U.S. patent application number 15/660616 was filed with the patent office on 2018-02-01 for multilayer disposable cartridge for ferrofluid-based assays and method of use.
The applicant listed for this patent is Ancera Corp.. Invention is credited to Hur KOSER, G. Thomas ROTH, William M. SUTTER.
Application Number | 20180029033 15/660616 |
Document ID | / |
Family ID | 61011494 |
Filed Date | 2018-02-01 |
United States Patent
Application |
20180029033 |
Kind Code |
A1 |
KOSER; Hur ; et al. |
February 1, 2018 |
MULTILAYER DISPOSABLE CARTRIDGE FOR FERROFLUID-BASED ASSAYS AND
METHOD OF USE
Abstract
The disclosed embodiments relate to method, system and apparatus
for assay testing. In an exemplary embodiment, the disclosure
relates to a cartridge for testing an assay. The cartridge includes
a sample reservoir to receive a mixture of a plurality of target
particles and a ferrofluidic solution; a capture region formed on
the cartridge; a fluidic channel to communicate the mixture between
the sample reservoir and the capture region; a magnetic
ferrofluidic solution positioned inside the fluidic channel; and at
least one pneumatic valve to communicate a quantity of the mixture
from the sample reservoir. The magnetic ferrofluidic solution is
excitable in response to an externally applied electromagnetic
field to affect the ferrofluidic solution in the mixture.
Inventors: |
KOSER; Hur; (Wellingford,
CT) ; ROTH; G. Thomas; (Fairfield, CT) ;
SUTTER; William M.; (Philadelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ancera Corp. |
Branford |
CT |
US |
|
|
Family ID: |
61011494 |
Appl. No.: |
15/660616 |
Filed: |
July 26, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62369163 |
Jul 31, 2016 |
|
|
|
62369151 |
Jul 31, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/502707 20130101;
B01L 2300/10 20130101; B01L 2300/0627 20130101; B03C 1/288
20130101; G01N 15/1056 20130101; B01L 2400/043 20130101; B01D
19/0031 20130101; B01L 3/502761 20130101; B01L 2400/0487 20130101;
B03C 1/32 20130101; G01N 15/1031 20130101; G01N 35/1097 20130101;
G01N 2015/0065 20130101; B01L 2200/0652 20130101; B03C 2201/18
20130101; B03C 1/0335 20130101; B01L 2400/082 20130101; B01L
2400/0622 20130101; G01N 2015/1081 20130101; B01L 2300/0681
20130101; B01L 3/50273 20130101; B03C 2201/20 20130101; G01N
2015/1006 20130101; B03C 2201/26 20130101; B07B 1/4636 20130101;
G01N 21/6452 20130101; B01L 3/502738 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; B07B 1/46 20060101 B07B001/46; B03C 1/32 20060101
B03C001/32; G01N 15/10 20060101 G01N015/10; B01D 19/00 20060101
B01D019/00 |
Claims
1. A biological particle capture device, comprising: a sample
reservoir to receive a mixture of a plurality of target particles
and a ferrofluidic solution; a capture region formed on the
cartridge; a fluidic channel to communicate the mixture between the
sample reservoir and the capture region, the fluidic channel
configured to receive a magnetic ferrofluidic solution; and at
least one pneumatic valve to communicate a quantity of the mixture
from the sample reservoir; wherein the magnetic ferrofluidic
solution is excitable in response to an externally applied
electromagnetic field to affect the ferrofluidic solution in the
mixture.
2. The device of claim 1, wherein the magnetic ferrofluidic
solution is excitable in response to an externally applied
electromagnetic field to attract the ferrofluidic solution to a
proximal region of the fluidic channel.
3. The device of claim 1, wherein the at least one pneumatic valve
is responsive to an external pressure to communicate the mixture
from the reservoir to the fluidic channel.
4. The device of claim 1, further comprising a secondary reservoir
to receive a secondary solution.
5. The device of claim 1, wherein the sample reservoir further
comprises a plurality of reservoir wells and wherein each well is
configured to receive an independent mixture.
6. The device of claim 1, further comprising a filter mesh
positioned between the sample reservoir and the fluidic
channel.
7. The device of claim 6, further comprising a controller to cause
application of a magnetic field to the filter mesh to dynamically
change a threshold filter particle size.
8. The device of claim 1, further comprising a degasser region to
remove gas from the one or more fluidic channels.
9. The device of claim 1, further comprising a plurality of capture
molecules proximate to the capture region to capture at least some
of the plurality of target particles through proximity with the
capture molecules.
10. A method to sort biological particle in a cartridge, the method
comprising: communicating a mixture of a plurality of target
particles and a ferrofluidic solution from a reservoir to a capture
region through a fluidic channel; activating the ferrofluidic
solution inside the fluidic channel by applying an electromagnetic
field; substantially localizing a quantity of the ferrofluidic
solution to a region influenced by the electromagnetic field while
directing target particles toward the capture region; and
identifying target particles at the capture region; wherein the
ferrofluidic solution is activated in response to an externally
applied electromagnetic field to affect the ferrofluidic solution
in the mixture.
11. The method of claim 10, further comprising communicating a
quantity of the mixture from the reservoir to the fluidic channel
using a pneumatic valve integrated into the fluidic channel.
12. The method of claim 10, wherein the directing of target
particles toward the capture regions comprises pneumatically moving
the particles toward the capture region.
13. The method of claim 11, wherein the pneumatic valve is
responsive to an external pressure to communicate the mixture from
the reservoir to the fluidic channel.
14. The method of claim 10, wherein applying the electromagnetic
field comprises applying an external electromagnetic field to
attract the ferrofluidic solution to a proximal region of the
fluidic channel.
15. The method of claim 10, further comprising introducing a dye to
the reservoir.
16. The method of claim 10, further comprising filtering the
mixture through a filter to capture at least a first particle
before communicating the mixture from the reservoir to the fluidic
channel.
17. The method of claim 16, further comprising electromagnetically
tuning the filter to capture the at least first particle.
18. The method of claim 10, further comprising degassing the
mixture.
19. The method of claim 10, further comprising positioning the
cartridge proximal to an external excitation source to align an
excitation source electrode with the fluidic channel to provide an
externally applied electromagnetic force to the ferrofluidic
solution positioned inside the fluidic channel.
20. An integrated cartridge to separate particles from a mixture,
the cartridge comprising: a sample reservoir to receive a mixture
of a plurality of target particles and a ferrofluidic solution; a
capture region formed on the cartridge; a fluidic channel to
communicate the mixture between the sample reservoir and the
capture region; a filter positioned between the sample reservoir
and the fluidic channel, the filter having at least one aperture
configured to retain particles larger than a threshold size; and a
fluidic pump to convey the mixture from the filter to the capture
region.
21. The cartridge of claim 20, wherein the filter comprises an
electromagnetic filter.
22. The cartridge of claim 21, wherein the electromagnetic filter
communicates with an external source to dynamically tune the at
least one aperture size.
23. The cartridge of claim 20, wherein the fluidic pump comprises a
movable diaphragm responsive to an external pressure and wherein
the diaphragm is integrated with the cartridge.
24. The cartridge of claim 20, wherein the fluidic channel
comprises a smooth surface to communicate the mixture.
25. The cartridge of claim 20, wherein the fluidic channel
comprises a pattern to communicate the mixture.
26. The cartridge of claim 20, wherein the capture region further
comprises a piezoelectric sensor.
27. The cartridge of claim 20, wherein the capture region further
comprises an integrated electrode.
Description
BACKGROUND
[0001] The present application claims priority to U.S. Provisional
Application Ser. No. 62/369,151, filed Jul. 31, 2016, and entitled
"Systems, Devices and Methods for Cartridge Securement," and U.S.
Provisional Application Ser. No. 62/369,163, filed Jul. 31, 2016,
and entitled "Multilayer Disposable Cartridge for Ferrofluid-Based
Assays and Method of Use." The disclosures of each of these
applications are incorporated herein by reference in their
entireties.
FIELD
[0002] The disclosure generally relates to a multilayered
disposable cartridge for ferrofluid based assays and method for
using same.
BACKGROUND
[0003] Conventional laboratory testing and measurement systems are
comprised of at least two components: an instrument and a
cartridge. The instrument provides power and excitation signals to
perform a given assay and measures generated signals to ultimately
quantify the result of the said assay. The cartridge can be
inserted into the instrument and provides an interface between the
instrument and the assay.
[0004] The cartridge may be replaced and/or disposed of at the end
of each assay; a new cartridge may be inserted into the instrument
at the beginning of the next assay. In most applications, the
disposable cartridge is inserted into the instrument through an
opening at the beginning of the new assay. This opening can be a
door, a slot or a compartment built into the instrument to receive
the cartridge. In some assays, reagents flow within channels inside
the cartridge. The reagents transport biological and/or chemical
moieties relevant to the assay from input reservoirs into different
compartments within that cartridge. The fluid motion leads to
pressure variations between different segments or channels of the
cartridge. The fluid motion also leads to pressure differences
between the inside of the cartridge and the ambient pressure. As
such, cartridge walls are normally built so that they are thick
enough to withstand and tolerate pressure differences between its
channels and the ambient pressure.
[0005] Conventional fluidic devices make use of physical phenomena
that apply controlled forces on a stationary collection or a stream
of non-biological and biological particles, molecules, cells, or
microbeads to manipulate them in the context of a given assay.
Examples of such approaches include microfluidic devices that
utilize dielectrophoresis or acoustophoresis for cell separation
and capture, as well as immune-magnetic separation devices that use
functionalized magnetic microbeads and externally applied magnetic
fields to enrich cell populations. In the case of highly localized
forces (e.g. electrostatic, dielectrophoretic, or acoustophoretic),
the force transducer typically needs to be integrated within the
body of the fluidic cartridge. Hence, the cartridge requires
electrical ports in addition to fluidic and pneumatic ports. These
requirements substantially increase the cost of the cartridge.
[0006] Conventional ferrofluid-based cellular and biological
particle manipulation scheme rely on current-carrying electrodes on
an industrial printed circuit board (PCB). The magnetic fields
generated in such devices may be short-range and limited by
electrode spacing on the PCB traces (e.g., 250 microns or less). In
order to keep the design of the fluidic cartridge simple and
low-cost, the excitation PCB resides outside the cartridge
volume.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] These and other embodiments of the disclosure will be
discussed with reference to the following exemplary and
non-limiting illustrations, in which like elements are numbered
similarly, and where:
[0008] FIG. 1 is a perspective view of an exemplary embodiment of a
cartridge;
[0009] FIG. 2 is a schematic illustration of a multi-layer
cartridge according to one embodiment of the disclosure;
[0010] FIG. 3 is a top view of an exemplary cartridge showing
components according to some embodiments of the disclosure;
[0011] FIG. 4 illustrates one or more layers having a pumping valve
and degassing chambers;
[0012] FIG. 5A provides a simplified schematic of the fluidic
network;
[0013] FIGS. 5B-5D illustrate an exemplary pumping sequence for a
multilayer cartridge; and
[0014] FIG. 6 schematically illustrates an exemplary layer of the
multi-layered cartridge containing the main assay channel.
DETAILED DESCRIPTION
[0015] In some embodiments, the design of a fluidic cartridge can
be simplified and production cost can be reduced by, for example,
placing an excitation printed circuit board (PCB) outside the
cartridge volume. The excitation PCB may comprise one or more
excitation electrodes to generate an electromagnetic field. A
disposable fluidic cartridge for ferrofluid-based assays according
to some embodiments is schematically illustrated in FIG. 1. The
integrated cartridges allow ferrofluid and sample mixtures to run
in self-contained fluidic networks for performing biological
assays. The cartridge architecture provides main channels in which
biological particle manipulation, concentration, separation,
sorting and/or capture takes place. Such biological particles may
include cells, microbeads, bacteria, fungi, algae, viruses, etc.
The main channels may be in intimate proximity to the magnetic
excitation fields that are generated off the cartridge. For
example, the magnetic excitation fields may be generated by an
instrument which receives the cartridge. Such instrument may be
stand-alone instrument with a cavity or a receptacle configured to
receive the fluidic cartridge.
[0016] The cartridge may be configured with a plurality of capture
molecules that are configured for capturing capture particles. The
capture particles may be one or more of a biological or a
non-biological particle. The capture molecules (which may be, e.g.,
at least one receptor, antibody, lectin, etc.) may be configured
downstream of the main channels and are configured to bind with the
capture particles. The capture molecules may be affixed to a
capture zone of the cartridge. As such, these main channels may
reside just inside one of the faces of the disposable cartridge
(typically the bottom face). In some embodiments, the main channels
may be capped by a very thin (typically 25-50 microns) layer of
film. The film may comprise PET, PMMA, or some other flexible
material. As used herein, the term particle may include: latex
beads; any bead ranging in size from 0.1 micron to 20 microns;
cells; bacteria; algae; parasites; cysts; viruses; spores;
macromolecular biological assemblies; cellular organelles; oocytes;
sperm cells; and/or the like.
[0017] In some exemplary embodiments, the magnetic field excitation
is generated by current-carrying electrode traces on a PCB, which
can be a part of the instrument. The cartridge may be placed
directly over the PCB with the main channels lined up directly (or
approximately) over the electrodes.
[0018] Fluid flow within the cartridge creates a positive pressure
(with respect to ambient pressure) inside the flow channels, which
may cause bulging or inflation of the thin bottom channels during
an assay. In order to account for this, the disclosed cartridge can
be configured to work in conjunction with a lid that applies
controlled pressure over the top surface of the cartridge in order
to maintain dimensional integrity of the main channels. In one
embodiment, the lid is a mechanical door or a clamp that closes
over the cartridge and restrains it with sufficient pressure to
prevent channel inflation. In another embodiment, an inflatable
bladder may be used to provide a substantially even pressure to the
channels, as discussed in Applicant's U.S. Provisional Patent
Application No. 62/369,151, which has been incorporated by
reference herein. For example, the lid may incorporate an air
bladder that can be pneumatically and/or electronically actuated to
inflate over and conformally cover the top side of the cartridge to
apply a uniform, tunable pressure over the cartridge.
[0019] FIG. 1 is a perspective depiction of an exemplary embodiment
of a cartridge that is configured to perform eleven independent,
parallel assays. Other embodiments of a cartridge may be configured
to perform a different number of parallel assays (such as 8 or 12),
or they may be configured to run a single assay. The width of the
cartridge 100 may change depending on the total number of assays
supported.
[0020] Cartridge 100 may comprise multiple layers integrated into a
unitary or an integrated cartridge. In an alternative embodiment,
cartridge 100 may comprise a single construction with various
features discussed below integrated therein. Cartridge 100 may
include base layer 102, cartridge-instrument alignment features
118, a reagent spotting mask 114, pump valves 120 and a reservoir
stack 108. Reservoir stack 108 may further include main reservoirs
112, return chimneys 122 and a plurality of secondary (and, in some
implementations, tertiary, etc.) reservoirs 110. The cartridge may
also comprise internal alignment features 104 and 116 that may be
used to ensure proper registration between the internal layers
during its construction.
[0021] Cartridge-instrument alignment features 118 enable aligning
placement of cartridge 100 within an assay instrument (not shown).
The alignment may ensure, in part, that the cartridge main channels
can align directly (or approximately) over the electrodes of the
excitation PCB. This may also ensure that any other interface to
the cartridge (such as pneumatic input ports for pumping fluid
reagents within the cartridge) are aligned with the corresponding
output from the instrument. Cartridge 100 may be inserted into an
instrument slot (not shown) or may be placed at a designated space
(such as a dedicated receptacle) within the assay instrument (not
shown).
[0022] A plurality of cartridge analysis windows (or viewing ports)
106 may correspond with each of a plurality of reaction channels
(not shown). As described below, the reaction channels (not shown)
may be embedded or formed over base 102. Cartridge analysis windows
106 provide optical viewing ports to each of the reaction
channels.
[0023] The reagent spotting mask 114 may optionally be added to
accommodate, for example, the precise positioning and spotting of
assay reagents (e.g., capture reagents such as antibodies,
aptamers, DNA fragments, other proteins or molecules used for
surface modification or detection, etc.). The mask may consist of a
matrix of patterned openings over an adhesive or a soft gasket
(e.g., silicone rubber, PDMS, etc.) that is temporarily affixed
over one of the bounding surfaces of the main assay channels. The
assay reagents may thus be coated (or spotted) over that surface of
the cartridge through the mask openings, either during the assembly
of the cartridge or prior to running the assay by the end-user.
Following an optional incubation period, the coated (or spotted)
windows might be washed and/or dried, and the reagent spotting mask
114 may be removed (e.g., peeled off the cartridge surface) prior
to capping the main assay channels with the final capping layer of
the multi-stack assembly.
[0024] The internal alignment features 104 and 116 may optionally
be used to assist in the assembly of the cartridge internal layers
in order to ensure that each layer is properly aligned with and
registered to its neighbors within a given positional tolerance. In
some embodiments, the alignment features may be holes of a given
shape (e.g., circular, square, hexagonal, diamond, etc.) that mate
with alignment posts on an alignment jig.
[0025] In some embodiments, the cartridge may have pneumatic input
ports 120. These ports may lead into pneumatic lines integrated
into the cartridge. Together, they relay pressure and/or vacuum
signals from the instrument to membrane valves (not shown)
integrated into the body of the cartridge.
[0026] Reservoir stack 108, as described below, can retain the
cartridge input fluids. For example, the reservoir stack 108 may
receive and retain assay reagents which are then directed to the
fluidic network (not shown in FIG. 1) of cartridge 100. Main
reservoirs 112 typically receive ferrofluid and/or input sample
reagents that are intended for the ferrofluidic assay. They may
also be configured to receive additional reagents, as needed.
[0027] In some embodiments, reservoir stack 108 may support more
than one set of reservoir wells per independent assay. Secondary
reservoirs 110 may be configured to receive secondary reagents used
for an assay under study. The secondary reagents may include
labels, dyes, secondary antibodies, PCR reagents required for DNA
amplification after cell capture, etc. In some implementations, the
secondary reservoirs may be left blank or empty.
[0028] In certain embodiments, assay cartridge 100 may include
multiple, patterned, alternating layers of double-sided adhesive
tapes and plain plastic film that are laminated to each other in a
specific sequence. One such embodiment is illustrated in FIG. 2.
The multi-layered cartridge provides ease of manufacturing and
reduced cost. In the multi-layered cartridge, each layer may be
independently patterned by a subtractive process (such as laser or
die cutting, etc.) and subsequently laminated to its neighbors
through a manual and/or automated lamination procedure. When
pressure-sensitive adhesive (PSA) layers are used, the lamination
process may involve a roll-laminator or a hydraulic linear press.
Other types of adhesives (such as heat-activated or UV-activated)
may also be used, depending on compatibility with the assay. In
some embodiments, neighboring layers are bonded using thermal
compression or solvent-based bonding techniques in place of using
adhesives.
[0029] FIG. 2 is a schematic illustration of a multi-layer
cartridge according to some embodiments. Specifically, FIG. 2 is an
exploded view of cartridge layers that may be combined to form
cartridge 100 of FIG. 1. In FIG. 2, layer 1 may be a base layer
(e.g., base layer 102, FIG. 1). Layers 2, 3-6 and 7-13 provide
integral and optional components of the cartridge. Layer M may be
an optional masking layer that may be used for coating reagents
directly on Layer 3. Layer D may be used as the degasser component.
In an exemplary manufacturing process, the layers with sequential
numbers in FIG. 2 may be laminated to each other, with the largest
number being on top and in descending order towards the bottom.
[0030] In some embodiments, certain neighboring functional layers
of the cartridge may be combined into single, injection-molded
segments that can be patterned on both sides with the desired
features. In this hybrid approach, the total number of layers (and,
hence, the assembly complexity of the cartridge) may be reduced,
which lowers manufacturing costs. For example, layers 4-6 of FIG. 2
may be combined into a single injection-molded layer with the
corresponding flow channels patterned on either side. The
connecting holes of the middle layer may be patterned through the
molded layer.
[0031] In certain embodiments, the cartridge architecture can be
based on defining the outlines of flow channels and chambers within
the adhesive layers. The plain plastic layers (interchangeably,
`end layers`) may support flow connections and via holes between
neighboring channel levels. Here, the channels can be capped from
the top and bottom by the end layers and from the side by the cut
boundaries of adhesive tape (not shown). By using different
materials or coatings for the end layers, it is possible to exert
substantial control on the wetting and filling properties of the
channels in various levels. Another advantage of such designs is
that channel depths can be controlled with relative precision,
since each channel is defined by the tightly controlled thickness
values of roll adhesive films to provide better than 5% variation
in thickness.
[0032] Since fluid flow within the cartridge creates a positive
pressure inside the flow channels, the thin bottom capping layer
(i.e., Layer 1 in FIG. 2) may bulge or inflate during operation. To
address this, in certain implementations, the cartridge may operate
in concert with a lid that applies controlled pressure over the top
surface of the cartridge to maintain dimensional integrity of the
channels. The lid can be a mechanical door or a clamp that closes
over the cartridge and restrains the cartridge with sufficient
pressure to prevent channel inflation. In other embodiments, the
lid may incorporate an inflatable bladder which can be
pneumatically actuated to inflate over the cartridge. The
inflatable bladder can conformally cover the top side of the
cartridge to apply a substantially uniform tunable pressure over
the cartridge.
[0033] FIG. 3 is a top view of an exemplary cartridge showing
components according to some embodiments of the disclosure.
Cartridge 300 of FIG. 3 shows cartridge alignment features 318,
capture and analysis region 350, degasser component 330, integrated
pneumatic pumping system 340, reservoir tank 308, secondary
reservoir 310, main reservoir 312, return chimney 322, pump valve
320, pneumatic line 344, pneumatic port 342, degasser vent 332,
capture/analysis windows 306 and optical viewing port 307.
[0034] It should be noted that FIG. 3 (as well as other Figures)
are produced for illustrative purposes. The configuration and the
layout of the various components shown in the cartridge 300 of FIG.
3 may be rearranged or removed without departing from the disclosed
principles.
[0035] Reservoir stack 308 may be configured to receive
sample-ferrofluid mixtures prior to the assay. The
sample-ferrofluid mixtures may be introduced to main reservoir 312,
for example, via pipetting by a technician or through an
independent robotic system (not shown). While not shown, a
filtering region may be included before or after reservoir section
stack 308 to exclude particles above a certain size from the
downstream fluidic components.
[0036] Integrated pneumatic pumping system 340 may be configured to
introduce and circulate the sample and reagents through the fluidic
networks of cartridge 300. The integrated-pneumatic pumping section
may include pump valves 320, pneumatic line(s) 344 and pneumatic
port 342. Pump valves 320 may be used to facilitate movement of
sample and/or reagents from the reservoir stack into the fluidic
network downstream, as described in more detail below.
[0037] Degasser component 330 occupies a section of cartridge 300
and is shown with degasser vent 332. Degasser component 330 may be
optional and may be omitted when degassing is not required. In
certain embodiments, the degassing section selectively removes or
vents gas bubbles over a certain size from the recirculating fluid
in the fluidic network.
[0038] While not shown, cartridge 300 includes a main channel
section in which ferrofluid-mediated particle manipulation,
concentration, sorting and capture takes place.
[0039] Reservoir stack 308 of FIG. 3 may be used as a holding
vessel for the input liquid reagents that are introduced into the
downstream fluidic network. The number of reservoir wells 313 may
depend on the number of independent assays that cartridge 300
supports. In the exemplary embodiment of FIG. 3, eleven independent
assays are supported by cartridge 300; this is denoted by the
number of main reservoir wells 313. The number of assays supported
by a cartridge may depend on the particular needs of a given
application. Thus, for example, the cartridge may be constructed
with a particular number assays based on the application for that
cartridge. While not shown, each reservoir may communicate with a
respective main fluidic channel. The main fluidic channels are
illustrated, for example, in layer 2 of FIG. 2.
[0040] In some embodiments, the main reservoir wells may be
configured to receive the ferrofluid-sample mixture. The sample to
be placed in the main reservoir may be mixed with a corresponding
amount of ferrofluid, either outside the cartridge prior to
introduction into the reservoir well (e.g., in a micro-centrifuge
tube) or inside the reservoir well itself. In the latter
application, a given amount (an aliquot) of ferrofluid may be
previously stored and sealed inside the main reservoir well for
each assay on the cartridge. Alternatively, the ferrofluid may be
added to the reservoir well before, after, or simultaneously with
the sample.
[0041] In some implementations, a pipettor may be used to introduce
the sample (or ferrofluid-sample mixture) into the main reservoirs.
In certain embodiments, the reservoirs may be hermetically sealed
(e.g., with plastic or foil cover) and may be punctured with the
pipette tip during sample introduction. Thus, the inner volume of
the reservoirs and the fluidic network downstream can be kept clean
and sterile, evaporation of the reagents can be mitigated during
the run, and the puncture can provide a visual indicator to the
user that the well has been filled.
[0042] The reservoir stack 308 may be configured to support more
than one set of reservoir wells per independent assay. For example,
secondary reservoir 310 (including secondary wells 315) may be
configured to receive secondary reagents for a given assay. The
secondary reagents may include labels, dyes, secondary antibodies,
PCR reagents to run DNA amplification following cell capture, etc.
In certain applications, the secondary wells may be left blank or
empty.
[0043] In some embodiments, at least one valve may be used to
connect each reservoir well 313, 315 to the fluidic network
downstream. The valve may be part of the pumping system 340 and may
be integrated onto cartridge 300. In such embodiments, the
appropriate reservoir (e.g., main reservoir 312 or secondary
reservoir 310) to remove fluid is selected by actuating its
corresponding valve while the other reservoir valves are kept
closed.
[0044] In certain embodiments, the reservoir stack 308 may be
located at the end of the cartridge on a side that is configured to
be closest to the user (not shown).
[0045] In some embodiments, the stack may have return chimneys that
are configured to return the circulated reagents back into the main
reservoir. This way, the main reservoir reagents (i.e.,
ferrofluid-sample mixture) may be recirculated through the entirety
of the fluidic network as many times as necessary. The reservoir
stack may also be configured to receive a filter mesh directly
underneath (e.g., layer 10, FIG. 2). The filter may retain
particles larger than a certain size which can be tunable using
externally applied magnetic fields as described below, as well as
aggregates of particles, aggregates of extracellular matrix, fatty
globules, and other debris.
[0046] In some embodiments, an active filter system may be
incorporated into cartridge 300. The filter may be positioned at
the bottom of the reservoir stack. One such architecture is shown
in FIG. 2, where filter layer 10 is positioned below reservoir
stack layer 12. In the depicted embodiment, Layers 9 and 11 are
adhesive layers that affix the filter layer to its neighboring
layers.
[0047] The filter may prevent particles larger than a predetermined
threshold from leaving the input reservoirs and entering the flow
channels positioned downstream of the reservoir. When used in the
context of ferrofluid-mediated assays, the filter layer may be used
both in passive or active filtration modes.
[0048] The openings (interchangeably, `pores` or `apertures`) of
the filter may be configured to be substantially larger than the
biological and non-biological particles, including microbeads, of
interest. In passive filtration mode, the filter mesh removes large
particulate contaminants and debris present in the sample or in the
sample-ferrofluid mixture. For example, the target particles of the
assay may include bacteria (1-5 microns in length, typically less
than 1 micron in width), and the filter pore size may be selected
to be between 20 and 50 microns (i.e., much larger than the target
bacteria). In this embodiment, the filter mesh removes large
particulates, such as sand, small rocks, or large aggregates of
extracellular matrices, as well as aggregates of particles. The
filter pore size can be selected to be somewhat smaller than the
smallest dimension present in the fluidic network downstream (such
as the width or height of the smallest channel) to ensure that the
fluidic networks and channels would not be physically clogged by
particulate contaminants.
[0049] When used in the so-called active mode, the filter may be
electromagnetically tuned to allow passage of particles of a
desired size. In an exemplary implementation, when a
ferrofluid-sample mixture flows from the input reservoir through
the filter, application of an external magnetic field changes the
threshold particle size that ends up being filtered by the active
filter. Specifically, even if the applied magnetic field is uniform
(or locally-uniform around the filter mesh), the field lines in the
immediate vicinity of the filter go through the higher
susceptibility ferrofluid medium within the pores, as opposed to
going through the non-magnetic filter material. Thus, negative
magnetic field gradient forms around each pore, leading to a very
localized magnetic force acting on each non-magnetic particle that
attempts to travel through the filter mesh.
[0050] The magnetic force follows the direction of the field
gradient, so as to direct each particle away from the pore and
towards the filter material between the pores. Since the
ferrofluid-mediated magnetic force on a non-magnetic particle
suspended in ferrofluid is proportional to the volume of that
particle, larger particles will feel much larger diversion forces
and will tend to land on the material between the pores, while
smaller particles will succumb to hydrodynamic drag and travel
through the pores. The separated particles stay away from the
pores. Therefore, the filter does not clog in its active mode of
operation.
[0051] As the amplitude of the externally applied magnetic field
excitation is increased, the maximum particle size that ends up
passing through the filter is proportionally reduced. This
component is therefore an actively tunable filter in which the
threshold particle size can be adjusted (i.e., tuned) by varying
the applied magnetic fields. The magnetic fields may be adjusted
either at the beginning of an assay or in real-time during an
assay. In this manner, a filter that features a pore size of 30
microns can be utilized to effectively hold on to particles that
are 5 microns and larger. The threshold size can be changed in
real-time depending on the particular stage of a given assay.
Particles that have been held back on the filter can also be
released into the fluidic network downstream simply by lowering the
magnetic field amplitude.
[0052] Referring once again to FIG. 3, the exemplary cartridge 300
may include an integrated pneumatic pumping system. The pumping
system allows biological and/or non-biological particles that enter
the main channels (see e.g., layer 2, FIG. 2) of the cartridge to
be continually pushed towards the ceiling of those channels by an
external magnetic field generated directly (or approximately) below
the cartridge. The local flow rate near a channel wall can be much
lower than the average fluid flow rate within that channel. The
lower flow rate can be due to non-slip boundary conditions of the
fluid. The biological and non-biological particles rolling over the
channel ceiling travel much slower than the average linear flow
rate. Consequently, the ferrofluid medium may be recirculated
through the fluidic system to give the cells sufficient time to
reach the analysis regions located near the downstream end of the
main channel ceiling.
[0053] The cartridge may feature continuous, closed-loop
recirculating flow of ferrofluid-sample mixture, in from the
reservoir stack, through the internal components and fluidic
network, and back to the reservoir stack again. In this approach,
it may not be necessary to add any additional ferrofluid, sample or
other reagents into the cartridge after the initial loading of the
reservoir stack at the beginning of the assay. Thus, all reagents
necessary for the assay are conveniently confined to the inside of
the disposable cartridge, enabling easy disposal of potential
biohazards at the end of each assay. Further, the instrument
requires no reagent storage or dispense capabilities. This
simplifies the design and operation of the cartridge, and is
therefore more cost effective.
[0054] FIG. 4 illustrates layers 4-6 of FIG. 2, and more
specifically, FIG. 4 is a schematic representation of layers 4-6 of
FIG. 2, illustrating the pumping valves and the degassing chamber.
Cartridge 400 of FIG. 4 includes degassing compartment 430 with
multiple degassing chambers 432. Each degassing chamber 432 may
correspond to a respective fluidic channel in the fluidic network.
Cartridge 400 also shows valves 421, 422, 423 and 424. Valve 421
may correspond to the secondary reservoir control valve and valve
422 may correspond to the main reservoir control valve. In some
embodiments, the recirculating flow may be set up by the
peristaltic action of a series of integrated membrane valves
downstream of the reservoir stack (308, FIG. 3) and the active
filter mesh (layer 10, FIG. 2).
[0055] Valves 421-424 can be actuated by pneumatic input pulses
(for example, by alternating between pressure and vacuum) generated
by the instrument (not shown) and relayed to cartridge 400 through
pneumatic ports (342, FIG. 3) located on a surface of the
cartridge. When a relative negative pressure (i.e., vacuum) is
applied to a specific valve, the valve membrane is pulled up into
the open position to thereby fill the chamber with fluid. In
contrast, when a relative positive pressure is applied (e.g., to
about 20 psi or between 10-25 psi), the valve membrane is pushed
down into the closed position to thereby evacuate the liquid within
the volume of the valve chamber. Where the fluid is drawn from, or
where it is evacuated to, can be determined by which fluidic path
is available. This, in turn, can be determined by the position of
the neighboring valves. By actuating the valves in a specific,
repeating sequence, valves 421-424 can pump reagents from the
reservoir stack (308, FIG. 3) to the fluidic network downstream
(layer 2, FIG. 2) and eventually back into the reservoir stack
(308, FIG. 3).
[0056] FIG. 5A provides a simplified schematic of the fluidic
network depicting the main and secondary reservoirs ("S"), the
pumping valves and the main channel. FIG. 5B, 5C and 5D illustrate
an exemplary pumping sequence for an exemplary multilayer cartridge
as shown in FIGS. 2-4. In FIGS. 5B-5D, the numeral 1 indicates that
the valve is pressurized (i.e., closed). The numeral 0 means the
valve is open. Other valve sequences are possible and equally
applicable without departing from the disclosed principles. The
valve sequence may be configured depending on the tolerable levels
of flow pulsation versus back-flow that the pumping sequence
generates. Typically, the faster the valves cycle through the
predetermined set of switching states, the faster the recirculating
flow will be. Thus, average flow rate may be controlled via
changing the time period spent at each set of valve states (i.e.,
by changing the duration of the pressure and vacuum pulses sent to
the pneumatic ports). It is also possible to implement variable
valve timing (i.e., different set of valve states could have
different active durations) in order to minimize the impact of flow
pulsation and back-flow issues.
[0057] Referring to FIG. 5A, secondary reservoir (S) 502 is
directed to valve 1 (V1) which is also in fluidic communication
with valve 2 (V2). V2 is in fluid communication with main reservoir
503. The output of V2 is directed to valve 3 (V3), which is
serially connected to valve 4 (V4) downstream. The output of valve
4 (V4) leads eventually to the fluid channel 504. As shown in FIG.
5A, channel 504 is also connected to the main reservoir 503.
[0058] The pneumatic valves (V1-V4) integrated into the cartridge
can also act as stop valves. In the exemplary embodiments of FIGS.
4 and 5A, valve 2 (V2) connects directly to the main
sample-ferrofluid reservoir, while valve 1 (V1) connects to the
secondary (e.g., label, dye) reservoir 502. The main portion of the
assay circulates the sample-ferrofluid mixture through the fluidic
network. Hence, valves V2, V3, and V4 may be actuated in sequence
while valve 1 remains closed (i.e., pressurized). After particle
manipulation and/or capture is complete, V2 is closed, and V1, V3
and V4 are actuated in sequence to introduce the secondary reagent
(e.g., for a label or dye) into the channels. This process of
pumping from a different reservoir may be repeated as needed. The
approach can be flexible and can easily accommodate additional
input reservoirs (i.e. beyond the two exemplified here) as long as
one additional valve is added to the pump subsystem for each new
reservoir. The opening and closing sequences of valves V1-V4 of
FIG. 5A are illustrated in the tables of FIGS. 5B, 5C and 5D.
[0059] As stated, the cartridge may also incorporate a degasser
component downstream of the integrated pump. The degasser may
remove gas bubbles that may be either initially present or
subsequently generated within the input reagents (e.g., cavitation
around the pump valves). Gas bubbles may be removed from fluids
prior to the fluid introduction to the cartridge channels
downstream. An exemplary degasser component was illustrated in
FIGS. 3 (degasser 330) and 4 (degasser 430).
[0060] In some embodiments, the degassing functionality may be
achieved by forming a flow chamber where at least one wall is
comprised of a hydrophobic (or super-hydrophobic) porous membrane
on one side and open to the atmosphere on the other side. As a gas
bubble suspended in the fluid reagent flows through the degassing
chamber, the bubble makes contact with the hydrophobic membrane and
is pushed into the pores by the fluid pressure. As the residence
time inside the volume of the degassing chamber is increased, the
degassing becomes more effective and efficient. The smaller the
pore size of the hydrophobic membrane, the stronger the capillary
forces in the pores and the more fluid back pressure the membrane
can withstand before the fluid leaks through the pores.
[0061] In some embodiments, the membrane may be made of a
hydrophobic material. Such materials include
poly-tetrafluoro-ethylene (PTFE). In an exemplary embodiment, the
vent pore diameter may be in a range of about 100 nanometers (0.1
microns) or smaller. In such embodiments, the degasser can
withstand at least several tens of psi of fluid pressure. Pores of
about 0.1 microns wide or smaller may not let bacteria and larger
cells through when there is a fluid leak. Thus, if a spill out of
the cartridge occurs, the spill is sterile and/or cell-free. The
membrane may be made of different materials including plastics and
may be further coated to improve hydrophobic properties.
[0062] In an exemplary embodiment, the porous PTFE membrane may be
bonded to a polyester or a polypropylene mesh backbone. The thin
PTFE membrane may be fragile and difficult to handle without
wrinkling. Thus, in some embodiments, a mesh backbone may be added
to make it much easier to process, cut, and handle the degasser
film. The mesh also permits gas bubbles to vent laterally from the
top side of the PTFE membrane which allows proper operation of the
degasser even when it is covered from the mesh side by other
capping layers of the cartridge.
[0063] By way of illustration, FIG. 3 shows an exemplary embodiment
where the degasser film is covered by the top layer (i.e., the
molded plastic backbone of the cartridge) and vents to atmosphere
from either side of the cartridge 332.
[0064] In some embodiments, the degasser may vent into channels
that connect with overflow reservoirs that are part of the
reservoir stack. In such applications, any accidental leak through
the degasser may be contained without leaking outside the cartridge
volume.
[0065] FIG. 6 schematically illustrates an exemplary layer of the
multi-layered cartridge containing the main assay channel. Layer
600 may define a layer within the cartridge. For example, layer 600
may define layer 2 of FIG. 2. Layer 600 is shown with eleven
independent channels 610. It should be noted that the illustrated
number of channels is purely exemplary; more or fewer assay
channels may be included without departing from the disclosed
principles.
[0066] FIG. 6 also shows fluidic connector channels 612 and 614.
These small channels carry the outputs of the main and secondary
reservoirs (directly downstream of the reservoir stack and filter
components) to valves V2 and V1, respectively.
[0067] In some embodiments, main channel layer 600 may be located
downstream of the degasser component of the cartridge. The main
channel layer may be where the ferrofluid-mediated particle
sorting, separation, manipulation, concentration, enrichment,
specific capture and/or eventual quantification takes place.
Additional biochemical reactions may also take place in the channel
layer.
[0068] An exemplary cartridge may be positioned in an instrument
having excitation electrodes. The excitation electrodes generate
fields that can manipulate ferrofluidic material in channels 610.
Thus, the main channels may be configured to line up proximal to
excitation electrodes of the instrument PCB (not shown). Further,
the width of channels 610 may closely correlate to the width of
each PCB electrode (not shown). In an exemplary implementation, the
electrode set was about 4.00 millimeters wide and the main channel
width of the corresponding cartridge was about 3.85
millimeters.
[0069] The length of the main channels may also correlate with the
electrode length (not shown) of the PCB. This dimension may be
determined as a function of the particles traveling within the main
channels and the length needed to push up and/or sort the particles
prior to capture/analysis regions which are downstream and at the
end of the channels. By way of example, for a main channel depth of
around 85 microns and width of 3.85 millimeters, a channel length
on the order of about 5 cm is needed. This ensures that bacteria
suspended in a moderate strength ferrofluid mixture (i.e., magnetic
susceptibility on the order of 0.1-0.5) flowing at about 10-50
microliters/min can be focused between the two central electrode
traces (i.e., within a narrow central band of about 200 microns)
when the electrodes generate up to 10 mT of magnetic flux density
within the channel volume.
[0070] In some embodiments, the main channel inner walls may be
featureless and smooth. In another embodiment, the channel inner
walls may include micro-scale patterns that interact with the
ferrohydrodynamic flow to assist in particle sorting/separation and
particle capture. Such surface features may include micro posts or
chevron structures (patterns) to assist in hydrodynamic separation
of particles based on size. These features may act independently of
the magnetic fields applied to the cartridge or they may interact
with the fields to enhance or augment the intended function. Some
micro-structures within the main channels may interact with the
applied fields to act as secondary active filters. Microposts
functionalized with capture ligands (such as antibodies, aptamers,
single-strand DNA, etc.) may also be utilized in the
analysis/capture region.
[0071] Near the downstream end of the main channels 610, a
capture/analysis region may be positioned. In one embodiment, the
main channels feature antibody-coated capture windows. An exemplary
window is shown in FIG. 3 as capture/analysis window 306. The
cartridge layers directly above these windows are designed to be
optically transparent. The optical transparency can be accomplished
either by using layers that are transparent themselves or by simply
cutting out viewing ports through the otherwise opaque layers.
[0072] In some embodiments, the capture/analysis region may feature
integrated thin electrodes and quantification of the assay results
may be based on measuring impedance changes over the window at
various frequencies. In some implementations, the sensor integrated
in this region is a piezoelectric mass balance or an
electrochemical sensor. Such sensors can provide additional
information based on observations made at the capture/analysis
region. A piezoelectric mass balance, for example, can provide
information about the captured particles. The electrochemical
sensor can provide information about the captured particles' charge
or pH. In such non-optical sensor approaches, the cartridge may not
need additional optical viewing ports but may have other components
such as thin electrodes integrated or printed on top of thin sheets
of plastic film.
[0073] It should be noted that an exemplary cartridge may be
configured to include a number of assays without departing from the
disclosed principles. For example, the cartridge may be configured
to include as few as one or more assays. In one exemplary
embodiment, the cartridge includes up to twelve or more assays.
[0074] The following embodiments illustrate exemplary and
non-limiting embodiments of the disclosure. Example 1 is directed
to a biological particle capture device, comprising: a sample
reservoir to receive a mixture of a plurality of target particles
and a ferrofluidic solution; a capture region formed on the
cartridge; a fluidic channel to communicate the mixture between the
sample reservoir and the capture region; a magnetic ferrofluidic
solution positioned inside the fluidic channel; and at least one
pneumatic valve to communicate a quantity of the mixture from the
sample reservoir; wherein the a magnetic ferrofluidic solution is
excitable in response to an externally applied electromagnetic
field to affect the ferrofluidic solution in the mixture.
[0075] Example 2 is directed to the device of example 1, wherein
the a magnetic ferrofluidic solution is excitable in response to an
externally applied electromagnetic field to attract the
ferrofluidic solution to a proximal region of the fluidic
channel.
[0076] Example 3 is directed to the device of example 1, wherein
the pneumatic valve is responsive to an external pressure to
communicate the mixture from the reservoir to the fluidic
channel.
[0077] Example 4 is directed to the device of example 1, further
comprising a dye reservoir to receive a dye solution.
[0078] Example 5 is directed to the device of example 1, wherein
the reservoir further comprises a plurality of reservoir wells and
wherein each well is configured to receive an independent
assay.
[0079] Example 6 is directed to the device of example 1, further
comprising a filter mesh positioned between the sample reservoir
and the at least one fluidic channel.
[0080] Example 7 is directed to the device of example 6, further
comprising a controller to cause application of a magnetic field to
the filter mesh to dynamically change a threshold filter particle
size.
[0081] Example 8 is directed to the device of example 1, further
comprising a degasser region to remove gas from the one or more
fluidic channels.
[0082] Example 9 is directed to the device of example 1, further
comprising a plurality of capture molecules proximate to the
capture region to capture at least some of the plurality of target
particles through proximity with the capture molecules.
[0083] Example 10 is directed to a method to sort biological
particle in a cartridge, the method comprising: communicating a
mixture of a plurality of target particles and a ferrofluidic
solution from a reservoir to a capture region through a fluidic
channel; activating a magnetic ferrofluidic solution inside the
fluidic channel by applying an electromagnetic field; substantially
localizing a quantity of the ferrofluidic solution to a region
influenced by the electromagnetic field while directing target
particles toward the capture regions; and identifying target
particles at the capture region; wherein the magnetic ferrofluidic
solution is activated in response to an externally applied
electromagnetic field to affect the ferrofluidic solution in the
mixture.
[0084] Example 11 is directed to the method of example 10, further
comprising communicating a quantity of the mixture from the
reservoir to the fluidic channel using a pneumatic valve integrated
into the fluidic channel.
[0085] Example 12 is directed to the method of example 10, wherein
the step of directing target particles toward the capture regions
further comprises pneumatically moving the particles toward the
capture region.
[0086] Example 13 is directed to the method of example 12, wherein
the pneumatic valve is responsive to an external pressure to
communicate the mixture from the reservoir to the fluidic
channel.
[0087] Example 14 is directed to the method of example 10, wherein
activating the electrode further comprises applying an external
electromagnetic field to attract the ferrofluidic solution to a
proximal region of the fluidic channel.
[0088] Example 15 is directed to the method of example 10, further
comprising introducing a dye to the reservoir.
[0089] Example 16 is directed to the method of example 10,
filtering the mixture through a filter to capture a first particle
before communicating the mixture from the reservoir to the fluidic
channel.
[0090] Example 17 is directed to the method of example 16, further
comprising electromagnetically tuning the filer to capture the
first particle.
[0091] Example 18 is directed to the method of example 10, further
comprising degassing the mixture.
[0092] Example 19 is directed to the method of example 10, further
comprising positioning the cartridge proximal to an external
excitation source to align an excitation source electrode with the
fluidic channel to thereby provide an externally applied
electromagnetic force to the magnetic ferrofluidic solution
positioned inside the fluidic channel.
[0093] Example 20 is directed to an integrated cartridge to
separate particles from a mixture, the cartridge comprising: a
sample reservoir to receive a mixture of a plurality of target
particles and a ferrofluidic solution; a capture region formed on
the cartridge; a fluidic channel to communicate the mixture between
the sample reservoir and the capture region; a filter positioned
between the sample reservoir and the fluidic channel, the filter
having at least one aperture configured to retain particles larger
than a threshold size; and a fluidic pump to convey the mixture
from the filter to the capture region.
[0094] Example 21 is directed to the cartridge of example 20,
wherein the filter comprises an electromagnetic filter.
[0095] Example 22 is directed to the cartridge of example 21,
wherein the electromagnetic filter communicates with an external
source to dynamically tune the at least one aperture size.
[0096] Example 23 is directed to the cartridge of example 20,
wherein the fluidic pump comprises a movable diaphragm responsive
to an external pressure and wherein the diaphragm is integrated
with the cartridge.
[0097] Example 24 is directed to the cartridge of example 20,
wherein the fluidic channel comprises a smooth surface to
communicate the mixture.
[0098] Example 25 is directed to the cartridge of example 20,
wherein the fluidic channel comprises a pattern to communicate the
mixture.
[0099] Example 26 is directed to the cartridge of example 20,
wherein the capture region further comprises a piezoelectric
sensor.
[0100] Example 27 is directed to the cartridge of example 20,
wherein the capture region further comprises an integrated
electrode.
[0101] Exemplary embodiments of the devices, systems and methods
have been described herein. As noted elsewhere, these embodiments
have been described for illustrative purposes only and are not
limiting. Other embodiments are possible and are covered by the
disclosure, which will be apparent from the teachings contained
herein. Thus, the breadth and scope of the disclosure should not be
limited by any of the above-described embodiments but should be
defined only in accordance with claims supported by the present
disclosure and their equivalents. Moreover, embodiments of the
subject disclosure may include methods, systems and devices which
may further include any and all elements from any other disclosed
methods, systems, and devices, including any and all elements
corresponding cartridges and systems thereof. In other words,
elements from one or another disclosed embodiment may be
interchangeable with elements from other disclosed embodiments. In
addition, one or more features/elements of disclosed embodiments
may be removed and still result in patentable subject matter (and
thus, resulting in yet more embodiments of the subject disclosure).
Correspondingly, some embodiments of the present disclosure may be
patentably distinct from one and/or another reference by
specifically lacking one or more elements/features. In other words,
claims to certain embodiments may contain negative limitation to
specifically exclude one or more elements/features resulting in
embodiments which are patentably distinct from the prior art which
include such features/elements.
[0102] While the principles of the disclosure have been illustrated
in relation to the exemplary embodiments shown herein, the
principles of the disclosure are not limited thereto and include
any modification, variation or permutation thereof.
* * * * *