinase K, as indicated in
Table 1. As shown in FIG. 13C, reaction liquid 1322 preferably
exits chamber B1 via reagent transport aperture 556 and is drawn
along microfluidic channel 734 in a direction towards sample
transport needle tip location T2, as indicated by an arrow 1324.
Reaction liquid 1322 preferably enters the hollow pointed end 1312
of sample transport needle 174 located at sample transport needle
tip location T2 and is preferably drawn along an interior
passageway 1326 of sample transport needle 174, as indicated by an
arrow 1328. Interior passageway 1326 of sample transport needle 174
is preferably in fluid connection with an interior of sample
transport tube 190, along which sample transport tube 190 reaction
liquid 1322 is preferably drawn in a direction indicated by an
arrow 1330. The interior of sample transport tube 190 in turn
communicates with the interior volume 1302 of syringe 194 via luer
connector 192.
[0134] Chamber B1 is preferably vented by venting needle 164
located at venting needle tip location V1 and in communication with
chamber B1 via microfluidic channel 634 terminating at venting
aperture 554.
[0135] Following the transfer of reaction liquid 1322 from chamber
B1 to syringe 194, sample transport and venting needles 174, 164
are preferably further lowered, as illustrated in FIG. 13D. Venting
needle 164 is preferably lowered such that hollow pointed end 1310
thereof enters venting needle tip location V22, as indicated by an
arrow 1340. Sample transport needle 174 is preferably lowered such
that hollow pointed end 1312 thereof enters transport needle tip
location T23, as indicated by an arrow 1342.
[0136] FIG. 13E shows the lowering of piston 1300, as indicated by
an arrow 1344, thus forcing reaction liquid 1322 from syringe 194
into sample receiving chamber 610. As appreciated from
consideration of FIG. 13E, reaction liquid 1322 is preferably
forced from syringe 194, as indicated by an arrow 1345, along tube
190 in a direction indicated by an arrow 1346, through interior
passageway 1326 of sample transport needle 174, as indicated by an
arrow 1347, and into sample receiving chamber 610 via microfluidic
channel 740 interconnecting sample transport needle tip location
T23 and sample receiving chamber 610, as indicated by an arrow
1348. It is appreciated that sample receiving chamber 610 is thus a
particularly preferred embodiment of a sample insertion
subassembly, communicating with at least one of the plurality of
operational volumes A1-A13 and B1-B14.
[0137] Sample receiving chamber 610 is preferably vented by venting
needle 164 located at venting needle tip location V22 and in
communication with sample receiving chamber 610 via a microfluidic
channel 1349.
[0138] FIG. 13F shows the raising and lowering of piston 1300, as
indicated by an arrow 1350, thereby repeatedly drawing the sample
1204 and reaction liquid 1322 at least partially into the interior
volume 1302 of syringe 194 below piston 1300. FIG. 13F also shows
the repeated displacement of the sample 1204 and reaction liquid
1322 within interior volume 1302 of syringe 194, as indicated by an
arrow 1352; the repeated displacement of the sample 1204 and
reaction liquid 1322 within tube 190, as indicated by an arrow
1354; the repeated displacement of the sample 1204 and reaction
liquid 1322 within interior passageway 1326 of sample transport
needle 174, as indicated by an arrow 1355; and the repeated
displacement of the sample 1204 and reaction liquid 1322 into and
out of sample receiving chamber 610 via microfluidic channel 740,
as indicated by an arrow 1356. It is appreciated that the raising
and lowering of piston 1300 is preferably carried out multiple
times in order to mix sample 1204 and reaction liquid 1322.
[0139] Following the mixing of sample 1204 with reaction liquid
1322, the sample 1204 and reaction liquid 1322 are preferably fully
or near fully drawn into interior volume 1302 of syringe 194 by way
of the raising of piston 1300 into a fully extended position, as
indicated by an arrow 1358 in FIG. 13G. The mixture of reaction
liquid 1322 and sample 1204 is preferably drawn out of sample
receiving chamber 610 via microfluidic channel 740, as indicated by
an arrow 1360, through interior passageway 1326 of sample transport
needle 174, as indicated by an arrow 1361, and thereafter along
tube 190, as indicated by an arrow 1362, and into interior volume
1302 of syringe 194, beneath piston 1300, as indicated by an arrow
1364.
[0140] As described hereinabove in Table 1 and Table 1, the sample
1204 is preferably incubated at 56.degree. C. for 8-10 minutes in
the presence of reaction liquid 1322, prior to being transferred,
via piston 1300 and sample transport needle 174, to chamber B2, as
described hereinbelow with reference to FIG. 14A. It is appreciated
that the heating of the sample 1204 is achieved utilizing a heating
element which is not part of cartridge 100 of FIGS. 1A-2.
[0141] Reference is now made to FIGS. 14A-14E, which are simplified
illustrations of typical further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 14A shows an operational
state subsequent to that of FIG. 13G, FIGS. 14A-14E showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B2 thereof.
[0142] FIG. 14A shows the raising of venting needle 164, as
indicated by an arrow 1400, and of sample transport needle 174, as
indicated by an additional arrow 1402. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location V2
and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T3. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V2 and chamber
B2, which fluid venting path is preferably formed by microfluidic
channel 634 terminating at venting aperture 554 of chamber B2. A
fluid transport path is preferably present between pointed end 1312
of sample transport needle 174 at sample transport needle tip
location T3 and chamber B2, which fluid transport path is
preferably formed by microfluidic channel 734 terminating at
reagent transport aperture 556 of chamber B2.
[0143] Chamber B2 preferably contains an additional reaction liquid
1410, such as a lysis solution containing Guanidinium Thiocyanate,
ionic detergent in TRIS-HCL, as well as magnetic beads 1412,
preferably comprising modified Sera-Mag.TM. SpeedBeads Magnetic
particles, commercially available from GE Life Sciences.
[0144] FIG. 14B shows the lowering of piston 1300 of syringe 194,
as indicated by an arrow 1420, thus forcing sample 1204 and
reaction liquid 1322 out of interior volume 1302, as indicated by
an arrow 1421, via tube 190, interior passageway 1326 of sample
transport needle 174 and microfluidic channel 734, as indicated by
arrows 1422, into chamber B2.
[0145] FIG. 14C shows raising and lowering of piston 1300 of
syringe 194, as indicated by an arrow 1424, thereby repeatedly
drawing the sample 1204, the reaction liquids 1322 and 1410 and
beads 1412 at least partially into the interior volume 1302 of
syringe 194 below piston 1300, as indicated by an arrow 1425. It is
appreciated that the raising and lowering of piston 1300 is
preferably carried out multiple times to produce lysis of cells
forming part of the sample 1204, which is indicated symbolically by
breaking up of dots which represent the cells of sample 1204.
Nucleic acids released from the lysed cells preferably bind to
magnetic beads 1412 via a coating thereof.
[0146] FIG. 14D shows the raising of piston 1300 of syringe 194, as
indicated by an arrow 1428, thereby drawing the magnetic beads 1412
and the nucleic acids from sample 1204 bound thereto, as well as
reaction liquids 1322 and 1410, into the interior volume 1302 of
syringe 194 below piston 1300. Following the raising of piston
1300, a magnet 1430, which magnet 1430 is not part of cartridge 100
of FIGS. 1A-2, is brought into propinquity with interior volume
1302 of syringe 194, as indicated by an arrow 1432, such that
magnet 1430 attracts the magnetic beads 1412 to which nucleic acids
from sample 1204 are bound.
[0147] FIG. 14E shows partial lowering of piston 1300, in a
direction indicated by an arrow 1440, such as to separate most of
the reaction liquids 1322 and 1410 and the remainder of sample
1204, including cell debris, from the magnetic beads 1412 and the
nucleic acids bound thereto. Most of the reaction liquids 1322 and
1410 as well as the remainder of sample 1204, including cell
debris, is preferably returned to chamber B2 via tube 190 as
indicated by an arrow 1442, sample transport needle 174, as
indicated by an arrow 1444, and microfluidic channel 734, as
indicated by an arrow 1446, where it remains, while the magnetic
beads 1412 and the nucleic acids bound thereto are retained
congregated in volume 1302 by magnet 1430.
[0148] It is appreciated that virtually all of reaction liquids
1322 and 1410 are returned to chamber B2 and that only traces
thereof remain volume 1302 of syringe 194 together with magnetic
beads 1412 and the nucleic acids of sample 1204 bound thereto.
[0149] Reference is now made to FIGS. 15A-15E, which are simplified
illustrations of typical still further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 15A shows an operational
state subsequent to that of FIG. 14E, FIGS. 15A 15E showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B3 thereof.
[0150] FIG. 15A shows the lowering of venting needle 164, as
indicated by an arrow 1500, and of sample transport needle 174, as
indicated by an additional arrow 1502. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location V4
and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T4. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V4 and chamber
B3, which fluid venting path is preferably formed by microfluidic
channel 634 terminating at venting aperture 554 of chamber B3. A
fluid transport path is preferably present between pointed end 1312
of sample transport needle 174 at transport needle tip location
sample transport needle tip location T4 and chamber B3, which fluid
transport path is preferably formed by microfluidic channel 734
terminating at reagent transport aperture 556 of chamber B3.
[0151] Chamber B3 preferably contains an additional reaction liquid
1504, preferably comprising a wash buffer I, such as Guanidinium
Thiocyanate, ionic detergent in Tris-HCl (pH 7.4), isopropanol,
(DEPC)-Water.
[0152] FIG. 15B shows removal of magnet 1430 from syringe 194, as
indicated by an arrow 1505, and the raising of the piston 1300, as
indicated by an arrow 1506, thus drawing reaction liquid 1504 from
chamber B3 into the interior volume 1302 underlying piston 1300,
which interior volume 1302 additionally already holds magnetic
beads 1412 and the nucleic acids of sample 1204 bound thereto as
well as any remaining traces of reaction liquids 1322 and 1410 and
residual sample material, as described hereinabove with reference
to FIG. 14D.
[0153] As seen in FIG. 15B, reaction liquid 1504 is drawn from
chamber B3 through microfluidic channel 734, as indicated by an
arrow 1508, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 1510, thereafter
along tube 190, as indicated by an arrow 1512, and thereafter into
interior volume 1302 underlying piston 1300.
[0154] FIG. 15C shows repeated lowering and raising of the piston
1300, as indicated by an arrow 1520, thereby forcing the reaction
liquid 1504 together with beads 1412 and the nucleic acids bound
thereto as well as any remaining traces of reaction liquids 1322
and 1410 and sample material, repeatedly into and out of the
interior volume 1302 of syringe 194, below piston 1300 via the
interior passageway 1326 of sample transport needle 174.
[0155] FIG. 15C also shows the repeated displacement of the
reaction liquid 1504 together with beads 1412 and the nucleic acids
bound thereto as well as any remaining traces of reaction liquids
1322 and 1410 and sample material, within interior volume 1302 of
syringe 194, as indicated by an arrow 1522; the repeated
displacement of the reaction liquid 1504 together with beads 1412
and the nucleic acids bound thereto as well as any remaining traces
of reaction liquids 1322 and 1410 and sample material within tube
190, as indicated by an arrow 1524; the repeated displacement of
the reaction liquid 1504 together with beads 1412 and the nucleic
acids bound thereto as well as any remaining traces of reaction
liquids 1322 and 1410 and sample material within interior
passageway 1326 of sample transport needle 174, as indicated by an
arrow 1526, and the repeated displacement of the reaction liquid
1504 together with beads 1412 and the nucleic acids bound thereto
as well as any remaining traces of reaction liquids 1322 and 1410
and sample material within microfluidic channel 734, as indicated
by an arrow 1527. It is appreciated that the raising and lowering
of piston 1300 is preferably carried out multiple times in order to
wash beads 1412 and the nucleic acids bound thereto with reaction
liquid 1504.
[0156] FIG. 15D shows the raising of piston 1300 of syringe 194, as
indicated by an arrow 1528, such that reaction liquid 1504 together
with now washed beads 1412 and the nucleic acids bound thereto as
well as additional components as described hereinabove, are at
least near fully drawn into the interior volume 1302 of syringe 194
beneath piston 1300.
[0157] FIG. 15D also shows magnet 1430 being brought into
propinquity with interior volume 1302 of syringe 194, as indicated
by an arrow 1529, such that magnet 1430 attracts magnetic beads
1412 to which nucleic acids from sample 1204 are bound.
[0158] FIG. 15E shows partial lowering of the piston 1300, in a
direction indicated by an arrow 1530, such as to separate most of
the reaction liquid 1504 and any remaining traces of reaction
liquids of reaction liquids 1322 and 1410 and any remainder of
sample 1204, including cell debris, from the magnetic beads 1412
and the nucleic acids bound thereto. Most of the material other
than the beads 1412 and nucleic acids bound thereto is returned to
chamber B3, where it remains, while the magnetic beads 1412 and the
nucleic acids bound thereto are retained congregated in interior
volume 1302 by magnet 1430.
[0159] It is appreciated that virtually all of reaction liquid 1504
is returned to chamber B3, via tube 190 and sample transport needle
174, as indicated by an arrow 1532, and that only traces thereof
remain in volume 1302 together with magnetic beads 1412 and the
nucleic acids bound thereto.
[0160] Reference is now made to FIGS. 16A-16E which are simplified
illustrations of typical still further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 16A shows an operational
state subsequent to that of FIG. 15E, FIGS. 16A-16E showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B4 thereof.
[0161] FIG. 16A shows the lowering of venting needle 164, as
indicated by an arrow 1600, and of sample transport needle 174, as
indicated by an additional arrow 1602. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location V5
and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T6. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V5 and chamber
B4, which fluid venting path is preferably formed by microfluidic
channel 634 terminating at venting aperture 554 of chamber B4. A
fluid transport path is preferably present between pointed end 1312
of sample transport needle 174 at sample transport needle tip
location T6 and chamber B4, which fluid transport path is
preferably formed by microfluidic channel 734 terminating at
reagent transport aperture 556 of chamber B4.
[0162] Chamber B4 preferably contains an additional reaction liquid
1604, preferably comprising a wash buffer 11, such as KCl/Tris (pH
7), Ethanol, in Rnasc-free water.
[0163] FIG. 16B shows removal of magnet 1430 from syringe 194, as
indicated by an arrow 1605, and the raising of the piston 1300, as
indicated by an arrow 1606, thus drawing reaction liquid 1604 from
chamber B4 into the interior volume 1302 underlying piston 1300,
which interior volume 1302 additionally already holds magnetic
beads 1412 and the nucleic acids bound thereto of sample 1204, as
well as any remaining traces of reaction liquids 1322, 1410 and
1504 and any remainder of sample 1204, including cell debris.
[0164] As seen in FIG. 16B, reaction liquid 1604 is drawn from
chamber B4 through microfluidic channel 734, as indicated by an
arrow 1608, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 1610, further
thereafter along tube 190, as indicated by an arrow 1612, and still
further thereafter into interior volume 1302 underlying piston
1300, as indicated by an arrow 1614.
[0165] FIG. 16C shows repeated lowering and raising of the piston
1300, as indicated by an arrow 1620, thereby forcing the reaction
liquid 1604 together with beads 1412 and the nucleic acids bound
thereto as well as any remaining traces of reaction liquids 1322,
1410 and 1504 and sample material, repeatedly into and out of the
interior volume 1302 of syringe 194, below piston 1300 via the
interior passageway 1326 of sample transport needle 174.
[0166] FIG. 16C also shows the repeated displacement of the
reaction liquid 1604 together with beads 1412 and the nucleic acids
bound thereto as well as any remaining traces of reaction liquids
1322, 1410 and 1504 and sample material, within interior volume
1302 of syringe 194, as indicated by an arrow 1622; the repeated
displacement of the reaction liquid 1604 together with beads 1412
and the nucleic acids bound thereto as well as any remaining traces
of reaction liquids 1322, 1410 and 1504 and sample material within
tube 190, as indicated by an arrow 1624; the repeated displacement
of the reaction liquid 1604 together with beads 1412 and the
nucleic acids bound thereto as well as any remaining traces of
reaction liquids 1322, 1410 and 1504 and sample material within
interior passageway 1326 of sample transport needle 174, as
indicated by an arrow 1626; and the repeated displacement of the
reaction liquid 1604 together with beads 1412 and the nucleic acids
bound thereto as well as any remaining traces of reaction liquids
1322, 1410 and 1504 and sample material within microfluidic channel
734, as indicated by an arrow 1627. It is appreciated that the
raising and lowering of piston 1300 is preferably carried out
multiple times in order to wash beads 1412 and the nucleic acids
bound thereto with reaction liquid 1604.
[0167] FIG. 16D shows the raising of piston 1300 of syringe 194, as
indicated by an arrow 1628, such that reaction liquid 1604 together
with now washed beads 1412 and the nucleic acids bound thereto as
well as additional components as described hereinabove, are at
least near fully drawn into the interior volume 1302 of syringe 194
beneath piston 1300.
[0168] FIG. 16D also shows magnet 1430 being brought into
propinquity with interior volume 1302 of syringe 194, as indicated
by an arrow 1629, such that magnet 1430 attracts magnetic beads
1412 to which nucleic acids from sample 1204 are bound.
[0169] FIG. 16E shows partial lowering of the piston 1300, in a
direction indicated by an arrow 1630, such as to separate most of
the reaction liquid 1604 and any remaining traces of reaction
liquids 1322, 1410 and 1504 and any remainder of sample 1204,
including cell debris, from the magnetic beads 1412 and the nucleic
acids bound thereto. Most of the material other than the beads 1412
and nucleic acids bound thereto is returned to chamber B4, where it
remains, while the magnetic beads 1412 and the nucleic acids bound
thereto are retained congregated in interior volume 1302 by magnet
1430.
[0170] It is appreciated that virtually all of reaction liquid 1604
is returned to chamber B4, via tube 190 and sample transport needle
174, as indicated by an arrow 1632, and that only traces thereof
remain in volume 1302 together with magnetic beads 1412 and the
nucleic acids bound thereto.
[0171] Reference is now made to FIGS. 17A-17E, which are simplified
illustrations of typical still further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 17A shows an operational
state subsequent to that of FIG. 16E, FIGS. 17A-17E showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B5 thereof.
[0172] FIG. 17A shows the lowering of venting needle 164, as
indicated by an arrow 1700, and of sample transport needle 174, as
indicated by an additional arrow 1702. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location V7
and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T8. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V7 and chamber
B5, which fluid venting path is preferably formed by microfluidic
channel 634 terminating at venting aperture 554 of chamber B5. A
fluid transport path is preferably present between pointed end 1312
of sample transport needle 174 at sample transport needle tip
location T8 and chamber B5, which fluid transport path is
preferably formed by microfluidic channel 734 terminating at
reagent transport aperture 556 of chamber B5.
[0173] Chamber B5 preferably contains an additional reaction liquid
1704, preferably comprising a wash buffer III such as KCl/Tris (pH
7), in Rnase-free water, as detailed in Tables I and II.
[0174] FIG. 17B shows removal of magnet 1430 from syringe 194, as
indicated by an arrow 1705, and the raising of the piston 1300, as
indicated by an arrow 1706, thus drawing reaction liquid 1704 from
chamber B5 into the interior volume 1302 underlying piston 1300,
which interior volume 1302 additionally already holds magnetic
beads 1412 and the nucleic acids bound thereto of sample 1204, as
well as any remaining traces of reaction liquids 1322, 1410, 1504
and 1604 and any remainder of sample 1204, including cell
debris.
[0175] As seen in FIG. 17B, reaction liquid 1704 is drawn from
chamber B5 through microfluidic channel 734, as indicated by an
arrow 1708, into interior passageway 1326 of sample transport
needle 174, as indicated by an arrow 1710, thereafter along tube
190, as indicated by an arrow 1712, and thereafter into interior
volume 1302 underlying piston 1300, as indicated by an arrow
1714.
[0176] FIG. 17C shows repeated lowering and raising of the piston
1300, as indicated by an arrow 1720, thereby forcing the reaction
liquid 1704 together with beads 1412 and the nucleic acids bound
thereto as well as any remaining traces of reaction liquids 1322,
1410, 1504 and 1604 and sample material, repeatedly into and out of
the interior volume 1302 of syringe 194, below piston 1300 via the
interior passageway 1326 of sample transport needle 174.
[0177] FIG. 17C also shows the repeated displacement of the
reaction liquid 1704 together with beads 1412 and the nucleic acids
bound thereto as well as any remaining traces of reaction liquids
1322, 1410, 1504 and 1604 and sample material, within interior
volume 1302 of syringe 194, as indicated by an arrow 1722; the
repeated displacement of the reaction liquid 1704 together with
beads 1412 and the nucleic acids bound thereto as well as any
remaining traces of reaction liquids 1322, 1410, 1504 and 1604 and
sample material within tube 190, as indicated by an arrow 1724; the
repeated displacement of the reaction liquid 1704 together with
beads 1412 and the nucleic acids bound thereto as well as any
remaining traces of reaction liquids 1322, 1410, 1504 and 1604 and
sample material within interior passageway 1326 of sample transport
needle 174, as indicated by an arrow 1726; and the repeated
displacement of the reaction liquid 1704 together with beads 1412
and the nucleic acids bound thereto as well as any remaining traces
of reaction liquids 1322, 1410, 1504 and 1604 and sample material
within microfluidic channel 734, as indicated by an arrow 1727. It
is appreciated that the raising and lowering of piston 1300 is
preferably carried out multiple times in order to wash beads 1412
and the nucleic acids bound thereto with reaction liquid 1704.
[0178] FIG. 17D shows the raising of piston 1300 of syringe 194, as
indicated by an arrow 1728, such that reaction liquid 1704 together
with now washed beads 1412 and the nucleic acids bound thereto as
well as additional components as described hereinabove, are at
least near fully drawn into the interior volume 1302 of syringe 194
beneath piston 1300.
[0179] FIG. 17D also shows magnet 1430 being brought into
propinquity with interior volume 1302 of syringe 194, as indicated
by an arrow 1729, such that magnet 1430 attracts magnetic beads
1412 to which nucleic acids from sample 1204 are bound.
[0180] FIG. 17E shows partial lowering of the piston 13), in a
direction indicated by an arrow 1730, such as to separate most of
the reaction liquid 1704 and any remaining traces of reaction
liquids 1322, 1410, 1504 and 1604 and any remainder of sample 1204,
including cell debris, from the magnetic beads 1412 and the nucleic
acids bound thereto. Most of the material other than the beads 1412
and nucleic acids bound thereto is returned to chamber B5, where it
remains, while the magnetic beads 1412 and the nucleic acids bound
thereto are retained congregated in interior volume 1302 by magnet
1430.
[0181] It is appreciated that virtually all of reaction liquid 1704
is returned to chamber B5, via tube 190 and sample transport needle
174, as indicated by an arrow 1732, and that only traces thereof
remain in volume 1302 together with magnetic beads 1412 and the
nucleic acids bound thereto.
[0182] Reference is now made to FIGS. 18A-18D, which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 18A shows an operational
state subsequent to that of FIG. 17E, FIGS. 18A-18D showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber 86 thereof.
[0183] FIG. 18A shows the lowering of venting needle 164, as
indicated by an arrow 1800, and of sample transport needle 174, as
indicated by an additional arrow 1802. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location V9
and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T9. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V9 and chamber
B6, which fluid venting path is preferably formed by microfluidic
channel 634 terminating at venting aperture 554 of chamber B6. A
fluid transport path is preferably present between pointed end 1312
of sample transport needle 174 at sample transport needle tip
location T9 and chamber B6, which fluid transport path is
preferably formed by microfluidic channel 734 terminating at
reagent transport aperture 556 of chamber B6.
[0184] Chamber B6 preferably contains an additional reaction liquid
1804, preferably comprising an elution buffer, such as Tris based
buffer or Ultra-Pure DNase and RNase free water (DDW), commercially
available from Biological Industries of Beit Haemek, Israel.
[0185] FIG. 18B shows removal of magnet 1430 in a direction away
from interior volume 1302 of syringe 194, as indicated by an arrow
1805, and the subsequent raising of piston 1300, as indicated by an
arrow 1806, thus drawing reaction liquid 1804 from chamber B6 into
interior volume 1302 underlying piston 1300, via sample transport
needle 174 and tube 190. It is appreciated that interior volume
1302 already holds magnetic beads 1412 and the nucleic acids from
sample 1204 as well as any remaining traces of reaction liquids
1322, 1410, 1504, 1604 and 1704, as described hereinabove with
reference to FIG. 17E.
[0186] As seen in FIG. 18B, reaction liquid 1804 is drawn from
chamber B6 through microfluidic channel 734, as indicated by an
arrow 1808, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 1810, further
thereafter along tube 190, as indicated by an arrow 1812, and still
further thereafter into interior volume 1302 underlying piston
1300, as indicated by an arrow 1814, wherein reaction liquid 1804
engages with content already present in syringe 194.
[0187] FIG. 18C shows subsequent repeated lowering and raising of
piston 1300, as indicated by an arrow 1820, thereby forcing
reaction liquid 1804 and any remainder of sample 1204, including
cell debris, as well as magnetic beads 1412 and the nucleic acids
bound thereto, at least partially out of and into the interior
volume 1302 of syringe 194 below piston 1300. As a result, the
nucleic acids from sample 1204 are disengaged from magnetic beads
1412.
[0188] FIG. 18C also shows the repeated displacement of the
reaction liquid 1804 together with beads 1412 and the nucleic acids
bound thereto as well as any remaining traces of reaction liquids
1322, 1410, 1504, 1604 and 1704 and sample material, within
interior volume 1302 of syringe 194, as indicated by an arrow 1822;
the repeated displacement of the reaction liquid 1804 together with
beads 1412 and the nucleic acids bound thereto as well as any
remaining traces of reaction liquids 1322, 1410, 1504, 1604 and
1704 and sample material within tube 190, as indicated by an arrow
1824; the repeated displacement of the reaction liquid 1804
together with beads 1412 and the nucleic acids bound thereto as
well as any remaining traces of reaction liquids 1322, 1410, 1504,
1604 and 1704 and sample material within interior passageway 1326
of sample transport needle 174, as indicated by an arrow 1826; and
the repeated displacement of the reaction liquid 1804 together with
beads 1412 and the nucleic acids bound thereto as well as any
remaining traces of reaction liquids 1322, 1410, 1504, 1604 and
1704 and sample material within microfluidic channel 734, as
indicated by an arrow 1827. It is appreciated that the raising and
lowering of piston 1300 is preferably carried out multiple times in
order to disengage the nucleic acids from sample 1204 from magnetic
beads 1412.
[0189] FIG. 18D shows the nucleic acids from sample 1204 separated
from the magnetic beads 1412 in the solution containing the
reaction liquid 1804 located within volume 1302, underlying piston
1300, which piston 1300 is in a fully raised position in FIG. 18D
as indicated by an arrow 1830. The separated nucleic acids am
symbolically shown and designated by reference numeral 1840.
[0190] It is appreciated that virtually all of reaction liquid
1804, together with magnetic beads 1412 and the separated nucleic
acids 1840, is drawn from chamber B6 through microfluidic channel
734, as indicated by an arrow 1842, through interior passageway
1326 of sample transport needle 174, as indicated by an arrow 1844,
thereafter along tube 190, as indicated by an arrow 1846, and
thereafter into interior volume 1302 underlying piston 1300.
[0191] Reference is now made to FIGS. 19A-19D, which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 19A shows an operational
state subsequent to that of FIG. 18D, FIGS. 19A-19D showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber A3 thereof.
[0192] FIG. 19A shows the raising of venting needle 164, as
indicated by an arrow 1900, and of sample transport needle 174, as
indicated by an additional arrow 1902. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location V8
and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T7. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V8 and chamber
A3, which fluid venting path is preferably formed by microfluidic
channel 636 terminating at an opening of chamber A3. A fluid
transport path is preferably present between pointed end 1312 of
sample transport needle 174 at sample transport needle tip location
T7 and chamber A3, which fluid transport path is preferably formed
by microfluidic channel 736, terminating at another opening of
chamber A3.
[0193] A permanent magnet 1904 is preferably located in
juxtaposition to and exteriorly of chamber A3. Magnet 1904
preferably extends generally parallel to and along the length of
chamber A3, such that an interior of chamber A3 is preferably in
magnetic communication with magnet 1904. Magnet 1904 preferably
does not form a part of cartridge 100.
[0194] FIG. 19B shows lowering of the piston 1300, as indicated by
an arrow 1906, thus forcing the reaction liquid 1804, including
separated nucleic acids 1840 and beads 1412, into chamber A3.
[0195] As seen in FIG. 19B, reaction liquid 1804 including
separated nucleic acids 1840 and beads 1412, is forced out of
volume 1302 of syringe 194 into tube 190, as indicated by an arrow
1908, thereafter through tube 190, as indicated by an arrow 1909,
thereafter through interior passageway 1326 of sample transport
needle 174, as indicated by an arrow 1910, and thereafter through
microfluidic channel 736 into chamber A3, as indicated by an arrow
1912, which chamber A3 is vented by venting needle 164.
[0196] FIG. 19C shows magnetic attraction of beads 1412 by magnet
1904 such that only the unbound nucleic acids 1840 from sample 1204
remain free in the reaction liquid 1804 located in chamber A3.
[0197] FIG. 19D shows the partial raising of piston 1300 to an
intermediate position thereof, as indicated by an arrow 1920,
thereby drawing a portion of the unbound nucleic acids 1840 from
sample 1204 and the reaction liquid 1804 from chamber A3 through
microfluidic channel 736, as indicated by an arrow 1922, interior
passageway 1326 of sample transport needle 174, as indicated by an
arrow 1924, thereafter along tube 190, as indicated by an arrow
1926, and thereafter into the interior volume 1302 of syringe 194
below piston 1300, as indicated by an arrow 1928.
[0198] Reference is now made to FIGS. 20A-20D, which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 20A shows an operational
state subsequent to that of FIG. 19D, FIGS. 20A-20D showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B7 thereof.
[0199] FIG. 20A shows the lowering of venting needle 164, as
indicated by an arrow 2000, and of sample transport needle 174, as
indicated by an additional arrow 2002. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V10 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T10. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V10 and chamber
B7, which fluid venting path is preferably formed by microfluidic
channel 634 terminating at venting aperture 554 of chamber B7. A
fluid transport path is preferably present between pointed end 1312
of sample transport needle 174 at sample transport needle tip
location T10 and chamber B7, which fluid transport path is
preferably formed by microfluidic channel 734 terminating at
reagent transport aperture 556 of chamber B7.
[0200] Chamber B7 preferably contains an additional reaction liquid
2004, preferably comprising an elution dilution buffer, such as
Sample Buffer A (L-Histidine, 1-Thioglycerol in DDW) or DDW
(Ultra-Pure DNase and RNase free water), commercially available
from Biological Industries, of Beit Haemek, Israel.
[0201] FIG. 20B shows the raising of piston 1300 to a fully
extended position thereof, as indicated by an arrow 2006, thus
drawing at least a portion of reaction liquid 2004 from chamber B7
into interior volume 1302 underlying piston 1300, via microfluidic
channel 734, interior passageway 1326 of sample transport needle
174 and tube 190. It is appreciated that interior volume 1302
already holds unbound nucleic acids 1840 from sample 1204 and
reaction liquid 1804, as described hereinabove with reference to
FIG. 19D.
[0202] As seen in FIG. 20B, reaction liquid 2004 is drawn from
chamber B7 through microfluidic channel 734, as indicated by an
arrow 2008, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2010, thereafter
along tube 190, as indicated by an arrow 2012, and thereafter into
interior volume 1302 underlying piston 1300, as indicated by an
arrow 2014, wherein reaction liquid 2004 engages with content
already present in syringe 194.
[0203] FIG. 20C shows subsequent repeated lowering and raising of
piston 1300, as indicated by an arrow 2020, thereby forcing
reaction liquid 2004 and unbound nucleic acids 1840 from sample
1204 as well as remaining reaction liquid 1804, at least partially
into and out of and into the interior volume 1302 of syringe 194
below piston 1300. As a result, the unbound nucleic acids 1840 from
sample 1204 are mixed with reaction liquid 2004 and diluted
thereby.
[0204] FIG. 20C also shows the repeated displacement of the
reaction liquid 2004 together with unbound nucleic acids 1840 from
sample 1204 as well as remaining reaction liquid 1804, within
interior volume 1302 of syringe 194, as indicated by an arrow 2022;
the repeated displacement of the reaction liquid 2004 together with
unbound nucleic acids 1840 from sample 1204 as well as remaining
reaction liquid 1804 within tube 190, as indicated by an arrow
2024; the repeated displacement of the reaction liquid 2004
together with unbound nucleic acids 1840 from sample 1204 as well
as remaining reaction liquid 1804 within interior passageway 1326
of sample transport needle 174, as indicated by an arrow 2026; and
the repeated displacement of the reaction liquid 2004 together with
unbound nucleic acids 1840 from sample 1204 as well as remaining
reaction liquid 1804 within microfluidic channel 734, as indicated
by an arrow 2028. It is appreciated that the raising and lowering
of piston 1300 is preferably carried out multiple times in order to
tix nucleic acids 1840 and reaction liquid 2004.
[0205] FIG. 20D shows the raising of piston 1300 of syringe 194, as
indicated by an arrow 2030, such that the unbound nucleic acids
1840 from sample 1204 now diluted by reaction liquid 2004 as well
as remaining reaction liquid 1804, are at least near fully drawn
into the interior volume 1302 of syringe 194 beneath piston
1300.
[0206] It is appreciated that at least a portion of reaction liquid
2004, together with unbound nucleic acids 1840 from sample 1204 as
well as remaining reaction liquid 1804, is drawn from chamber B7
through microfluidic channel 734, as indicated by an arrow 2042,
interior passageway 1326 of sample transport needle 174, as
indicated by an arrow 2044, thereafter along tube 190, as indicated
by an arrow 2046, and thereafter into interior volume 1302 of
syringe 194 underlying piston 1300, as indicated by an arrow
2048.
[0207] Reference is now made to FIGS. 21A-21E, which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 21A shows an operational
state subsequent to that of FIG. 20D, FIGS. 21A-21E showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with a PCR amplification subsystem thereof.
[0208] FIG. 21A shows the lowering of sample transport needle 174,
as indicated by an arrow 2100, such that hollow pointed end 1312 of
sample transport needle 174 preferably enters sample transport
needle location T11. A fluid transport path is preferably present
between pointed end 1312 of sample transport needle 174 at sample
transport needle tip location T11 and PCR amplification subsystem
600, which fluid transport path is preferably formed by
microfluidic channel 752 interfacing with sample transport needle
tip location T11 and PCR amplification subsystem 600. Venting
needle 164 preferably remains stationary at venting needle tip
location V10.
[0209] PCR amplification subsystem 600 preferably produces
amplification of the unbound nucleic acids 1840. Generally, PCR
amplification subsystem 600 operates as follows: unbound nucleic
acids 1840 enter the PCR amplification subsystem 600 via
microfluidic channel 752. Depression of piston 1300 preferably
drives a solution comprising the unbound nucleic acids 1840 up
channel legs 760 of the PCR amplification subsystem 600 until the
solution reaches corresponding chambers A8-A13 having dried PCR
reaction mixture stored thereon. This arrangement facilitates
reconstitution of the dry PCR reaction mixture within chambers
A8-A13 and subsequent nucleic acid amplification within the
corresponding amplification chambers 770. Gas springs 772
preferably provide even distribution of the nucleic acids 1840
among the various PCR amplification chambers 770. Once PCR
amplification is completed, the resulting PCR products, or
amplicons, are returned to syringe 194 by way of the raising of
piston 1300. A valve (not shown) may optionally be present between
sample transport needle tip location T11 and PCR amplification
subsystem 600 in order to allow the release of pressure from PCR
amplification subsystem 600 during the amplification process.
[0210] FIG. 21B shows lowering of piston 1300, as indicated by an
arrow 2110, thus forcing the unbound nucleic acids 1840 of sample
1204 into operative engagement with PCR amplification subsystem 600
via microfluidic channel 752. As seen in FIG. 21B, a solution
comprising the unbound nucleic acids 1840 is preferably forced out
of internal volume 1302 of syringe 194, as indicated by an arrow
2112, thereafter through tube 190 in a direction towards sample
transport needle 174, as indicated by an additional arrow 2114, and
thereafter downwards through internal passageway 1326 of sample
transport needle 174 and into microfluidic channel 752, as
indicated by additional arrows 2116, 2118. The unbound nucleic
acids 1840 are then preferably distributed between reagent plug
chambers A8-A13 and amplification chambers 770 via channel legs
760, as indicated by an arrow 2119.
[0211] FIG. 21C shows repeated raising and lowering of piston 1300,
as indicated by an arrow 2120, thus repeatedly passing the solution
containing unbound nucleic acids 1840 over reagent plug chambers
A8-A13 and thereby reconstituting the dried PCR mix located on
chambers A8-A13, as indicated by an arrow 2122 and as described
hereinabove in Table II. The solution containing the unbound
nucleic acids 1840 is preferably displaced to and fro by the
repeated movement of piston 1300 along the fluid path formed by
interior volume 1302 of syringe 194, tube 190, internal passageway
1326 of sample transport needle 174, microfluidic channel 752 and
channel legs 760, as indicated by arrows 2124, 2126, 2128, 2130 and
2132, respectively.
[0212] FIG. 21D shows lowering of piston 1300, as indicated by an
arrow 2140, thus forcing unbound nucleic acids 1840 into
amplification chambers 770. The solution containing the unbound
nucleic acids 1840 is preferably forced along the fluid path formed
by interior volume 1302 of syringe 194, tube 190, internal
passageway 1326 of sample transport needle 174, microfluidic
channel 752 and channel legs 760, as indicated by a series of
arrows 2142, 2144, 2146, 2148 and 2150, respectively. The
amplification of unbound nucleic acids 1840 is symbolically
represented in FIG. 24D by the high density of unbound nucleic
acids 1840 present in amplification chambers 770.
[0213] FIG. 21E shows the partial raising of piston 1300 to an
intermediate position thereof, as indicated by an arrow 2160, thus
drawing the amplified unbound nucleic acids 1840 of sample 1204 out
of amplification chambers 770, as indicated by an arrow 2162. The
amplified unbound nucleic acids 1840 are preferably drawn along
channel legs 760, thereafter through microfluidic channel 752,
further thereafter through interior passageway 1326 of sample
transport needle 174, and still further thereafter through tube 190
into interior volume 1302 of syringe 194 beneath piston 1300, as
respectively indicated by a series of arrows 2164, 2166, 2168, 2170
and 2172.
[0214] Reference is now made to FIGS. 22A-220, which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 22A shows an operational
state subsequent to that of FIG. 21E. FIGS. 22A-22G showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B8 thereof.
[0215] FIG. 22A shows the lowering of venting needle 164, as
indicated by an arrow 2200, and of sample transport needle 174, as
indicated by an additional arrow 2202. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V11 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T12. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V11 and chamber
B8, which fluid venting path is preferably formed by microfluidic
channel 634 terminating at venting aperture 554 of chamber B8. A
fluid transport path is preferably present between pointed end 1312
of sample transport needle 174 at sample transport needle tip
location T12 and chamber B8, which fluid transport path is
preferably formed by microfluidic channel 748 terminating at
reagent transport aperture 556 of chamber B8.
[0216] As appreciated from consideration of FIG. 22A, an unsealed
reagent plug 572 is preferably present along microfluidic channel
748. Reagent plug 572 is preferably empty in the case of cartridge
100 being used in conjunction with PCR subsystem 600 and simply
forms a passive part of the fluid pathway between sample transport
needle 174 and chamber B8. In other possible embodiments of the
present invention, in which an RCA amplification system may be
employed in conjunction with cartridge 100, reagent plug 572 may be
used to stow a dried RCA reagent.
[0217] Chamber B8 preferably contains an additional reaction liquid
2204, preferably comprising an amplicon dilution buffer, such as
Sample Buffer A (L-Histidine l-Thioglycerol in DDW) or DDW
(Ultra-Pure DNase and RNase free water), commercially available
from Biological Industries of Beit Haemek, Israel.
[0218] FIG. 22B shows the raising of piston 1300 to a fully
extended position thereof, as indicated by an arrow 2206, thus
drawing reaction liquid 2204 from chamber B8 into interior volume
1302 underlying piston 1300, via microfluidic channel 748, sample
transport needle 174 and tube 190. It is appreciated that interior
volume 1302 already holds amplified nucleic acids 1840 of sample
1204, as described hereinabove with reference to FIG. 21E.
[0219] As seen in FIG. 22B, reaction liquid 2204 is drawn from
chamber B8 through microfluidic channel 748, as indicated by an
arrow 2208, into interior passageway 1326 of sample transport
needle 174, as indicated by an arrow 2210, thereafter along tube
190, as indicated by an arrow 2212, and thereafter into interior
volume 1302 underlying piston 1300, as indicated by an arrow
2214.
[0220] FIG. 22C shows repeated lowering and raising of the piston
1300, as indicated by an arrow 2220, thereby forcing the reaction
liquid 2204 together with amplified nucleic acids 1840 of sample
1204, repeatedly into and out of the interior volume 1302 of
syringe 194, below piston 1300 via the interior passageway 1326 of
sample transport needle 174.
[0221] FIG. 22C also shows the repeated displacement of the
reaction liquid 2204 together with amplified nucleic acids 1840 of
sample 1204, within interior volume 1302 of syringe 194, as
indicated by an arrow 2222; the repeated displacement of the
reaction liquid 2204 together with amplified nucleic acids 1840
within tube 190, as indicated by an arrow 2224; the repeated
displacement of the reaction liquid 2204 together with amplified
nucleic acids 1840 within interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2226; and the
repeated displacement of the reaction liquid 2204 together with
amplified nucleic acids 1840 within microfluidic channel 748, as
indicated by an arrow 2227. It is appreciated that the raising and
lowering of piston 1300 is preferably carried out multiple times in
order to dilute amplified nucleic acids 1840 with reaction liquid
2204.
[0222] FIG. 22D shows the raising of piston 1300 of syringe 194, as
indicated by an arrow 2228, such that reaction liquid 2204 together
with now diluted nucleic acids 1840 are at least near fully drawn
into the interior volume 1302 of syringe 194 beneath piston
1300.
[0223] It is appreciated that virtually all of reaction liquid
2204, together with diluted nucleic acids 1840, is drawn from
chamber B8 through microfluidic channel 734, as indicated by an
arrow 2230, interior passageway 1326 of sample transport needle
174, as indicated by an arrow 2232, thereafter along tube 190, as
indicated by an arrow 2234, and thereafter into interior volume
1302 underlying piston 1300, as indicated by an arrow 2236.
[0224] FIG. 22E shows the lowering of venting needle 164, as
indicated by an arrow 2240, and of sample transport needle 174, as
indicated by an additional arrow 2242. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V21 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T21. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V21 and carbon
array 440, which fluid venting path is preferably formed by
microfluidic channel 634 terminating at venting aperture 554 of
chamber B14, which chamber B14 is preferably in turn connected to
carbon array 440 via venting aperture 784 which is aligned with
carbon array outlet aperture 462. A fluid transport path is
preferably present between pointed end 1312 of sample transport
needle 174 at sample transport needle tip location T21 and carbon
array 440, which fluid transport path is preferably formed by
microfluidic channel 782 which terminates at aperture 783, which is
aligned with carbon array inlet aperture 460 of carbon array
440.
[0225] FIG. 22F shows the partial lowering of piston 1300 of
syringe 194 to an intermediate position thereof, as indicated by an
arrow 2250, such that a portion of the diluted amplified nucleic
acids 1840 passes over carbon array 440 in order to wash off a
layer of Raffinose present on the carbon array 440, as described
hereinabove in Table II. It is appreciated that the Raffinose
washing step illustrated in FIG. 22F may be repeated multiple times
with aliquots of the diluted amplified nucleic acids 1840,
depending on the washing requirements of carbon array 440.
[0226] As seen in FIG. 22F, the portion of the diluted amplified
nucleic acids 1840 flows from interior volume 1302 of syringe 194,
as indicated by an arrow 2252; through tube 190, as indicated by an
arrow 2254, through interior passageway 1326 of sample transport
needle 174, as indicated by an arrow 2256, through microfluidic
channel 782, as indicated by an arrow 2258, through carbon array
440 to chamber B14. The draining passage of the portion of diluted
amplified nucleic acids 1840 from carbon array 440 via carbon array
outlet aperture 462 aligned with aperture 784 of chamber B14 is
indicated in FIG. 22F by an arrow 2259.
[0227] FIG. 22G shows further lowering of piston 1300, as indicated
by an arrow 2260, thus forcing the remainder of the diluted
amplified nucleic acids 1840 of sample 1204 at least partially into
operative engagement with carbon array 440. The diluted amplified
nucleic acids 1840, also known as amplicons, become attached to
predetermined locations on the carbon array 440, as described in
detail, inter alia, in the following U.S. Pat. Nos. 5,605,662;
6,238,624; 6,303,082; 6,403,367; 6,524,517; 6,960,298; 7,101,661;
7,601,493 and 8,288,155, the disclosures of which are hereby
incorporated by reference.
[0228] As seen in FIG. 22G, the remainder of the diluted amplified
nucleic acids 1840 flows from interior volume 1302 of syringe 194,
as indicated by an arrow 2262; through tube 190, as indicated by an
arrow 2264, through interior passageway 1326 of sample transport
needle 174, as indicated by an arrow 2266, through microfluidic
channel 782, as indicated by an arrow 2268, into carbon array 440
via aperture 783 and carbon array inlet aperture 462 aligned
therewith.
[0229] Amplified nucleic acids 1840 which do not become attached to
carbon array 440 are preferably drained from carbon array 440 into
chamber B14 via carbon array outlet aperture 462 aligned with
aperture 784 of chamber B14, is indicated by an arrow 2270.
Reference is now made to FIGS. 23A and 23B, which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 23A shows an operational
state subsequent to that of FIG. 22G, FIGS. 23A and 23B showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber A4 thereof.
[0230] FIG. 23A shows the raising of venting needle 164, as
indicated by an arrow 2300, and of sample transport needle 174, as
indicated by an additional arrow 2302. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V14 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T14. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V14 and chamber
A4, which fluid venting path is preferably formed by microfluidic
channel 636 terminating at an opening of chamber A4. A fluid
transport path is preferably present between pointed end 1312 of
sample transport needle 174 at sample transport needle tip location
T14 and chamber A4, which fluid transport path is preferably formed
by microfluidic channel 736 terminating at another opening of
chamber A4.
[0231] Chamber A4 preferably contains an additional reaction liquid
2304, preferably comprising a first discriminator mix containing
nucleic acids which are complementary to specific ones of the
diluted amplified nucleic acids 1840 of sample 1204.
[0232] FIG. 23B shows the partial raising of piston 1300 to an
intermediate position thereof, as indicated by an arrow 2306, thus
drawing reaction liquid 2304 from chamber A4 into interior volume
1302 underlying piston 1300, via microfluidic channel 736, interior
passageway 1326 of sample transport needle 174 and tube 190.
[0233] As seen in FIG. 23B, reaction liquid 2304 is drawn from
chamber A4 through microfluidic channel 736, as indicated by an
arrow 2308, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2310, further
thereafter along tube 190, as indicated by an arrow 2312, and still
further thereafter into interior volume 1302 underlying piston
1300, as indicated by an arrow 2314.
[0234] Reference is now made to FIGS. 24A-24F, which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-1 IF wherein FIG. 24A shows an operational
state subsequent to that of FIG. 23B, FIGS. 24A-24F showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B10 and a sensor array thereof.
[0235] FIG. 24A shows the raising of venting needle 164, as
indicated by an arrow 2400, and the lowering of sample transport
needle 174, as indicated by an additional arrow 2402. Hollow
pointed end 1310 of venting needle 164 preferably enters venting
needle tip location V13 and hollow pointed end 1312 of sample
transport needle 174 preferably enters sample transport needle tip
location TIS. A fluid venting path is preferably present between
pointed end 1310 of venting needle 164 at venting needle tip
location V13 and chamber B10, which fluid venting path is
preferably formed by microfluidic channel 634 terminating at
venting aperture 554 of chamber B10. A fluid transport path is
preferably present between pointed end 1312 of sample transport
needle 174 at sample transport needle tip location T15 and chamber
B10, which fluid transport path is preferably formed by
microfluidic channel 748 terminating at reagent transport aperture
556 of chamber B10.
[0236] As appreciated from consideration of FIG. 24A, unsealed
reagent plug 572 is preferably present along microfluidic channel
748. Reagent plug 572 is preferably empty in the case of cartridge
100 being used in conjunction with PCR subsystem 600 and simply
forms a passive part of the fluid pathway between sample transport
needle 174 and chamber B10. In other possible embodiments of the
present invention, in which an RCA amplification system may be
employed in conjunction with cartridge 100, reagent plug 572 may be
used to stow a dried RCA reagent.
[0237] Chamber B10 preferably contains an additional reaction
liquid 2404, preferably comprising a buffer for discriminator mix
dilution. Reaction liquid 2404 preferably comprises a High Salt
Buffer (HSB) such as NaPO4, NaCl, Triton, pH 7.4.
[0238] FIG. 24B shows the raising of piston 1300 to a fully
extended position thereof, as indicated by an arrow 2406, thus
drawing a portion of reaction liquid 2404 from chamber B10 into
interior volume 1302 of syringe 194 underlying piston 1300, via
microfluidic channel 748, interior passageway 1326 of sample
transport needle 174 and tube 190, which interior volume 1302 of
syringe 194 already holds reaction liquid 2304.
[0239] As seen in FIG. 24B, reaction liquid 2404 is drawn from
chamber B10 through microfluidic channel 748, as indicated by an
arrow 2408, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2410, further
thereafter along tube 190, as indicated by an arrow 2412, and still
further thereafter into interior volume 1302 underlying piston
1300, as indicated by an arrow 2414, wherein reaction liquid 2404
engages with previously present reaction liquid 2304.
[0240] FIG. 24C shows repeated lowering and raising of the piston
1300, as indicated by an arrow 2420, thereby forcing the reaction
liquids 2304 and 2404 containing the first discriminator mix and
buffer therefore, repeatedly into and out of the interior volume
1302 of syringe 194, below piston 1300 via the tube 190, interior
passageway 1326 of sample transport needle 174 and microfluidic
channel 748.
[0241] FIG. 24C also shows the repeated displacement of the
reaction liquids 2304 and 2404 within interior volume 1302 of
syringe 194, as indicated by an arrow 2422; the repeated
displacement of reaction liquids 2304 and 2404 within tube 190, as
indicated by an arrow 2424; the repeated displacement of reaction
liquids 2304 and 2404 within interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2426; and the
repeated displacement of reaction liquids 2304 and 2404 within
microfluidic channel 748, as indicated by an arrow 2427. It is
appreciated that the raising and lowering of piston 1300 is
preferably carried out multiple times in order to mix reaction
liquids 2304 and 2404 and thus dilute the first discriminator mix,
comprising reaction liquid 2304, by the discriminator buffer,
comprising reaction liquid 2404.
[0242] FIG. 24D shows the raising of piston 130 of syringe 194, as
indicated by an arrow 2428, such that reaction liquid 2304, now
diluted by reaction liquid 2404, is at least near fully drawn into
the interior volume 1302 of syringe 194 beneath piston 1300.
[0243] As seen in FIG. 24D, reaction liquid 2304, now diluted by
reaction liquid 2404, is drawn from chamber B10 through
microfluidic channel 748, as indicated by an arrow 2430, thereafter
through interior passageway 1326 of sample transport needle 174, as
indicated by an arrow 2432, thereafter along tube 190, as indicated
by an arrow 2434, and thereafter into interior volume 1302 of
syringe 194 underlying piston 1300, as indicated by an arrow
2436.
[0244] FIG. 24E shows the lowering of venting needle 164, as
indicated by an arrow 2440, and of sample transport needle 174, as
indicated by an additional arrow 2442. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V21 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T21. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V21 and carbon
array 440, which fluid venting path is preferably formed by
microfluidic channel 634 terminating at venting aperture 554 of
chamber B14, which chamber B14 is preferably in turn connected to
carbon array 440 via venting aperture 784 (FIG. 6B) and carbon
array outlet aperture 462. A fluid transport path is preferably
present between pointed end 1312 of sample transport needle 174 at
sample transport needle tip location T21 and carbon array 440,
which fluid transport path is preferably formed by microfluidic
channel 782 terminating at aperture 783 (FIG. 6B) which is aligned
with carbon array inlet aperture 460 of carbon array 440.
[0245] FIG. 24F shows the lowering of piston 1300 of syringe 194,
as indicated by an arrow 2444, such that reaction liquid 2304, now
diluted by reaction liquid 2404, is forced into operative
engagement with carbon array 440 and more specifically, with the
diluted amplified nucleic acids 1840 which are attached to
predetermined locations on carbon array 440. To the extent that the
nucleic acids in the diluted first discriminator mix are
complementary to the diluted amplified nucleic acids 1840,
hybridization occurs.
[0246] As seen in FIG. 24F, the reaction liquid 2304, now diluted
by reaction liquid 2404, flows from interior volume 1302 of syringe
194, as indicated by an arrow 2450; through tube 190, as indicated
by an arrow 2452, through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2454, through
microfluidic channel 782, as indicated by an arrow 2456, into
carbon array 440 via aperture 783 (FIG. 6B) and carbon array inlet
aperture 462 aligned therewith.
[0247] Nucleic acids contained in the diluted first discriminator
mix 2304 which are not complementary to the diluted amplified
nucleic acids 1840 and which therefore do not become attached to
carbon array 440, are preferably drained from carbon array 440 into
chamber B14 via carbon array outlet aperture 462 aligned with
aperture 784 (FIG. 6B) of chamber B14, as indicated by an arrow
2460.
[0248] Reference is now made to FIGS. 25A and 25B, which are
simplified illustrations of typical yet further steps in the
operation of a cartridge such as that shown in FIGS. 1A-2 including
the core assembly of FIGS. 4A-11F, wherein FIG. 25A shows an
operational state subsequent to that of FIG. 24F, FIGS. 25A and 258
showing the microfluidic base portion of FIGS. 6A-6H and operative
engagement with chamber A5 thereof.
[0249] FIG. 25A shows the raising of venting needle 164, as
indicated by an arrow 2500, and of sample transport needle 174, as
indicated by an additional arrow 2502. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V16 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T16. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V16 and chamber
A5, which fluid venting path is preferably formed by microfluidic
channel 636 terminating at an opening of chamber A5. A fluid
transport path is preferably present between pointed end 1312 of
sample transport needle 174 at sample transport needle tip location
T16 and chamber A5, which fluid transport path is preferably formed
by microfluidic channel 736 terminating at another opening of
chamber A5.
[0250] Chamber A5 preferably contains an additional reaction liquid
2504, preferably comprising a second discriminator mix containing
nucleic acids which are complementary to other specific ones of the
diluted amplified nucleic acids 1840 of sample 1204.
[0251] FIG. 25B shows the partial raising of piston 1300 to an
intermediate position thereof, as indicated by an arrow 2506, thus
drawing reaction liquid 2504 from chamber A5 into interior volume
1302 underlying piston 1300, via microfluidic channel 736, interior
passageway 1326 of sample transport needle 174 and tube 190.
[0252] As seen in FIG. 25B, reaction liquid 2504 is drawn from
chamber A5 through microfluidic channel 736, as indicated by an
arrow 2508, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2510, further
thereafter along tube 190, as indicated by an arrow 2512, and still
further thereafter into interior volume 1302 underlying piston
1300, as indicated by an arrow 2514.
[0253] Reference is now made to FIGS. 26A-26F, which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 26A shows an operational
state subsequent to that of FIG. 25B, FIGS. 26A-26F showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B10 and a sensor array thereof.
[0254] FIG. 26A shows the raising of venting needle 164, as
indicated by an arrow 2600, and the raising of sample transport
needle 174, as indicated by an additional arrow 2602. Hollow
pointed end 1310 of venting needle 164 preferably enters venting
needle tip location V13 and hollow pointed end 1312 of sample
transport needle 174 preferably enters sample transport needle tip
location T15. A fluid venting path is preferably present between
pointed end 1310 of venting needle 164 at venting needle tip
location V13 and chamber B10, which fluid venting path is
preferably formed by microfluidic channel 634 terminating at
venting aperture 554 of chamber B10. A fluid transport path is
preferably present between pointed end 1312 of sample transport
needle 174 at sample transport needle tip location T15 and chamber
B10, which fluid transport path is preferably formed by
microfluidic channel 748 terminating at reagent transport aperture
556 of chamber B10.
[0255] As appreciated from consideration of FIG. 26A, unsealed
reagent plug 572 is preferably present along microfluidic channel
748. Reagent plug 572 is preferably empty in the case of cartridge
100 being used in conjunction with PCR subsystem 600 and simply
forms a passive part of the fluid pathway between sample transport
needle 174 and chamber B10. In other possible embodiments of the
present invention, in which an RCA amplification system may be
employed in conjunction with cartridge 100, reagent plug 572 may be
used to store a dried RCA reagent.
[0256] As described hereinabove with reference to FIG. 24A, chamber
B10 preferably contains reaction liquid 2404, preferably comprising
a buffer for Discriminator mix dilution. Reaction liquid 2404
preferably comprises a High Salt Buffer (HSB) such as NaPO4, NaCl,
Triton, pH 7.4.
[0257] FIG. 26B shows the raising of piston 1300 to a fully
extended position thereof, as indicated by an arrow 2606, thus
drawing an additional portion of reaction liquid 2404 from chamber
B10 into interior volume 1302 underlying piston 1300, via sample
transport needle 174 and tube 190, which interior volume 1302
already holds reaction liquid 2504.
[0258] As seen in FIG. 26B, reaction liquid 2404 is drawn from
chamber B10 through microfluidic channel 748, as indicated by an
arrow 2608, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2610, further
thereafter along tube 190, as indicated by an arrow 2612, and still
further thereafter into interior volume 1302 underlying piston
1300, as indicated by an arrow 2614, wherein reaction liquid 2404
engages with previously present reaction liquid 2504.
[0259] FIG. 26C shows repeated lowering and raising of the piston
1300, as indicated by an arrow 2620, thereby forcing the reaction
liquids 2504 and 2404 containing the second discriminator mix and
buffer therefore, repeatedly into and out of the interior volume
1302 of syringe 194, below piston 1300 via the interior passageway
1326 of sample transport needle 174.
[0260] FIG. 26C also shows the repeated displacement of the
reaction liquids 2504 and 2404 within interior volume 1302 of
syringe 194, as indicated by an arrow 2622; the repeated
displacement of reaction liquids 2504 and 2404 within tube 190, as
indicated by an arrow 2624; the repeated displacement of reaction
liquids 2504 and 2404 within interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2626; and the
repeated displacement of reaction liquids 2504 and 2404 within
microfluidic channel 734, as indicated by an arrow 2627. It is
appreciated that the raising and lowering of piston 1300 is
preferably carried out multiple times in order to mix reaction
liquids 2504 and 2404 and thus dilute the second discriminator mix
comprising reaction liquid 2504 by the discriminator buffer
comprising reaction liquid 2404.
[0261] FIG. 26D shows the raising of piston 1300 of syringe 194, as
indicated by an arrow 2628, such that reaction liquid 2504 now
diluted by reaction liquid 2404 is at least near fully drawn into
the interior volume 1302 of syringe 194 beneath piston 1300.
[0262] As seen in FIG. 26D, reaction liquids 2404 and 2504 are
drawn from chamber B10 through microfluidic channel 748, as
indicated by an arrow 2630, thereafter through interior passageway
1326 of sample transport needle 174, as indicated by an arrow 2632,
further thereafter along tube 190, as indicated by an arrow 2634,
and still further thereafter into interior volume 1302 underlying
piston 1300, as indicated by an arrow 2636.
[0263] FIG. 26E shows the lowering of venting needle 164, as
indicated by an arrow 2640, and of sample transport needle 174, as
indicated by an additional arrow 2642. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V21 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T21. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V21 and carbon
array 440, which fluid venting path is preferably formed by
microfluidic channel 634 terminating at venting aperture 554 of
chamber B14, which chamber B14 is preferably in turn connected to
carbon array 440 via venting aperture 784 (FIG. 6B) and carbon
array outlet 462. A fluid transport path is preferably present
between pointed end 1312 of sample transport needle 174 at sample
transport needle tip location T21 and carbon array 440, which fluid
transport path is preferably formed by microfluidic channel 782
terminating at inlet 460 of carbon array 440.
[0264] FIG. 26F shows the lowering of piston 1300 of syringe 194,
as indicated by an arrow 2644, such that reaction liquids 2504 and
2404 are forced into operative engagement with carbon array 440 and
more specifically, with the diluted amplified nucleic acids 1840
which are attached to predetermined locations on carbon array 440.
Reaction liquids 2504 and 2404 displace previously present reaction
liquids 2304 and 2404, which displaced reaction liquids 2304 and
2404 preferably drain from carbon array 440 into chamber B14 via
carbon array outlet 462 and venting aperture 784 (FIG. 6B), as
indicated by an arrow 2660. To the extent that the nucleic acids in
the diluted second discriminator mix 2504 are complementary to the
diluted amplified nucleic acids 1840, hybridization occurs.
[0265] Reference is now made to FIGS. 27A and 27B, which are
simplified illustrations of typical yet further steps in the
operation of a cartridge such as that shown in FIGS. 1A-2 including
the core assembly of FIGS. 4A-11F, wherein FIG. 27A shows an
operational state subsequent to that of FIG. 26F, FIGS. 27A and 27B
showing the microfluidic base portion of FIGS. 6A-6H and operative
engagement with chamber A6 thereof.
[0266] FIG. 27A shows the raising of venting needle 164, as
indicated by an arrow 2700, and of sample transport needle 174, as
indicated by an additional arrow 2702. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V18 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T18. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V18 and chamber
A6, which fluid venting path is preferably formed by microfluidic
channel 636 terminating at an opening of chamber A6. A fluid
transport path is preferably present between pointed end 1312 of
sample transport needle 174 at sample transport needle tip location
T18 and chamber A6, which fluid transport path is preferably formed
by microfluidic channel 736 terminating at another opening of
chamber A6.
[0267] Chamber A6 preferably contains yet an additional reaction
liquid 2704, preferably comprising a third discriminator mix
containing nucleic acids which are complementary to yet other
specific ones of the diluted amplified nucleic acids 1840 of sample
1204.
[0268] FIG. 27B shows the partial raising of piston 1300 to an
intermediate position thereof, as indicated by an arrow 2706, thus
drawing reaction liquid 2704 from chamber A6 into interior volume
1302 underlying piston 1300, via sample transport needle 174 and
tube 190.
[0269] As seen in FIG. 27B, reaction liquid 2704 is drawn from
chamber A6 through microfluidic channel 736, as indicated by an
arrow 2708, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2710, further
thereafter along tube 190, as indicated by an arrow 2712, and still
further thereafter into interior volume 1302 underlying piston
1300, as indicated by an arrow 2714.
[0270] Reference is now made to FIGS. 28A-28F, which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 28A shows an operational
state subsequent to that of FIG. 27B, FIGS. 28A-28F showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B10 and a sensor array thereof. FIG. 28A shows the
raising of venting needle 164, as indicated by an arrow 2800, and
the raising of sample transport needle 174, as indicated by an
additional arrow 2802. Hollow pointed end 1310 of venting needle
164 preferably enters venting needle tip location V13 and hollow
pointed end 1312 of sample transport needle 174 preferably enters
sample transport needle tip location TIS. A fluid venting path is
preferably present between pointed end 1310 of venting needle 164
at venting needle tip location V13 and chamber B10, which fluid
venting path is preferably formed by microfluidic channel 634
terminating at venting aperture 554 of chamber B10. A fluid
transport path is preferably present between pointed end 1312 of
sample transport needle 174 at sample transport needle tip location
T15 and chamber B10, which fluid transport path is preferably
formed by microfluidic channel 734 terminating at reagent transport
aperture 556 of chamber B10.
[0271] As appreciated from consideration of FIG. 28A, unsealed
reagent plug 572 is preferably present along microfluidic channel
748. Reagent plug 572 is preferably empty in the case of cartridge
100 being used in conjunction with PCR subsystem 600 and simply
forms a passive part of the fluid pathway between sample transport
needle 174 and chamber B10. In other possible embodiments of the
present invention, in which an RCA amplification system may be
employed in conjunction with cartridge 100, reagent plug 572 may be
used to stow a dried RCA reagent.
[0272] As described hereinabove with reference to FIG. 24A, chamber
B10 preferably contains reaction liquid 2404, preferably comprising
a buffer for Discriminator mix dilution. Reaction liquid 2404
preferably comprises a High Salt Buffer (HSB) such as NaPO4, NaCl,
Triton, pH 7.4.
[0273] FIG. 28B shows the raising of piston 1300 to a fully
extended position thereof, as indicated by an arrow 2806, thus
drawing a further portion of reaction liquid 2404 from chamber B10
into interior volume 1302 underlying piston 1300, via sample
transport needle 174 and tube 190, which chamber B10 already holds
reaction liquid 2704.
[0274] As seen in FIG. 28B, reaction liquid 2404 is drawn from
chamber B10 through microfluidic channel 734, as indicated by an
arrow 2808, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2810, further
thereafter along tube 190, as indicated by an arrow 2812, and still
further thereafter into interior volume 1302 underlying piston
1300, as indicated by an arrow 2814, wherein reaction liquid 2404
engages with previously present reaction liquid 2704.
[0275] FIG. 28C shows repeated lowering and raising of the piston
1300, as indicated by an arrow 2820, thereby forcing the reaction
liquids 2704 and 2404 containing the third discriminator mix and
buffer therefore, repeatedly into and out of the interior volume
1302 of syringe 194, below piston 1300 via the interior passageway
1326 of sample transport needle 174.
[0276] FIG. 28C also shows the repeated displacement of the
reaction liquids 2704 and 2404 within interior volume 1302 of
syringe 194, as indicated by an arrow 2822; the repeated
displacement of reaction liquids 2704 and 2404 within tube 190, as
indicated by an arrow 2824; the repeated displacement of reaction
liquids 2704 and 2404 within interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2826; and the
repeated displacement of reaction liquids 2704 and 2404 within
microfluidic channel 734, as indicated by an arrow 2827. It is
appreciated that the raising and lowering of piston 1300 is
preferably carried out multiple times in order to mix reaction
liquids 2704 and 2404 and thus dilute the third discriminator mix
comprising reaction liquid 2704 by the discriminator buffer
comprising reaction liquid 2404.
[0277] FIG. 28D shows the raising of piston 130 of syringe 194, as
indicated by an arrow 2828, such that reaction liquid 2704 now
diluted by reaction liquid 2404 is at least near fully drawn into
the interior volume 1302 of syringe 194 beneath piston 1300.
[0278] As seen in FIG. 28D, reaction liquids 2704 and 2404 are
drawn from chamber B10 through microfluidic channel 748, as
indicated by an arrow 2830, thereafter through interior passageway
1326 of sample transport needle 174, as indicated by an arrow 2832,
further thereafter along tube 190, as indicated by an arrow 2834,
and still further thereafter into interior volume 1302 underlying
piston 1300, as indicated by an arrow 2836.
[0279] FIG. 28E shows the lowering of venting needle 164, as
indicated by an arrow 2840, and of sample transport needle 174, as
indicated by an additional arrow 2842. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V21 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T21. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V21 and carbon
array 440, which fluid venting path is preferably formed by
microfluidic channel 634 terminating at venting aperture 554 of
chamber B14, which chamber B14 is preferably in turn connected to
carbon array 440 via venting aperture 784 (FIG. 6B) and carbon
array outlet 462. A fluid transport path is preferably present
between pointed end 1312 of sample transport needle 174 at sample
transport needle tip location T21 and carbon array 440, which fluid
transport path is preferably formed by microfluidic channel 782
terminating at inlet 460 of carbon array 440.
[0280] FIG. 28F shows the lowering of piston 1300 of syringe 194,
as indicated by an arrow 2844, such that reaction liquids 2704 and
2404 are forced into operative engagement with carbon array 440 and
mom specifically, with the diluted amplified nucleic acids 1840
which are attached to predetermined locations on carbon array 440.
Reaction liquids 2704 and 2404 displace previously present reaction
liquids 2504 and 2404, which displaced reaction liquids 2504 and
2404 preferably drain from carbon array 440 into chamber B14 via
carbon array outlet 462 and venting aperture 784 (FIG. 68), as
indicated by an arrow 2860. To the extent that the nucleic acids in
the diluted third discriminator mix 2704 are complementary to the
diluted amplified nucleic acids 1840, hybridization occurs.
[0281] Reference is now made to FIGS. 29A and 29B, which are
simplified illustrations of typical yet further steps in the
operation of a cartridge such as that shown in FIGS. 1A-2 including
the core assembly of FIGS. 4A-11F, wherein FIG. 29A shows an
operational state subsequent to that of FIG. 28F, FIGS. 29A and 29B
showing the microfluidic base portion of FIGS. 6A-6H and operative
engagement with chamber A7 thereof.
[0282] FIG. 29A shows the raising of venting needle 164, as
indicated by an arrow 2900, and of sample transport needle 174, as
indicated by an additional arrow 2902. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V20 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T20. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V20 and chamber
A7, which fluid venting path is preferably formed by microfluidic
channel 636 terminating at an opening of chamber A7. A fluid
transport path is preferably present between pointed end 1312 of
sample transport needle 174 at sample transport needle tip location
T20 and chamber A7, which fluid transport path is preferably formed
by microfluidic channel 736 terminating at another opening of
chamber A7.
[0283] Chamber A7 preferably contains yet an additional reaction
liquid 2904, preferably comprising a fourth discriminator mix
containing nucleic acids which are complementary to still other
specific ones of the diluted amplified nucleic acids 1840 of sample
1204.
[0284] FIG. 29B shows the partial raising of piston 1300 to an
intermediate position thereof, as indicated by an arrow 2906, thus
drawing reaction liquid 2904 from chamber A7 into interior volume
1302 underlying piston 1300, via sample transport needle 174 and
tube 190.
[0285] As seen in FIG. 29B, reaction liquid 2904 is drawn from
chamber A7 through microfluidic channel 736, as indicated by an
arrow 2908, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 2910, further
thereafter along tube 190, as indicated by an arrow 2912, and still
further thereafter into interior volume 1302 underlying piston
1300, as indicated by an arrow 2914.
[0286] Reference is now made to FIGS. 30A-30F, which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 30A shows an operational
state subsequent to that of FIG. 29B. FIGS. 30A-30F showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B10 and a sensor array thereof.
[0287] FIG. 30A shows the raising of venting needle 164, as
indicated by an arrow 3000, and the raising of sample transport
needle 174, as indicated by an additional arrow 3002. Hollow
pointed end 1310 of venting needle 164 preferably enters venting
needle tip location V13 and hollow pointed end 1312 of sample
transport needle 174 preferably enters sample transport needle tip
location T15. A fluid venting path is preferably present between
pointed end 1310 of venting needle 164 at venting needle tip
location V13 and chamber B10, which fluid venting path is
preferably formed by microfluidic channel 634 terminating at
venting aperture 554 of chamber B10. A fluid transport path is
preferably present between pointed end 1312 of sample transport
needle 174 at sample transport needle tip location T15 and chamber
B10, which fluid transport path is preferably formed by
microfluidic channel 748 terminating at reagent transport aperture
556 of chamber B10.
[0288] As appreciated from consideration of FIG. 30A, unsealed
reagent plug 572 is preferably present along microfluidic channel
748. Reagent plug 572 is preferably empty in the case of cartridge
100 being used in conjunction with PCR subsystem 600 and simply
forms a passive part of the fluid pathway between sample transport
needle 174 and chamber B10. In other possible embodiments of the
present invention, in which an RCA amplification system may be
employed in conjunction with cartridge 100, reagent plug 572 may be
used to store a dried RCA reagent.
[0289] As described hereinabove with reference to FIG. 24A, chamber
B10 preferably contains reaction liquid 2404, preferably comprising
a buffer for Discriminator mix dilution. Reaction liquid 2404
preferably comprises a High Salt Buffer (HSB) such as NaPO4, NaCl,
Triton, pH 7.4.
[0290] FIG. 30B shows the raising of piston 1300 to a fully
extended position thereof, as indicated by an arrow 3006, thus
drawing yet an additional portion of reaction liquid 2404 from
chamber B10 into interior volume 1302 underlying piston 1300, via
sample transport needle 174 and tube 190, which chamber B10 already
holds reaction liquid 2904.
[0291] As seen in FIG. 30B, reaction liquid 2404 is drawn from
chamber B10 through microfluidic channel 734, as indicated by an
arrow 3008, thereafter through interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 3010, further
thereafter along tube 190, as indicated by an arrow 3012, and still
further thereafter into interior volume 1302 underlying piston
1300, as indicated by an arrow 3014, wherein reaction liquid 2404
engages with previously present reaction liquid 2904.
[0292] FIG. 30C shows repeated lowering and raising of the piston
1300, as indicated by an arrow 3020, thereby forcing the reaction
liquids 2904 and 2404 containing the fourth discriminator mix and
buffer therefore, repeatedly into and out of the interior volume
1302 of syringe 194, below piston 1300 via the interior passageway
1326 of sample transport needle 174.
[0293] FIG. 30C also shows the repeated displacement of the
reaction liquids 2904 and 2404 within interior volume 1302 of
syringe 194, as indicated by an arrow 3022; the repeated
displacement of reaction liquids 2904 and 2404 within tube 190, as
indicated by an arrow 3024; the repeated displacement of reaction
liquids 2904 and 2404 within interior passageway 1326 of sample
transport needle 174, as indicated by an arrow 3026; and the
repeated displacement of reaction liquids 2904 and 2404 within
microfluidic channel 734, as indicated by an arrow 3027. It is
appreciated that the raising and lowering of piston 1300 is
preferably carried out multiple times in order to mix reaction
liquids 2904 and 2404 and thus dilute the fourth discriminator mix
comprising reaction liquid 2904 by the discriminator buffer
comprising reaction liquid 2404.
[0294] FIG. 30D shows the raising of piston 1300 of syringe 194, as
indicated by an arrow 3028, such that reaction liquid 2904 now
diluted by reaction liquid 2404 is at least near fully drawn into
the interior volume 1302 of syringe 194 beneath piston 1300.
[0295] As seen in FIG. 30D, reaction liquids 2904 and 2404 are
drawn from chamber B10 through microfluidic channel 748, as
indicated by an arrow 3030, thereafter through interior passageway
1326 of sample transport needle 174, as indicated by an arrow 3032,
further thereafter along tube 190, as indicated by an arrow 3034,
and still further thereafter into interior volume 1302 underlying
piston 1300, as indicated by an arrow 3036.
[0296] FIG. 30E shows the lowering of venting needle 164, as
indicated by an arrow 3040, and of sample transport needle 174, as
indicated by an additional arrow 3042. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V21 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T21. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V21 and carbon
array 440, which fluid venting path is preferably formed by
microfluidic channel 634 terminating at venting aperture 554 of
chamber B14, which chamber B14 is preferably in turn connected to
carbon array 440 via venting aperture 784 (FIG. 6B) and carbon
array outlet 462. A fluid transport path is preferably present
between pointed end 1312 of sample transport needle 174 at sample
transport needle tip location T21 and carbon array 440, which fluid
transport path is preferably formed by microfluidic channel 782
terminating at inlet 460 of carbon array 440.
[0297] FIG. 30F shows the lowering of piston 1300 of syringe 194,
as indicated by an arrow 3044, such that reaction liquids 2904 and
2404 are forced into operative engagement with carbon array 440 and
more specifically, with the diluted amplified nucleic acids 1840
which are attached to predetermined locations on carbon array 440.
Reaction liquids 2904 and 2404 displace previously present reaction
liquids 2704 and 2404, which displaced reaction liquids 2704 and
2404 preferably drain from carbon array 440 into chamber B14 via
carbon array outlet 462 and venting aperture 784 (FIG. 68), as
indicated by an arrow 3060. To the extent that the nucleic acids in
the diluted fourth discriminator mix 2904 are complementary to the
diluted amplified nucleic acids 1840, hybridization occurs.
[0298] Reference is now made to FIGS. 31A-31R which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 31A shows an operational
state subsequent to that of FIG. 30F, FIGS. 31A-31F showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B9 thereof and with a sensor array thereof.
[0299] FIG. 31A shows the raising of venting needle 164, as
indicated by an arrow 3100, and of sample transport needle 174, as
indicated by an additional arrow 3102. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V12 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T13. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V12 and chamber
B9, which fluid venting path is preferably formed by microfluidic
channel 634 terminating at venting aperture 554 of chamber B9. A
fluid transport path is preferably present between pointed end 1312
of sample transport needle 174 at sample transport needle tip
location T15 and chamber B9, which fluid transport path is
preferably formed by microfluidic channel 748 communicating with
reagent transport aperture 556 of chamber B9 via reagent plug
572.
[0300] As appreciated from consideration of FIG. 31A, unsealed
reagent plug 572 is preferably present along microfluidic channel
748. Reagent plug 572 preferably has stored thereon a dried
reporter 3103, which contains nucleic acids bound to fluorescent
dyes which hybridize to any one or more of the complementary
nucleic acids contained in first-fourth discriminator mixes 2304,
2504, 2704 and 2904. Detection of the fluorescent dyes at
predetermined locations in carbon array 440 indicates which of the
discriminators is complementary to a predetermined nucleic acid
target site and thus provides an indication of the presence of a
particular nucleic acid in the sample 1204.
[0301] Chamber B9 preferably contains an additional reaction liquid
3104, preferably comprising a buffer for reporter reconstitution.
Reaction liquid 3104 preferably comprises a High Salt Buffer (HSB)
(NaPO4, NaCl, Triton, pH 7.4) if reporter 3103 is dried in DDW, or
DDW if reporter 3103 is dried in HSB.
[0302] FIG. 31B shows the raising of piston 1300 to a fully
extended position thereof, as indicated by an arrow 3106, thus
drawing reaction liquid 3104 from chamber B9 into interior volume
1302 underlying piston 1300, via sample transport needle 174 and
tube 190.
[0303] As seen in FIG. 31B, reaction liquid 3104 is drawn from
chamber B9 through microfluidic channel 748, as indicated by an
arrow 3108, and over reagent plug 572 having dried reporter 3103
thereon, as indicated by an arrow 3109. Reaction liquid 3104
preferably dissolves at least a portion of dried reporter 3103 upon
contact therewith. Reaction liquid 3104 together with the dissolved
portion of reporter 3103 is preferably drawn thereafter through
interior passageway 1326 of sample transport needle 174, as
indicated by an arrow 3110, further thereafter along tube 190, as
indicated by an arrow 3112, and still further thereafter into
interior volume 1302 underlying piston 1300, as indicated by an
arrow 3114.
[0304] It is appreciated that the flow of reaction liquid 3104 over
reagent plug 572 having dried reporter 3103 thereon is illustrated
in a highly simplified manner in FIG. 31B, in order to
schematically represent the passage of reaction liquid 3104 with
respect to reagent plug 572.
[0305] FIG. 31C shows repeated lowering and raising of the piston
1300, as indicated by an arrow 3120, thereby forcing the reaction
liquid 3104 containing the reporter reconstitution buffer
repeatedly into and out of the interior volume 1302 of syringe 194
via the interior passageway 1326 of sample transport needle 174 and
by way of reagent plug 572 having remaining dried reporter 3103
thereon.
[0306] FIG. 31C also shows the repeated displacement of the
reaction liquid 3104 and dissolved reporter 3103 within interior
volume 1302 of syringe 194, as indicated by an arrow 3122; the
repeated displacement of reaction liquid 3104 and dissolved
reporter 3103 within tube 190, as indicated by an arrow 3124; the
repeated displacement of reaction liquid 3104 and dissolved
reporter 3103 within interior passageway 1326 of sample transport
needle 174, as indicated by an arrow 3126; and the repeated
displacement of reaction liquid 3104 and dissolved reporter 3103
within microfluidic channel 748 and over reagent plug 572, as
indicated by an arrow 3127.
[0307] It is appreciated that the raising and lowering of piston
1300 is preferably carried out multiple times in order to both
dissolve all reporter 3103 contained in reagent plug 572 by
reaction liquid 3104, as well as to mix reaction liquid 3104 with
the reporter 3103 dissolved therein, thereby reconstituting
reporter 3103.
[0308] FIG. 31D shows the raising of piston 1300 of syringe 194, as
indicated by an arrow 3128, such that reaction liquid 3104 having
reporter 3113 mixed therewith is at least near fully drawn into the
interior volume 1302 of syringe 194 beneath piston 1300.
[0309] It is appreciated that virtually all of reaction liquid 3104
together with reporter 3103, is drawn from chamber B9 through
microfluidic channel 748, as indicated by an arrow 3130, thereafter
through interior passageway 1326 of sample transport needle 174, as
indicated by an arrow 3132, thereafter along tube 190, as indicated
by an arrow 3134, and thereafter into interior volume 1302
underlying piston 1300, as indicated by an arrow 3136.
[0310] FIG. 31E shows the lowering of venting needle 164, as
indicated by an arrow 3140, and of sample transport needle 174, as
indicated by an additional arrow 3142. Hollow pointed end 1310 of
venting needle 164 preferably enters venting needle tip location
V21 and hollow pointed end 1312 of sample transport needle 174
preferably enters sample transport needle tip location T21. A fluid
venting path is preferably present between pointed end 1310 of
venting needle 164 at venting needle tip location V21 and carbon
array 440, which fluid venting path is preferably formed by
microfluidic channel 634 terminating at venting aperture 554 of
chamber B14, which chamber B14 is preferably in turn connected to
carbon array 440 via venting aperture 784 (FIG. 6B) which is
aligned with carbon array outlet aperture 462. A fluid transport
path is preferably present between pointed end 1312 of sample
transport needle 174 at sample transport needle tip location T21
and carbon array 440, which fluid transport path is preferably
formed by microfluidic channel 782, which terminates at aperture
783, which is aligned with carbon array inlet aperture 460 of
carbon array 440.
[0311] FIG. 31F shows the lowering of piston 1300 of syringe 194,
as indicated by an arrow 3150, thus forcing the reconstituted
reporter comprising reaction liquid 3104 and reporter 3103 into
operative engagement with the interior of carbon array 440, via
carbon array inlet aperture 460, and specifically with the nucleic
acids contained in first-fourth discriminators attached to
predetermined locations on the carbon array 440. To the extent that
the nucleic acids in the reconstituted reporter are complementary
to the nucleic acids in one or more of the first fourth
discriminators, hybridization occurs at the location of the
relevant discriminator. The reconstituted reporter comprising
reaction liquid 3104 mixed with reporter 3103 displaces previously
present reaction liquids 2904 and 2404, which displaced reaction
liquids 2904 and 2404 preferably drain from carbon array 440 into
chamber B14 via carbon array outlet aperture 462 and venting
aperture 784 (FIG. 6B), as indicated by an arrow 3151.
[0312] As seen in FIG. 31F, the reconstituted reporter comprising
reaction liquid 3104 and reporter 3103 flows from interior volume
1302 of syringe 194, as indicated by an arrow 3152; through tube
190, as indicated by an arrow 3154, through interior passageway
1326 of sample transport needle 174, as indicated by an arrow 3156,
through microfluidic channel 782, as indicated by an arrow 3158,
and into carbon array 440 via carbon array inlet aperture 460.
[0313] Reference is now made to FIGS. 32A-32D, which are simplified
illustrations of typical yet further steps in the operation of a
cartridge such as that shown in FIGS. 1A-2 including the core
assembly of FIGS. 4A-11F, wherein FIG. 32A shows an operational
state subsequent to that of FIG. 31F. FIGS. 32A-32D showing the
microfluidic base portion of FIGS. 6A-6H and operative engagement
with chamber B13 thereof and with a sensor array thereof.
[0314] FIG. 32A shows the raising of venting needle 164, as
indicated by an arrow 3200, and the lowering of sample transport
needle 174, as indicated by an additional arrow 3202. Hollow
pointed end 1310 of venting needle 164 preferably enters venting
needle tip location V19 and hollow pointed end 1312 of sample
transport needle 174 preferably enters sample transport needle tip
location 122. A fluid venting path is preferably present between
pointed end 1310 of venting needle 164 at venting needle tip
location V19 and chamber B13, which fluid venting path is
preferably formed by microfluidic channel 634 terminating at
venting aperture 554 of chamber B13. A fluid transport path is
preferably present between pointed end 1312 of sample transport
needle 174 at sample transport needle tip location T22 and chamber
B13, which fluid transport path is preferably formed by
microfluidic channel 734 terminating at reagent transport aperture
556 of chamber B13.
[0315] Chamber B13 preferably contains an additional reaction
liquid 3204, preferably comprising a sensor wash buffer, such as a
Low Salt Buffer (LSB)(NaPO4, Triton, pH 7.4).
[0316] FIG. 32B shows the raising of piston 1300 to a fully
extended position thereof, as indicated by an arrow 3206, thus
drawing reaction liquid 3204 from chamber B13 into interior volume
1302 underlying piston 1300, via microfluidic channel 734, sample
transport needle 174 and tube 190.
[0317] As seen in FIG. 32B, reaction liquid 3204 is drawn from
chamber B13 through microfluidic channel 734, as indicated by an
arrow 3208, into interior passageway 1326 of sample transport
needle 174, as indicated by an arrow 3210, thereafter along tube
190, as indicated by an arrow 3212, and thereafter into interior
volume 1302 underlying piston 13), as indicated by an arrow
3214.
[0318] FIG. 32C shows the lowering of venting needle 164, as
indicated by an arrow 3240, and the raising of sample transport
needle 174, as indicated by an additional arrow 3242. Hollow
pointed end 1310 of venting needle 164 preferably enters venting
needle tip location V21 and hollow pointed end 1312 of sample
transport needle 174 preferably enters sample transport needle tip
location T21. A fluid venting path is preferably present between
pointed end 1310 of venting needle 164 at venting needle tip
location V21 and carbon array 440, which fluid venting path is
preferably formed by microfluidic channel 634 terminating at
venting aperture 554 of chamber B14, which chamber B14 is
preferably in turn connected to carbon array 440 via venting
aperture 784 (FIG. 6B) which is aligned with carbon array outlet
aperture 462. A fluid transport path is preferably present between
pointed end 1312 of sample transport needle 174 at sample transport
needle tip location T21 and carbon array 440, which fluid transport
path is preferably formed by microfluidic channel 782 which
terminates at aperture 783 (FIG. 6B), which is aligned with carbon
array inlet aperture 460 of carbon array 440.
[0319] FIG. 32D shows the lowering of piston 1300 of syringe 194,
as indicated by an arrow 3250, thus forcing reaction liquid 3204
into operative engagement with the interior of carbon array 440,
thereby washing carbon array 440. Reaction liquid 3204 displaces
previously present reaction liquid 3104, which displaced reaction
liquid 3104 preferably drains from carbon array 440 into chamber
B14 via carbon array outlet aperture 462 and venting aperture 784
(FIG. 6B) as indicated by an arrow 3251.
[0320] As seen in FIG. 32D, reaction liquid 3204 flows from
interior volume 1302 of syringe 194, as indicated by an arrow 3252;
through tube 190, as indicated by an arrow 3254, through interior
passageway 1326 of sample transport needle 174, as indicated by an
arrow 3256, through microfluidic channel 782, as indicated by an
arrow 3258, and into carbon array 440 via carbon array inlet
aperture 460.
[0321] It is understood that carbon array 440 may be washed by the
entire volume of reaction liquid 3204 in a single step, as
illustrated in FIG. 32D, or may be washed by aliquots of reaction
liquid 3204 in multiple steps, wherein piston 1300 is progressively
incrementally lowered between such steps.
[0322] Carbon array 440 is preferably imaged following the washing
thereof.
[0323] It is appreciated that the heating of the sample 1204,
described hereinabove with reference to FIG. 13F, as well as
movement of magnet 1430, venting and sample transport needles 164
and 174 and piston 1300 described hereinabove, are preferably
provided by a computerized controller and mechanism which is not
part of cartridge 100.
[0324] It is also appreciated that those contents of the chambers
described with reference to Tables I-II and FIGS. 13A-32D which am
described as being in the form of concentrated fluid requiring
dilution may alternatively be in the form of a powder or solid
requiring reconstitution.
[0325] It is further appreciated that the volume of fluid shown in
the various chambers, sample transport needle 174 and syringe 194
at the various stages throughout the process described hereinabove
with reference to FIGS. 13A-32D is not shown to scale and may
illustrate more or less than the actual volume of fluid
present.
[0326] It will be appreciated by persons skilled in the art that
the present invention is not limited by what has been specifically
described hereinabove and includes both combinations and
subcombinations of the features described hereinabove as well as
equivalents and modifications thereof which would occur to persons
skilled in the art upon reading the foregoing and which are not in
the prior art.
* * * * *