U.S. patent application number 09/917649 was filed with the patent office on 2002-03-14 for fluidics system.
Invention is credited to Feldstein, Mark J..
Application Number | 20020031836 09/917649 |
Document ID | / |
Family ID | 22869693 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020031836 |
Kind Code |
A1 |
Feldstein, Mark J. |
March 14, 2002 |
Fluidics system
Abstract
The present invention provides a fluidics system and a method
for selectively drawing fluid from at least one selected reservoir
into a channel by providing a negative pressure source downstream
of the fluid and channel and selectively back filling the selected
reservoir with a gas.
Inventors: |
Feldstein, Mark J.;
(Washington, DC) |
Correspondence
Address: |
Naval Research Laboratory, Code 1008.2
4555 Overlook Ave., S.W.
Washington
DC
20375-5320
US
|
Family ID: |
22869693 |
Appl. No.: |
09/917649 |
Filed: |
July 31, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60231548 |
Sep 11, 2000 |
|
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|
Current U.S.
Class: |
436/180 ;
422/400; 422/82.05; 422/82.11; 436/164 |
Current CPC
Class: |
Y10T 137/0352 20150401;
B01L 2300/0654 20130101; B01L 2400/0622 20130101; B01L 2300/0887
20130101; B01L 2400/049 20130101; B01L 3/5027 20130101; B01L
2300/0864 20130101; Y10T 436/2575 20150115; B01L 2300/0816
20130101; B01L 3/50273 20130101; B01L 3/502738 20130101; B01L 3/502
20130101; B01L 2400/084 20130101; B01L 2400/0694 20130101; B01L
2400/0633 20130101; B01L 2400/0655 20130101; B01L 2400/082
20130101; B01L 2300/168 20130101; B01L 2300/0867 20130101 |
Class at
Publication: |
436/180 ;
436/164; 422/82.05; 422/82.11; 422/100 |
International
Class: |
G01N 035/10 |
Claims
What is claimed is:
1. A fluidics system, comprising; a primary fluid channel
comprising an input and an output; an enclosed first reservoir
connected to said primary fluid channel input and comprising a
first adjustable vent; an enclosed second reservoir connected to
said primary fluid channel input and comprising a second adjustable
vent; a negative pressure connect to said primary fluid channel
output; wherein the fluidics system is configured to selectively
draw at least one fluid from at least one of the first and second
reservoirs into the primary fluid channel when the negative
pressure source is activated and the respective reservoir is
unsealed.
2. The fluidics system of claim 1, further comprising: an
analytical device associated with said primary fluid channel.
3. The fluidics system of claim 1, wherein said primary fluid
channel is at least 10% larger in cross section than any particle
in said first and second fluids.
4. The fluidics system of claim 1, further comprising: more than
one secondary fluid channels configured parallel and/or serial to
each other.
5. The fluidics system of claim 4, further comprising: more than
one negative pressure sources downstream of said secondary fluid
channels.
6. The fluidics system of claim 4, further comprising: a manifold
connecting said secondary fluid channels to said negative pressure
source.
7. The fluidics system of claim 1, wherein said first reservoir
comprises more than one chamber.
8. The fluidics system of claim 1, further comprising: a valve
associated with said first vent; and a valve associated with said
second vent.
9. The fluidics system of claim 1 further comprising: an auxiliary
fluid reservoir and a connection valve, wherein the auxiliary fluid
reservoir is connected through the connection valve to an auxiliary
input of at least one the first and second reservoirs; and the
system is configured to selectively draw fluid from the auxiliary
fluid reservoir into at least one of the first and second
reservoirs when the negative pressure source is activated, the
connection valve is open, and the respective reservoir is not
vented to a pressure source having a pressure less than a pressure
of the negative pressure source.
10. The fluidics system of claim 1, further comprising: a second
primary fluid channel; and a second manifold connecting said
primary fluid channels to said negative pressure source down stream
of said primary fluid channels.
11. The fluidics system of claim 1, further comprising: a waveguide
for surface-sensitive optical detection of an analyte in said first
or second fluid.
12. The fluidics system of claim 11, further comprising: a
waveguide sensing system; wherein said waveguide sensing system
comprises: a plurality of waveguides; wherein each of said
waveguides has a first surface, a second surface opposing said
first surface, and an end surface essentially perpendicular to said
first and second surfaces, and wherein said first surface of each
of said waveguides has a analyte recognition element thereon; a
waveguide holder to which each of said waveguides is secured; and
an optical detector positioned opposite said end surface of at
least one of said waveguides.
13. The fluidics system of claim 1, wherein said first and second
vents are adjustable so that first and second fluids from said
first and second reservoirs, respectively, move at a first and a
second flow rate to said primary fluid channel; and wherein a
difference between said first and second flow rates is proportional
to a difference in adjustments of said first and second vents.
14. The fluidics system of claim 1, wherein first or second fluid
moves from said first or second reservoir, respectively, at a first
and second flow rate, wherein a difference between said first and
second flow rates is proportional to a differential fluid flow
resistance, and wherein said differential fluid flow resistance is
adjusted by said first and second fluid vents.
15. The fluidics system of claim 1, wherein said primary fluid
channel has a cross section greater than 1 micron.
16. The fluidics system of claim 1, wherein said system is a
portable analysis system configured to perform at least one of a
biological and chemical analysis.
17. A portable analysis system for conduction of biochemical and/or
chemical analysis, comprising: a three-dimensional fluid circuit; a
first enclosed reservoir having a first adjustable vent; a second
enclosed reservoir having a second adjustable vent; a first
passageway for receiving a first fluid from said first reservoir; a
second passageway for receiving a second fluid from said second
reservoir; a primary fluid channel; a first connecting channel
connecting said first passageway to said primary channel; a second
connecting channel connecting said second passageway to said
primary channel; a multimode waveguide; a barrier configured to
prevent fluid flow between said first and second connecting
channels; and a negative pressure source downstream of said primary
fluid channel; wherein said first and second reservoirs and
passageways are elements in said fluid circuit; wherein said fluid
circuit comprising elements and a series of layers and at least one
of said elements is formed using molding techniques and at least
partial elements are formed by molding and mechanical, chemical,
thermal or optical etching, wherein each layer of a series of
layers is at least a partial element of said fluid circuit, wherein
said layers are fused together to form complete elements of said
fluid circuit, and wherein said negative pressure source being
configured for moving said first fluid but not said second fluid to
said primary fluid channel when said first adjustable lent is not
in a closed position and said second adjustable vent is in a closed
position; for moving said second fluid but not said first fluid to
said primary fluid channel when said second adjustable vent is not
closed and said first adjustable vent is closed; and for moving
said first and second fluids to said primary fluid channel when
said first and second adjustable vents are not closed.
18. The fluidics system of claim 17, wherein said fluidics system
is configured to conduct analysis of at least one of said first and
second fluid.
19. The fluidics system of claim 18, wherein said first and second
fluids are analyzed in said primary fluid channel.
20. The fluidics system of claim 17, wherein said first fluid is a
sample and said second fluid is a reagent.
21. A method of controlling fluid flow, comprising the steps of:
moving a first fluid in a first reservoir having an adjustable
first vent to a primary fluid channel when said first adjustable
vent is not in a closed position and not moving a second fluid in a
second reservoir having a second adjustable vent in a closed
position when a negative pressure source is activated downstream of
said primary fluid channel.
22. A method of performing a biochemical analysis, comprising the
steps of: moving a first fluid in a first reservoir having an
adjustable first vent to a primary fluid channel when said first
adjustable vent is not in a closed position and not moving a second
fluid in a second reservoir having a second adjustable vent in a
closed position when a negative pressure source is activated
downstream of said primary fluid channel; and analyzing a first
fluid.
23. The method of claim 22, wherein said analyzing step is
performed in said primary fluid channel, and wherein in said
primary channel an internal surface is configured to a least one of
capture, recognize, respond to, and detect an analyte.
24. The method of claim 23, wherein said primary fluid channel
comprises a waveguide, wherein said waveguide is adapted for
transmitting optical signals to a detector, and wherein said
optical signal indicates presence or absence of an analyte.
25. The method of claim 24, wherein said waveguide comprises a
multimode waveguide having a surface bearing patterned, reflective
coating, wherein said coating defines a reflectively coated region
and a first optically exposed region on said surface, wherein said
first optically exposed region is configured to produce an
alteration indicative of the presence of a first analyte, wherein
said alteration is detectable by launching a light wave into said
waveguide to generate an evanescent field at said patterned
surface, and then detecting an interaction of said first optically
exposed region with said evanescent wave.
26. A method of controlling fluid flow, comprising: selectively
drawing at least one fluid from at least one of a first and a
second reservoir into a primary fluid channel, the selectively
drawing comprising activating a negative pressure source and
unsealing one of the reservoirs, wherein the primary fluid channel
comprises an input and an output; the first reservoir comprises a
first fluid output fluidically connected to the primary fluid
channel input, and a first vent configured to selectively seal and
unseal said first reservoir; the second reservoir comprises a
second fluid output fluidically connected to the primary fluid
channel input, and a second vent configured to selectively seal and
unseal said second reservoir; and the negative pressure source is
connected to the primary fluid channel output.
27. The system of claim 1, wherein the system is configured such
that fluid does not flow from said reservoirs into said primary
fluid channel unless both said negative pressure source is
activated and said at least one reservoir is unsealed.
28. The system of claim 1, wherein the system further comprises a
system relief vent connected to said primary flow channel, said
system relief vent being configured to seal and unseal said primary
flow channel from contact with an external atmosphere.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/231,548, filed on Sep. 11, 2000.
FIELD OF THE INVENTION
[0002] This invention relates to methods and systems of controlling
fluid flow. This invention also relates to methods and systems of
fluid flow control for sample analysis and methods, and systems of
fluid flow control in portable fluidics systems.
BACKGROUND
[0003] Fluid control is necessary for many systems capable of
automated chemical and biochemical analysis. These systems
typically require liquid samples, reagents, and buffers to be
dispensed in a controlled manner. Making these analysis systems
portable presents unique demands on fluidics systems that have not
been successfully met by currently available technology. These
demands stem from the combined requirements of automation, compact
size and compatibility with unprocessed samples, especially for
field operations or point-of-care applications.
[0004] For laboratory-scale devices, there is an assortment of
mechanical valves suitable for fluid handling and control. However,
the size of these components makes them impractical for portable
analysis systems. While small valves of analogous design have been
developed and are commercially available, as the valve size is
reduced, clogs by the components of complex sample matrices become
an important limitation.
[0005] Micro-total analysis systems (F-TAS) perform integrated
chemical analysis and fluid control on the micron scale. Many of
these systems are capable of valveless fluid control by means of
electrokinetic pumping and switch-driving pressures. (Manz, A. et
al. in Micro Total Analysis Systems; and van den Berg, A. et al.,
Academic Publishers, Dordrecht, 1995, pp. 5-27). However,
micron-scale channels can become clogged when unprocessed
environmental and clinical samples are used. In addition, materials
can be adsorbed onto channel walls and interfere with osmotic
pumping. Furthermore, these devices have a relatively low-volume
throughput making them impractical for the analysis of milliliter
volumes, as may be required for accurate measurement of trace
constituents or analysis of inhomogeneous samples.
[0006] The need for intermediate scale fluid handling systems has
been identified. (VerLee, D. et al., Technical Digest, Solid-State
Sensor and Actuator Workshop, 1996, pp. 9-14) Among the
developments in this area are pneumatic diaphragm valves integrated
directly into the device's fluidics channels. This approach
provides fluid regulation while adding only slightly to the overall
size of the system. However, diaphragm-based valves can suffer from
sticking, clogging, and performance loss due to diaphragm
aging.
[0007] Valveless fluid control has also been developed, thus
eliminating the problem of valve clogging by suspended
contaminants. For example, pressure control and pressure
differentials can switch fluid flow between micro-channels. (Brody,
J. P., 1998, U.S. Pat. No. 5,726,404) This method of fluid control
is based on the application and regulation of differential
pressures to each fluid channel and is only applicable in the low
Reynolds number regime. The regulation of differential pressures
makes the design inherently complex and, further, the requirement
for pressure sources and regulators limits the feasibility of this
method for portable instrumentation. The limitation with regard to
the low Reynolds numbers regime makes the method impractical for
the control of aqueous fluids in channels greater than
approximately 100 microns. (Brody J. P. et al., Technical Digest,
Solid-State Sensor and Actuator Workshop, 1996, pp.105-108; and
Brody J. P., Biophysical Journal, 1996, 71, pp. 3430-3441).
Although valves may not be clogged with these approaches, the fluid
channels themselves are likely to be clogged by suspended
contaminants. Electrokinetic pumping and switching systems have
also accomplished valveless fluid control in micron-scale devices.
(Manz et al., Advances in Chromatography, 1993, 33, pp. 1-67.)
Similarly, however, these designs are limited to the low Reynolds
number regime, where micron-scale channels are prone to clogging.
Further, these methods require large driving potentials, typically
on the order of a kilovolt, and fluid flow that can be drastically
affected by sample components adhering to the wall of the
channel.
SUMMARY
[0008] According to certain embodiments, the present invention
provides a fluidics system and a method for selectively drawing
fluid from at least one reservoir into a channel by providing a
negative pressure source downstream of the fluid and channel and
simultaneously back filling the reservoir with a gas. For example,
the present invention may comprise a fluidics system comprising an
enclosed first reservoir having a first adjustable vent; an
enclosed second reservoir having a second adjustable vent; a
primary fluid channel; a first passageway for receiving a first
fluid from the first reservoir and connected to the primary fluid
channel; a second passageway for receiving a second fluid from the
second reservoir and connected to the primary fluid channel; and a
negative pressure source downstream of the primary fluid channel.
The negative pressure source is configured for moving the first
fluid but not the second fluid to the primary fluid channel when
the first adjustable vent is not in a closed position and the
second adjustable vent is in a closed position; for moving the
second fluid but not the first fluid to the primary fluid channel
when the second adjustable vent is not closed and the first
adjustable vent is closed; and for moving the first and second
fluids to the primary fluid channel when the first and second
adjustable vents are not closed.
[0009] According to certain embodiments, the present invention
provides a fluidics system and a method for selectively drawing
fluid from at least one reservoir. The fluidics system may comprise
a primary fluid channel comprising an input and an output; a first
sealable reservoir comprising a first fluid output fluidically
connected to the primary fluid channel input, and a first vent
configured to selectively seal and unseal said first reservoir; a
second sealable reservoir comprising a second fluid output
fluidically connected to the primary fluid channel input, and a
second vent configured to selectively seal and unseal said first
reservoir; and a negative pressure source connected to the primary
fluid channel output. The system can be configured to selectively
draw at least one fluid from at least one of the first and second
reservoirs into the primary fluid channel when the negative
pressure source is activated and the respective reservoir is
unsealed.
[0010] The present invention also involves a portable analysis
system for conduction of biochemical and/or chemical analysis that
contains a three-dimensional fluid circuit; a first enclosed
reservoir having a first adjustable vent; a second enclosed
reservoir having a second adjustable vent; a first passageway for
receiving a first fluid from the first reservoir; a second
passageway for receiving a second fluid from the second reservoir;
a primary fluid channel; a first connecting channel connecting the
first passageway to the primary channel; a second connecting
channel connecting the second passageway to the primary channel; a
multimode waveguide; a barrier configured to prevent fluid flow
between the first and second connecting channels; and a negative
pressure source downstream of the primary fluid channel. The first
and second reservoirs and passageways are elements of the fluid
circuit. The fluid circuit has elements and a series of layers and
at least one of the elements is formed using molding techniques,
and at least partial elements are formed by molding and mechanical,
chemical, thermal and optical etching. Each layer of a series of
layers is at least a partial element of the fluid circuit. The
layers are fused together to form a complete element of the fluid
circuit. The negative pressure source is configured for moving the
first fluid but not the second fluid to the primary fluid channel
when the first adjustable vent is not in a closed position and the
second adjustable vent is in a closed position; for moving the
second fluid but not the first fluid to the primary fluid channel
when the second adjustable vent is not closed and the first
adjustable vent is closed; and for moving the first and second
fluids to the primary fluid channel when the first and second
adjustable vents are not closed.
[0011] The present invention involves a method of performing a
biochemical analysis, having the steps of moving a first fluid in a
first reservoir having an adjustable first vent to a primary fluid
channel when said first adjustable vent is not in a closed position
and not moving a second fluid in a second reservoir having a second
adjustable vent in a closed position when a negative pressure
source is activated downstream of said primary fluid channel; and
analysing a first fluid.
BRIEF DESCRIPTION OF THE INVENTION
[0012] The accompanying drawings, are incorporated in and
constitute a part of this specification, and illustrate several
embodiments of the invention.
[0013] FIG. 1 is a schematic representation of a two-reservoir
fluidics system according to the present invention.
[0014] FIG. 2 is a schematic representation of a multi-positioned
valve for connecting a reservoir to a given source.
[0015] FIG. 3 is a schematic representation of a two-reservoir
fluidics system with serial and parallel fluid channels, multiple
negative pressure sources, and an auxiliary fluid reservoir.
[0016] FIG. 4 is a schematic representation of a three-reservoir
fluidics system with venting valves controlled by a controller.
[0017] FIG. 5 is a schematic representation of a three-sample
reservoir/chamber, two-reagent reservoir, and multiple fluid
channel fluidics system with a system relief vent.
[0018] FIG. 6 shows fluorescent signals corresponding to the
switching of a two-reservoir fluidics system.
[0019] FIG. 7 is a schematic representation of a series of layers
of a fluidics cube.
[0020] FIG. 8 is a three-dimensional perspective view of a
simplified two-sample reservoir, two-reagent reservoir fluidics
system.
[0021] FIG. 9 shows an image of an analysis performed with a
fluidics system.
[0022] FIG. 10 illustrates a summary of the analysis results from
FIG. 9.
DESCRIPTION OF CERTAIN EMBODIMENTS
[0023] Reference will now be made in detail to certain embodiments
of the invention, examples of which are illustrated in the
accompanying drawings.
[0024] The section headings used herein are for organizational
purposes only, and are not to be construed as limiting the subject
matter described. All documents cited in this application,
including, but not limited to patents, patent applications,
articles, books, and treatises, are expressly incorporated by
reference in their entirety for any purpose.
[0025] According to certain embodiments, the present invention
provides a fluidics system and a method for selectively drawing
fluid from at least one reservoir. As shown in FIG. 1, a fluidics
system 100 can include a primary fluid channel 110 having an input
end 112 and an output end 114; a first enclosed and sealable
reservoir 116 having a first fluid input duct 118 fluidically
connected to the primary fluid channel input end 112, and a first
vent 120 configured to selectively seal or unseal (open or close)
the first reservoir; a second sealable and enclosed reservoir 122
having a second fluid input duct 124 fluidically connected to the
primary fluid channel input end 112, and a second vent 126
configured to selectively seal or unseal (open or closed) the
second reservoir; and a negative pressure source 128 connected to
the primary fluid channel output end 114. The system can be
configured to selectively draw at least one fluid, 130 or 132, from
the first and/or second reservoir, 116 or 122. The fluid is drawn
through the first or second input duct, passageways, 118 or 124,
into the primary fluid channel, 110, when the negative pressure
source, 128, is activated and the selected reservoir is unsealed by
opening its vent, 120, 126. Gas can occupy space 134 above the
fluids, 130, 132 within the reservoirs, 116, 122. The reservoir is
enclosed except for an adjustable vent which can be associated with
a valve, 136.
[0026] It should be understood that, as used herein, "sealable",
"sealed", "unsealed", "open", "opened", "close", and "closed" and
grammatical variants thereof refer to the ability of a fluid to
flow in or out of an element, such as a reservoir, or to the state
of the vent that permits or prevents fluid flow. A fluidics
element, such as a reservoir, is understood to be sealed when fluid
can not readily flow out of the reservoir without, for example,
creating a (at least partial) vacuum in the reservoir.
[0027] For example, as shown in FIG. 2, reservoir 216 having a
fluid 230, a gas space above the fluid, 234, an input fluid duct
218 and a vent 220 connected to a valve 236 that is sealed when the
valve 236 is closed. As discussed further below, the reservoir 216
can also be in a sealed position when it is connected, via, for
example, by the vent 220 and valve 236, to a negative pressure
source 228. The reservoir 216 is unsealed when it is connected,
via, for example, by the vent 220 and valve 236 to the atmosphere.
As discussed further below, the reservoir 216 can also be in an
unsealed (or vented) position when it is connected, via, for
example, by the vent 220 and valve 236, to a positive pressure
source 238.
[0028] According to certain embodiments, sealed and unsealed can be
relative terms, when the sealed reservoir is connected to a
relatively low pressure source, and the unsealed reservoir is
connected to a relatively high pressure source. Further, as
discussed below, a vent can be connected not only to pressure
sources, but to additional fluid sources, such as an auxiliary
fluid source, as well. Moreover, as discussed below, a single
multi-positioned valve can be used to regulate the connection of
multiple reservoirs or chambers.
[0029] It should be understood that, as used herein, the
characterization of a pressure source as positive or negative is in
reference to atmospheric pressure. Additionally, "negative" and
"positive" can be considered relative terms when used together to
differentiate between multiple pressure sources. For example, a
negative pressure source is a pressure source that has or provides
a pressure of less than atmospheric pressure or less than a
positive pressure or provides a suction. A positive pressure source
is a pressure source that has or provides a pressure of greater
than atmospheric pressure or greater than a negative pressure.
Atmospheric pressure (also understood to be a pressure source) is
understood to be the local pressure of the atmosphere, and is not
necessarily limited to standard atmospheric pressure, and can be
either naturally occurring or artificially generated.
[0030] According to certain embodiments, the system can have
multiple fluid channels, such as more than one primary and/or
multiple secondary fluid channels, when a given primary fluid
channel includes at least two secondary fluid channels. For
example, the primary fluid channel can deliver fluid into a first
and a second secondary fluid channel, each secondary channel having
an input end and an output end or the secondary fluid channels can
function as the primary fluid channel. The multiple fluid channels
can be connected in serial fashion for serial fluid flow, in
parallel fashion for parallel fluid flow, or any combination
thereof, such that some of the multiple fluid channels are
connected in parallel while others are connected in serial.
[0031] For example, as shown in FIG. 3, the primary fluid channel
310 can be connected to secondary channels. The first 340 and
second 342 fluid channels can be fluidically connected in series or
the fluid channels 344, 346, 348 can be connected in parallel. The
first 316 and second 322 reservoirs can be fluidically connected to
input ends of the fluid channels. The connections can either be
direct connection, or can be indirect, such as, for example, via a
manifold 350, conduit, or other connection element.
[0032] According to certain embodiments, the system can be
configured to selectively draw fluid from at least one of the first
and second reservoirs into both the first and second secondary
fluid channels when the negative pressure source is activated and
the respective reservoir is unsealed. For example, in the case of
fluid flow from the first reservoir, the first reservoir duct can
be connected via a 1-2 manifold. The manifold connects to the
output end of the first reservoir duct and, on the other end,
connects to both the first and second primary fluid channels.
According to certain embodiments, the inverse connection scheme,
where two reservoirs are connected via a 2-1 manifold to a single
fluid channel, is also possible. Thus, according to certain
embodiments, the system can further include a manifold, to connect
the output ends of the first and second secondary fluid channels to
the negative pressure source. The manifold is not limited to 1-2 or
2-1 manifold, but can include any number of input and output
connections.
[0033] According to certain embodiments, including, for example,
embodiments containing multiple primary and/or secondary fluid
channels, the negative pressure source can include multiple
negative pressure sources. For example, as shown in FIG. 3, the
negative pressure source can contain first 328 and second 352
negative pressure sources. The first 328 and second 352 negative
pressure sources can be connected, for example, to the output ends
of the fluid channel (downstream) 344, 346, and 348 as shown in
FIG. 3, respectively. According to certain embodiments, the first
and second negative pressure sources can or can not be independent
negative pressure sources, and can or can not be configured to
operate sequentially and/or simultaneously.
[0034] According to certain embodiments of the present invention,
the system can be configured to selectively draw fluid from the
first and/or the second reservoir into the first and/or the second
secondary fluid channel when the negative pressure source that is
connected (downstream) to the secondary fluid channels is activated
and one or both of the reservoir's vent is unsealed, open.
[0035] For example, as shown in FIG. 3, if the first reservoir were
unsealed and the second reservoir were sealed, and if the negative
pressure source, 352, were activated, fluid in the first reservoir
would be selectively drawn into both fluid channels, 346, 348. The
secondary fluid channels, 346 and 348, are in a parallel
position.
[0036] As another example, if the first reservoir were sealed and
the second reservoir were unsealed, and if the negative pressure
source, (connected to two parallel secondary fluid channel output
ends ) were activated, fluid from the second reservoir would be
selectively drawn into at least one of the secondary fluid
channels.
[0037] As yet another example, the first negative pressure source
328 is connected to the output end of the first fluid channel 344
and the second negative pressure source 352 is connected to the
output end of a second fluid channel, 346 and 348. If the first
reservoir were unsealed and the second reservoir were sealed, and
if the first negative pressure source 328 were activated, fluid
from the first reservoir would be selectively drawn from the
reservoir into the first secondary fluid channel 344 but not into
fluid channels 346, 348. Depending on whether or not the second
negative pressure source functions as a closed valve when not
activated (thereby restricting fluid flow through the pressure
source), fluid in the second fluid channel can be drawn into the
first fluid channel. However, when it is desirable to prevent any
flow, a shut-off valve 354 and/or one-way valve 356 and/or a
negative pressure source 352 that functions as a closed valve,
e.g., a peristaltic pump, (when not activated) can be included in
the case of multiple fluid channels. The valve is positioned to
allow flow into the input end of the fluid channel and to restrict
fluid flow out of that input end.
[0038] Alternatively, it can be desirable to allow fluid transfer
between parallel fluid channels. For example, it can be desirable
to draw fluid from a reservoir into a first fluid channel, and then
draw the same fluid sample into a second parallel fluid channel.
This can be accomplished, for example, by effectively unsealing the
reservoirs, effectively unsealing the output end of the first fluid
channel, and activating a negative pressure source connected to the
output end of the second parallel fluid channel. This flow
arrangement could be used, for example, to allow for the sequential
analysis of a single sample in multiple parallel fluid channels
configured to analyze a sample.
[0039] According to certain embodiments, the system can include
multiple flow channels arranged in a serial arrangement. As shown
in FIG. 3, the first 316 and second 322 reservoir output ducts can
be fluidically connected to an input end of a first fluid channel
340. An output end of the first fluid channel 340 can be
fluidically connected to an input end of a second fluid channel
342, and an output end of the second fluid channel 342 can be
fluidically connected (directly or indirectly) to the negative
pressure sources 328, 352. According to certain embodiments, fluid
can be selectively drawn into the first secondary fluid channel,
and allowed to stop (by, for example, deactivating the negative
pressure source) in the first secondary fluid channel before being
subsequently drawn into the second secondary channel connected in
series. This flow arrangement could be used, for example, to allow
for the sequential analysis, including fixed and/or variable
incubation and/or analysis periods, of a single sample in multiple
parallel fluid channels configured to analyze a sample.
[0040] According to certain embodiments, the system can be
configured such that fluid does not flow into the primary fluid
channel unless both the negative pressure source is activated and
at least one reservoir is unsealed. In the case of multiple
negative pressure sources, the system can thus include auxiliary
cut-off valves and one-way valves, as discussed above. These
auxiliary elements can be contained in other embodiments as
well.
[0041] Additionally, unwanted fluid flow can be controlled by using
gravity in order to maintain it in a desired location, e.g., in a
given reservoir(s). For example, the connection path between a
given reservoir and a given fluid channel can include the fluid
being drawn to a height above the fluid level in the reservoir,
such that inadvertent or unwanted fluid flow is eliminated or
minimized absent the activation of the negative pressure source and
venting, opening, of the appropriate vent. Unwanted fluid flow can
also be minimized and/or eliminated in certain embodiments by
including a barrier between or extending the separation of the
connections of the multiple reservoirs into a common path leading
to the fluid channel.
[0042] The negative pressure source used to selectively draw fluid
into the fluid channel can be any pressure source capable of
drawing/moving a fluid. The negative pressure source can be, e.g.,
a pump, including a pump chosen from a peristaltic pump, a suction
pump, a syringe pump, and an adsorption pump; an evacuated
receptacle or cylinder; or a negative pressure source resulting
from a chemical reaction, e.g., a reaction that yields a net
reduced volume, such as the condensation reaction,
2H.sub.2(g)+0.sub.2(g).fwdarw.2H.sub.2O(1). The selection of a
negative pressure source can depend, among other things, on the
compatibility of the negative pressure source with the fluid, the
viscosity of the fluid, the overall resistance of the fluidics
system, the required flow rate, the capacity, the size and/or
weight of the negative pressure source, the electrical requirements
of the negative pressure source, and/or the reliability of the
negative pressure source. The flow rate of the fluid can be, for
example, nl/min to ml/min.
[0043] According to certain embodiments, the reservoirs can contain
multiple sub-reservoirs. For example, a first sealable reservoir
can include a first and a second fluid chamber. Each chamber can
have a fluid input duct for withdrawing the fluid from the
reservoir and an output end connected to the primary fluid channel
input end The vent can be configured to selectively seal or unseal
both fluid chambers. The system can be configured to selectively
draw fluid into the primary fluid channel from at least one of (1)
the first and second fluid chambers and (2) the second reservoir,
when the negative pressure source connected to the secondary fluid
channel is activated and the respective chambers or reservoir is
unsealed. In FIG. 5, reference number 516 designates chambers.
[0044] According to certain embodiments, the primary fluid channel
can include a first and a second secondary fluid channel. Each of
the secondary fluid channels can contain input and an output end.
The first sealed reservoir can contain first and second fluid
chambers. Each of the chambers can contain a fluid input and output
duct connected to the first and second secondary fluid channel
input ends. The vent can be configured to selectively seal or
unseal both of the fluid chambers. The system can be configured to
selectively draw fluid into at least one of the secondary fluid
channels from at least one of (1) the first and second fluid
chambers and (2) the second reservoir, when the negative pressure
source is activated and the respective chambers or reservoir is
unsealed.
[0045] The negative pressure source can include first and second
negative pressure sources connected, respectively, to the first and
second secondary fluid channel output ends, when, for example, the
secondary fluid channels are arranged parallel to each other.
According to certain embodiments, the system can be configured to
selectively allow fluid to be drawn into one or more of the
secondary fluid channels from the first and/or second fluid
chamber, and/or (2) the second reservoir when the negative pressure
source connected to the secondary fluid channel is activated and
the respective chamber or reservoir is unsealed, open.
[0046] According to certain embodiments, the system can be an
analysis system. For example, the system can be configured to
analyze at least one sample, such as a fluid sample or a sample
dispersed in a fluid, for the presence or absence of a given
analyte. For example, the fluid channel can be configured to be
responsive or sensitive to the presence or absence of the analyte.
Thus, according to certain embodiments, the sample fluid can be
selectively drawn into the fluid channel, where it can interact
with a surface or species sensitive to its presence or absence, or
otherwise be probed, such as probed optically, magnetically,
chemically, radioactively, and/or electrically. According to
certain embodiments, at least one of the first and second
reservoirs is configured to contain at least one sample fluid. For
example, the sample fluid can be introduced into and/or stored in a
reservoir prior to being selectively drawn into the primary fluid
channel. The primary fluid channel can be the location of a
detector for analyte detection and/or identification.
[0047] According to certain embodiments, the analysis can further
involve selectively introducing at least one reagent fluid into
said primary fluid channel. For example, the reagent fluid can
contain a rinse solution to remove excess sample; or a reactive
solution to react with residual sample or species in the primary
fluid channel. According to certain embodiments, reagents can be
introduced into the fluid channel any of prior to, simultaneously
with, or subsequent to (and combinations thereof) the introduction
of the sample into the fluid channel. According to certain
embodiments, at least one of the first and second reservoirs can be
configured to contain at least one reagent fluid. For example, the
reagent fluid can be introduced into and/or stored in a reservoir
prior to being selectively drawn into the primary fluid
channel.
[0048] According to certain embodiments, the system can contain a
waveguide. For example, at least one internal side of the primary
fluid channel can be a waveguide, such as a single mode or
multi-mode waveguide. For example, waveguides as disclosed in U.S.
Pat. Nos. 6,192,168 and 6,137,117, the disclosures of which are
incorporated herein by reference, can be used. According to certain
embodiments, the system can further contain a waveguide for
surface-sensitive optical detection of an analyte in a fluid
sample. For example, at least one internal side of the primary
fluid channel can be a waveguide.
[0049] According to certain embodiments, the system can further
include a multi-mode waveguide for surface-sensitive optical
detection of an analyte in a fluid sample. The multi-mode waveguide
can have a surface having a patterned reflective coating. The
patterned reflective coating defines a reflectively coated region,
e.g., and having an optically exposed region on the surface. The
optically exposed region can be sensitive to the analyte so as to
produce an alteration of the optically exposed region which is
indicative of the presence of the analyte in the sample. The
alteration is detectable by launching a light wave into the
waveguide to generate an evanescent field on the patterned surface,
and then detecting an interaction of the first optically exposed
region with the evanescent wave. According to certain embodiments,
the optically exposed region of the waveguide can define at least
part of at least one surface of the primary fluid channel.
[0050] According to certain embodiments, the system can further
include a waveguide sensing system. The waveguide sensing system
can contain, for example, a plurality of waveguides, each waveguide
having a first surface, a second surface opposing the first
surface, and an end surface essentially perpendicular to the first
and second surfaces. The first surface of each of the waveguides
can have analyte recognition elements thereon. This system can
further include a waveguide holder to which each of the waveguides
are secured, and an optical detector positioned opposite the end
surface of at least one of the waveguides. According to certain
embodiments, at least one of the first surfaces can define at least
part of at least one surface of the primary fluid channel.
[0051] According to certain embodiments, at least one of the
reservoirs of the system can contain an internal cavity configured
in such a manner as to be sealed from contact with an external
atmosphere. For example, according to certain embodiments, the at
least one internal cavity can be connected to a vent that is
configured to selectively connect and disconnect the respective
internal cavity from contact with the external atmosphere.
According to certain embodiments, the vent can be configured to
switch, in a binary fashion, between an "opened" and a "closed"
position. Valves can be configured to be fully opened, partially
opened, and fully closed and variation (including temporal) and
combination thereof.
[0052] According to certain embodiments, valves can be chosen from
one-way valves, two-way values, multi-way valves, and proportional
valves, and combinations thereof. For example, if the valve is a
one-way valve, it can be switched between an opened and closed
position. Two-way and multi-way valves can be used, for example, to
connect a reservoir or cavity to multiple external pressures,
including atmospheric, positive, and negative, and/or to additional
fluid supplies. Valves can also be configured to open and close
multiple reservoirs or cavities. For example, an input of a two-way
valve can be connected to a given pressure source, one of the two
valve outputs can be connected to one reservoir or cavity, and the
second valve output can be connected to another reservoir. Then,
for example, the valve can be used to selectively connect either of
the two (or more) reservoirs to the pressure source.
[0053] According to certain embodiments, a valve V comprising one
input I and two (or more) outputs O1 and O2 can be used to
selectively seal and/or unseal two (or more) reservoirs, R1 and R2.
For example, input I can be connected to the atmosphere (or a
positive pressure source) with outputs O1 and O2 connected to
reservoirs R1 and R2, respectively. When valve V is configured to
connect I to O1 but not O2, R1 will be unsealed and R2 will be
sealed. Likewise, when valve V is configured to connect I to O2 but
not O1, R2 will be unsealed and R1 will be sealed
[0054] According to certain embodiments, the system can be
configured to simultaneously have fluid drawn from the first and/or
second reservoir into the primary fluid channel at a first and/or a
second flow rate, respectively, when the difference between the
first and second flow rates is proportional to a difference in the
unsealing of the first and second vents. According to certain
embodiments, the system can be configured to selectively have fluid
drawn from the first and second reservoirs into the primary fluid
channel at first and second flow rates, respectively, when the
difference between the first and second flow rates is proportional
to the differential fluid flow resistance. The differential fluid
flow resistance is adjusted by the sealing and unsealing of the
first and second vents.
[0055] According to certain embodiments, at least one of the vents
or valves can be a proportional valve configured to partially or
fully unseal a reservoir. For example, to favor fluid flow from a
first reservoir, a proportional valve can be connected to the first
reservoir which can then be opened to a relatively greater degree
to a given pressure source. At the same time a second reservoir
connected to a second reservoir can be opened to the same pressure
source to a relatively lesser degree. According to certain
embodiments, the differential fluid flow can be at least partially
controlled by the relative pressure of the pressure sources to
which the reservoirs are connected. For example, to favor fluid
flow from a first reservoir, it can be vented (opened) to a
relatively high pressure source while a second reservoir can be
connected to a relatively low pressure source. According to certain
embodiments, differential fluid flow can be controlled by any
combination of proportional valves, relative vent source pressures,
fluid viscosities, fluid channel diameters, fluid channel surfaces
(e.g, rough, smooth, hydrophobic, hydrophillic, chemically
derivatized, biologically derivatized, etc.), and pressure and
current of the one or multiple negative pressure sources.
[0056] According to certain embodiments, the system can further
include a system relief vent connected to the primary flow channel.
For example, the system relief vent can be configured to seal or
unseal, open or close, the primary flow channel from contact with
an external atmosphere. According to certain embodiments, when the
system relief vent is in a closed or an open position, fluid flow
from the reservoirs/chambers into the primary fluid channel is
respectively enabled or disabled. According to certain embodiments,
the system relief vent can be configured to allow a fluid, such as
air and/or any of its component gases, to fill the primary fluid
channel, and/or displace a volume of the fluid previously contained
therein. The previous fluid can be, for example, a sample or
reagent fluid, as discussed further herein.
[0057] According to certain embodiments, a reservoir can be
selectively connected to atmospheric pressure or a positive
pressure source that is configured to apply pressure greater than
atmospheric pressure or a negative pressure source, that is
configured to have a pressure less than atmospheric pressure to the
unsealed reservoir.
[0058] According to certain embodiments, the system can further
contain an auxiliary fluid reservoir and a connection valve. The
auxiliary fluid reservoir 335 can be connected through the
connection valve 337 to an auxiliary input duct 339 of at least one
of the first and second reservoirs. According to certain
embodiments, the system can be configured to selectively have a
fluid drawn from the auxiliary fluid reservoir into the first
and/or second reservoir when the negative pressure source is
activated, the connection valve is open, and the respective
reservoir is closed or not vented to the atmosphere.
[0059] The connection valve can be a multi-way connection valve,
configured to selectively connect the auxiliary input to a source
chosen from the atmosphere, a positive pressure source, a negative
pressure source, and a fluid reservoir. According to certain
embodiments, a single valve can be used to seal or unseal a
reservoir, as well as the connection valve to connect the reservoir
to the auxiliary fluid reservoir.
[0060] According to certain embodiments, the sizes and dimensions
of the fluid channels, including the primary and secondary fluid
channels, can be configured to control a range of dynamic and
static parameters, including, for example, the fluid flow rate,
capacity, resistance, and turbulence. According to certain
embodiments, the primary fluid channel and/or the connecting
channels and/or other fluid channels in the system can be
configured to have minimal cross-sectional dimensions such that the
selective fluid drawing can be turbulent fluid flow.
[0061] According to certain embodiments, the primary fluid channel
and/or the connecting channels and/or other fluid channels in the
system may be configured to have minimal cross-sectional dimensions
such that the selective fluid drawing may or may not be a low
Reynolds number fluid flow.
[0062] According to certain embodiments, the primary fluid channel
and/or the connecting channels and/or other fluid channels in the
system may be configured have minimal cross-sectional dimensions
such that the selective fluid drawing may or may not be a low
Reynolds number fluid flow when the fluid has a density less than
five times the density of water.
[0063] According to certain embodiments, the primary fluid channel
and the connecting channels are configured to have minimal
cross-sectional dimensions such that the selective fluid drawing
may or may not be a low Reynolds number fluid flow when the fluid
is an aqueous fluid.
[0064] According to certain embodiments, the system can further
include a first connecting channel and a second connecting channel,
wherein first and second reservoirs are connected to the primary
fluid channel input by first and second connecting channels,
respectively. The connecting channels, the primary fluid channels,
the secondary channels and reservoir/chamber output ducts can have
minimum cross-sectional dimensions greater than 1 micron. For
example, the range of the cross-sectional size of any of the ducts
and/or channels in which a fluid moves can be at least 10% greater
than the largest particle size found in any of the fluids, e.g.,
whether a sample, a reagent or a indicator.
[0065] According to certain embodiments, the system can include a
three-dimensional fluid circuit (or fluid cube) comprising at least
one of the first and second reservoirs and the primary fluid
channel. According to certain embodiments, the fluid circuit can
include a series of layers, where the individual layers comprise at
least partial elements of the fluid circuit, such that, when some
or all of the series of layers are fused (or joined) together,
complete elements of the fluid circuit are formed.
[0066] According to certain embodiments, at least partial elements
are formed by at least one of molding and mechanical, chemical,
thermal, and optical etching. For example, the fluid circuit can
include elements formed using injection molding techniques, as well
as elements formed using other molding techniques, including blow
molding.
[0067] According to certain embodiments, the fluid circuit can
further include a first connecting channel and a second connecting
channel, wherein the first and second reservoir output duct ends
are connected to the primary fluid channel input ends by first and
second connecting channels, respectively. The fluid circuit can
also be configured, for example, so that the first and second
connecting channels have first and second input ends. These input
ends of the connecting channels can be connected to the first and
second fluid output ducts, respectively. The common channel can
have a first and a second end, where the first end can be connected
to the second ends of the connecting channels, and the second end
be connected to the input of the primary fluid channel input
ends.
[0068] According to certain embodiments, the first and second
connecting channels can further be J-shaped connecting channels,
where the lower ends of the J-shaped connecting channels can be
connected to each of the reservoirs and the upper ends of the
connecting channels can be connected to the common primary channel
and/or secondary channels. The connecting channels can further
include a barrier. The barrier is configured to inhibit fluid flow
between the first and second connecting channels.
[0069] According to certain embodiments, the fluid circuit can
further include at least one of a first pressure duct and a second
pressure duct, where the first and second pressure ducts connect
the first and second vents to the first and second reservoirs,
respectively.
[0070] According to certain embodiments, the system can be a
portable analysis system, that includes a three dimensional fluid
circuit. The fluid circuit can include a first sealed reservoir.
The first sealed reservoir can include a first fluid output duct
that is fluidically connected to the primary fluid channel input,
and a first vent configured to selectively seal and/or unseal (open
or close) the first reservoir. The fluid circuit can further
include a second sealed reservoir having a second fluid output duct
fluidically connected to the primary fluid channel input, and a
second vent configured to selectively seal and/or unseal (open or
close) the second reservoir. A negative pressure source can be
connected to the primary fluid channel output end. The system can
be configured, for example, to selectively draw at least one fluid
from at least one of the first and second reservoirs into the
primary fluid channel when the negative pressure source is
activated and the respective reservoir vent is unsealed.
[0071] According to certain embodiments, the system can be
configured to perform biological and/or chemical analysis. The
system can also comprise a three dimensional fluid circuit that has
a first reservoir having a first fluid output duct fluidically
connected to a primary fluid channel input, and a first vent
configured to selectively seal and/or unseal the first reservoir.
The system can further include a second sealed reservoir having a
second fluid output duct fluidically connected to the primary fluid
channel input, and a second vent configured to selectively seal
and/or unseal the first reservoir. A negative pressure source can
be connected to the primary fluid channel output. The system can be
configured, for example, to selectively draw at least one fluid
from at least one of the first and second reservoirs into the
primary fluid channel when the negative pressure source is
activated and the respective reservoir is unsealed. The fluid
circuit elements can be formed using molding or milling techniques.
The circuit can contain a series of layers, in which some or all of
the layers of the series have at least partial elements of the
fluid circuit. Some or all of the series of layers can be fused (or
otherwise joined, permanently or temporarily) together to form
completed elements of the fluid circuit. The at least partial
elements are formed by, for example, at least one of molding and
mechanical, chemical, thermal, and optical etching. The fluid
circuit can further include a first connecting channel and a second
connecting channel, where the first and second fluid outputs are
connected to the primary fluid channel input by first and second
connecting channels, respectively.
[0072] The system can further contain a common channel. It can be
configured such that the first ends of the connecting channels are
connected to the first and second fluid outputs, respectively, and
a first end of the common channel is connected to the second ends
of the output connecting channels, and a second end of the common
channel is connected to the input of the primary fluid channel
input. The first and second output connecting channels can also
have J-shaped connecting channels configured such that the lower
end of the J-shaped connecting channel is connected to the
reservoir, e.g., at the bottom of the reservoir. The upper end of
each connecting channels is connected to the common channel. The
connecting channels can further include a barrier configured to
inhibit fluid flow between the first and second output connecting
channels. Additionally, the fluid circuit can further include at
least one of a first pressure duct and a second pressure duct,
where the first and second pressure ducts connect the first and
second vents to the first and second reservoirs, respectively.
[0073] According to certain embodiments, the present invention
comprises a method of controlling fluid flow. The method can
comprise, for example, selectively drawing at least one fluid from
at least one of a first and a second reservoir into a primary fluid
channel. The selective drawing involves activating a negative
pressure source and unsealing one of the reservoirs. According to
certain embodiments, the first reservoir can contain a first fluid
output duct fluidically connected to the primary fluid channel
input, and a first vent configured to selectively seal and/or
unseal the first reservoir. According to certain embodiments, the
second reservoir can contain a second fluid output duct fluidically
connected to the primary fluid channel input, and a second vent
configured to selectively seal and/or unseal the second reservoir.
According to certain embodiments, the negative pressure source can
be connected to the primary fluid channel output.
[0074] According to certain embodiments, the selective drawing of a
fluid can involve (wholly, partially, and/or for a controlled
duration and/or cycle) sealing at least one of the unselected
reservoirs and/or (wholly, partially, and/or for a controlled
duration and/or cycle) unsealing at least one of the selected
reservoirs, and drawing at least one fluid from at least one
selected reservoir.
[0075] According to certain embodiments, the selective drawing can
involve partially unsealing at least one first selected reservoir
and partially unsealing at least one second selected reservoir, and
drawing fluid from both the first and second reservoirs. For
example, partial opening means partially unsealing (or opening) a
vent to partially open a reservoir to at least one of the
atmosphere and applied pressure.
[0076] According to certain embodiments, unsealing a selected
reservoir can involve connecting it to a first pressure source, and
sealing a selected reservoir can involve connecting it to a second
pressure source, where the pressure of the first pressure source is
greater than the pressure of the second pressure source. According
to certain embodiments, unsealing a selected reservoir can involve
opening a vent such that the selected reservoir is connected to
atmospheric pressure, e.g., by releasing the vacuum. According to
certain embodiments, unsealing a selected reservoir can involve
application of a pressure less than an atmospheric pressure to the
selected reservoir. According to certain embodiments, sealing a
selected reservoir can involve applying a pressure greater than an
atmospheric pressure to the selected reservoir.
[0077] According to certain embodiments, the present invention
comprises a method of performing an assay. The method can allow for
the, for example, selective drawing of a sample fluid from a sample
reservoir into a primary fluid channel. According to certain
embodiments, the selected sample (reagent) fluid that is drawn can
involve activating the negative pressure source, unsealing the
sample (reagent) reservoir, and sealing the reagent (sample)
reservoir.
[0078] According to certain embodiments, at least one side of the
primary fluid channel is configured to at least one of capture,
recognize, respond to, and detect at least one analyte. At least
one side can contain a waveguide that can, for example, have a
first optically exposed region sensitive to a first analyte so as
to produce an alteration of the first optically exposed region that
is indicative of the presence of the first analyte in the sample.
The alteration is detectable by launching a light wave into the
waveguide to generate an evanescent field at the patterned surface,
and then detecting an interaction of the first optically exposed
region with the evanescent wave.
[0079] According to certain embodiments, the waveguide can contain
a multimode waveguide having a surface bearing a patterned
reflective coating. The patterned reflective coating defining a
reflectively coated region and an optically exposed region on the
surface. The optically exposed region is configured to produce an
alteration that is indicative of the presence of an analyte. The
alteration is detectable by launching a light wave into the
waveguide to generate an evanescent field at the patterned surface,
and then detecting an interaction of the optically exposed region
with the evanescent wave.
[0080] According to certain embodiments, the at least one fluid has
a density not less than an atmospheric density. The fluid may, for
example, comprise a liquid, a gas having a density not less than an
atmospheric density, and/or a mixture wherein the density of the
mixture is not less than an atmospheric density.
[0081] According to certain embodiments, the at least one fluid can
be a dispersion, a solution, a suspension, or an emulsion.
According to certain embodiments, the at least one fluid can be an
aqueous fluid.
[0082] According to certain embodiments, at least one fluid can be
a biological or chemical specie. For example, at least one fluid
can contain an antibody, antigen, toxin, drug, metabolite,
polypeptide virus, protein, cell, amino acid, or amino acid
sequence. For example, the fluid can be a buffer, stabilizer,
preservative, enzyme, sugar or lack of a metabolite.
[0083] According to certain embodiments, the at least one fluid can
be tagged labels, including tagged labels selected from optically,
radioactively, magnetically, chemically, biologically, and
physically (such as mass and/or size and/or shape) tagged
labels.
[0084] According to certain embodiments, the invention pertains to
a method and apparatus for delivery a fluid to a selected
reservoir. For example, if the negative pressure source in FIG. 1
is a pump configured to push a fluid towards the reservoirs, and
one reservoir 116 is unsealed and the other reservoir 122 is
sealed, the fluid will be selectively delivered to the unsealed
reservoir 116.
[0085] The invention will be further clarified by the following
examples, which are intended to be purely exemplary of the
invention.
EXAMPLE I
[0086] A schematic illustration of an exemplary fluid flow control
arrangement 400 is depicted in FIG. 4. Arrangement 400 includes
three reservoirs 416, 422, and 460 in which respective fluids 430,
432, 462 and gas space 434 are contained. The reservoirs are all
fluid-tight (enclosed). Fluidly connected to each reservoir is a
respective pressure relief valve 464, 466, 468. Pressure relief
valves can be manually or remotely actuated to move between an open
position where pressure relief or air is provided to the respective
reservoir and a closed position where no pressure relief or air is
provided to the respective reservoir. In this arrangement, pressure
relief valves are each automatically actuated remotely by a
suitable control 470 as schematically shown, and are in a closed
(default) position when not actuated. Extending into or near a
bottom of each respective reservoir is a respective outlet pathway
or duct 418, 424, 472. The outlet ducts are connected by a
manifold, 450, to a primary fluid channel, 410, which is in turn
connected to a negative pressure, 428, as a pump. Any number of
reservoirs can be similarly connected to the manifold as long as
the manifold has sufficient branches.
[0087] When it is desired to draw a selected fluid from the
associated reservoir, such as fluid 430 from associated reservoir
416 the associated pressure relief valve 464 is actuated to move
from the closed position to the open position (as shown in FIG. 4).
With this opening of the pressure relief valve, atmospheric air is
now allowed to back fill the reservoir. At the same time, or
previously or subsequently, negative pressure source 428 is
actuated to exert a negative pressure, e.g. suction, on all fluids
in all reservoirs. However, as only reservoir 416 has an open
pressure relief valve 464, only fluid 430 is drawn from reservoir
416 into outlet duct 418 and through manifold 450 to the desired
delivery point. In this manner, fluid 430 is preferentially drawn
from reservoir 416 as air is permitted to flow into and back fill
reservoir 416 through open valve 464 while reservoirs 422 and 460
remained sealed from the atmosphere and hence comparatively
resistant to flow into the outlet ducts 424 and 472. If more than
one pressure relief valve is opened, then fluids from multiple
reservoirs can be drawn though manifold 450 simultaneously, and
combined at the common outlet of manifold and then conducted
towards the negative pressure source 428
[0088] It can thus be appreciated that the fluid flow control
arrangement 400 allows for selective is fluid flow from a selected
reservoir to a use point, e.g., the primary channel or a detector
in the primary channel. The necessity of passing the selected fluid
through any valves or the necessity of resorting to micro-scale
fluidics channels is eliminated. Thus, while the system can or can
not contain valves through which the fluids must pass, such valves
are not required for all embodiments and problems with valves and
channels clogging due to contaminants in the fluid are avoided. It
will further be appreciated that this arrangement is a reduction in
both the overall size and power consumption compared to other
fluidics arrangements as the pressure relief valves can be made
relatively small since normally only a gas passes through and such
a small valve requires very little power.
EXAMPLE II
[0089] Depicted schematically in FIG. 5 is a first embodiment of a
portable bio/chemical analysis system 500 incorporating a fluid
flow control arrangement as broadly discussed above whereby a
plurality of sample fluids can be first simultaneously analyzed and
then can be further simultaneously analyzed after addition of one
or more reagents. The system includes a bio/chemical analysis
device 574 having analyzing channels 576 in which analysis of a
fluid can be performed as is well known in the art. One surface of
the analyzing channels 576 can be a waveguide for performing
optical analysis. For example, a waveguide in the plane of the
figure co-extensive in area with the analysis device 574 could be
used. Each analyzing channel 576 includes an associated inlet 578
and an associated outlet 580 as shown. Associated with each
analyzing channel 576 is a sample reservoir/chamber 516 in which a
sample fluid 530 and air 534 are respectively provided. First
pathways or ducts 518 respectively connect a bottom of sample
reservoirs 516 to respective inlets 578 of analysis device 574 All
sample reservoirs 516 are connected to a common sample pressure
relief valve 536 as schematically shown. When sample pressure
relief valve 536 is opened, pressure relief (back fill air) is
provided above each sample in each reservoir.
[0090] Bio/chemical analysis system 500 also includes reagent
reservoirs 582 in which reagent fluids 584 and air space 534 are
respectively provided. Each reagent fluid is conducted through a
second pathway to the associated analyzing channel 576. This second
pathway includes second ducts 586 respectively connected to a lower
portion, i.e., below an upper level of each fluid 584 of each
reagent reservoir 582, and a common duct 588 connected to the tops
of sample reservoirs 516. In this embodiment where reagent fluid
584 is delivered to a selected reservoir, the second pathway
includes ducts 518 as well to complete the path to the analyzing
channels 576. Each reagent reservoir has connected thereto a
respective reagent pressure relief valve 590 as shown.
[0091] As shown in FIG. 5, the system further includes a pump 528
which serves as a source of negative pressure to draw fluids into
and through analyzing channels 576 of analysis device. Pump 528 is
connected to an outlet duct 592 of a suitable manifold 550, whose
inlet ducts 594 are respectively connected to outlets 580 of
analysis device. If desired, a system pressure relief valve 596 is
also connected to outlet duct 592 of manifold 550. System pressure
relief valve 596 is opened to feed gas to pump 528 and hence to
disable any flow of sample or reagent fluids in analysis system.
One or more system pressure relief vents can also be connected to
inlets 578, and can not only disable fluid 530 or 584 flow through
the channels 576, but also can be used to introduce air or gas into
the channels, e.g., 576 to displace the fluids 530 or 584 and/or to
dry interior surfaces of channels 576
[0092] With this system, it is possible to analyze sample fluids
530 simultaneously with analysis device 574, both before and then
after the addition of one or two reagent fluids 584 to the sample
fluid. Thus, in operation, pump 528 is initially actuated after
analysis device 574 is made ready to analyze any associated fluid
passing through respective analyzing channels 576. Sample pressure
relief valve 536 is then simultaneously (or subsequently or
previously) opened, allowing back fill air into all sample
reservoirs 516. This allows the pump 528 to draw the associated
sample fluid 530 from each respective reservoir 516 through the
associated analyzing channel 576, where analysis device 574
conducts all or part of the needed analysis for a reading or
analysis of each respective sample fluid. During this initial
analysis step, reagent pressure relief valves 590 are all closed,
so no reagent fluid is drawn into sample reservoirs.
[0093] After the first analysis step of the sample fluids is
accomplished, sample pressure relief valve 536 is closed and a
selected one (or both) of reagent pressure relief valves 590 is
opened. This causes the negative pressure created by pump 528 in
each sample reservoir 516 to cause a flow of reagent fluid from
whichever reagent reservoir 582 can be back filled with air due to
an open reagent pressure relief valve 590. Thus, after a small time
period of operation of pump 528 after opening of one or more
reagent pressure relief valves 590, a reagent fluid 584 is
delivered to the associated analyzing channels 576 for analysis by
analysis device 574. If the sample fluid had been substantially
depleted from the reservoirs, then relatively pure reagent may be
delivered to the channels. However, if the sample fluid has not
been substantially completely removed, according to the embodiment
shown in FIG. 5, the reagent could be mixed with the sample fluid
in reservoir. According to one mode of operation, one of the regent
fluids would be a wash fluid, such as a buffer fluid, to wholly or
partially rinse remaining sample fluid out of reservoirs and
channels. Then, for example, a second reagent fluid can be
delivered through reservoirs into channels without mixing with
sample fluids.
[0094] Where required, the amount of reagent fluid delivered to
each sample reservoir can be varied as desired where the rate of
flow of reagent fluid through ducts is known and the associated
open pressure relief valve is closed after the desired flow volume
is achieved (after which sample pressure relief valve is opened
again). Alternately, the amount of reagent fluid in each reagent
reservoir can be known, and flow maintained until the associated
reagent reservoir is emptied. Similarly, the amount of sample fluid
in each sample reservoir can be controlled by knowing the initial
volume as well as the flow rate through first ducts and inlets; and
this control can include emptying of the sample fluid therefrom so
that only a reagent fluid is then drawn to the analysis device.
[0095] When considering the range of fluid types, channel/duct
sizes, pump pressures, and substrate materials, the following may
also be considered. The relief valve control arrangement for the
fluidics system operates when the resistance to flow of a first
fluid in a first reservoir (due to surface tension, channel size,
channel material, etc.) is less than the resistance to flow of a
second fluid in the second reservoir that has been sealed-off from
the atmosphere. This difference in resistance between the flow of
the first and second fluids should be greater than the potential of
the negative pressure source at the flow rate used. Without being
bound by theory, a relation analogous to Ohm's law can be used to
express this requirement. That is, relief valve control will
operate under conditions such that:
R>P/I,
[0096] where:
[0097] R=R2-R1, where R2 is the resistance to fluid flow caused by
sealing the fluid from atmosphere and R1 is the resistance to fluid
flow due to factors such as fluid channel size, viscosity, channel
material, etc.; and
[0098] P is the pressure difference between the negative pressure
source and ambient or sealing pressure; and
[0099] I is the flow rate of the negative pressure source.
EXAMPLE III
[0100] As shown schematically in FIG. 1, two reservoirs 116, 122
were connected through a manifold 150 to a primary fluid channel
110 comprising fluorescence detector, 133. The fluorescence
detector was used to detect a fluorescent dye in one of the fluids
130, possibly water. The reservoir 116 contained water. Reservoir
122 contained a 60 nM aqueous solution of fluorescent dye Cy5, 132.
Each reservoir was sealed, closed, to the atmosphere except that
each was connected to vents 120, 126 to micro relief valves 136
("vent 1") and ("vent 2") (LFAA12034, The Lee Company),
respectively. The default closed position of the valve caused the
given reservoir to be sealed from the atmosphere. The relief valves
136 could be individually actuated (via a 12 volt signal) to open a
given reservoir to atmospheric pressure. The negative pressure
source 128 was a peristaltic pump, running at 1.5 ml/min. It was
used to draw fluid from each of the reservoirs and through the
fluid channel 110 comprising detector 133 to a waste collector (not
shown). In this configuration and as described above, when vent 120
was open the fluid in 116 (water) would be drawn through the
detector by the pump. The fluid, Cy5, 132 did not flow because of
its greater resistance to flow resulting from the inability of air
to replace back fill the fluid being withdrawn from the reservoir.
The fluid in 124 would flow, exclusively, when vent 120 was closed
and vent 126 was opened, and negative pressure source 128 was
activated.
[0101] As shown in FIG. 6, when vent 120 was opened and vent 126
was closed and the negative pressure source 128 was activated (see
control signal, solid line, right y-axis), the fluorescence
detector recorded a signal level of zero 698 (see fluorescence
signal, line with points, left y-axis). This indicated that the
water was pulled through the system. However, when vent 120 was
closed and vent 126 was opened (and negative pressure source was
128 was activated), the fluorescence signal 699 rose sharply (in
arbitrary units) since the Cy5 solution in 122 was drawn through
the system and detected. The slight delay of the signal rise as
compared to the opening of vent 126 was due to the finite distance
that the fluid needed to flow from the T-junction (manifold 150) to
the detector. The tailing of the signal level to zero when 120 was
open was attributed to the detection of residual Cy5 in the fluid
channels being washed out by the water.
EXAMPLE IV
[0102] Depicted in FIG. 7 is a simplified (for convenience of
illustration) fluid fluidics circuit which has been embodied in a
modular block or cube 700 formed of a series of layers 702, 704,
706, 708, 709 (from bottom to top). Cube 700 was designed to fit
into a preformed receptacle of a bio/chemical analysis device and
to have an overall small size of, for example, 75 cm.sup.3 where
six sample reservoirs 716 and six reagent reservoirs 782 were
provided for processing. Cube 700 can be designed for use in a
number of different assay formats (parallel, individually
selective, etc.), depending on the requirements. In this
embodiment, each sample reservoir 716 and corresponding (paired)
reagent reservoir 782 was each selectively connected to a
respective analyzing channel in the analysis device, with all
sample fluids or all reagent fluids being conducted at the same
time. Six different fluid samples were analyzed simultaneously.
Each sample can be analyzed for six different analytes when
combined with an array sensor, e.g., as disclosed by M. J.
Feldstein et al., Array Biosensor: Optical and Fluidics Systems,
Biomedical Microdevices 1(2) (1999), and Dodson et al., Fluidics
Cube for Biosensor Miniaturization, Analytical Chemistry, 2001,
(the disclosures of which are incorporated in their entireties by
reference). Alternately, cube 700 could be suitable for use with
other assay methodologies as desired.
[0103] Cube 700 is essentially a passive fluid circuit in that it
operates without the use of any internal valves or meters. Internal
valves and/or meters could, of course, be added. Instead, the fluid
circuit operates by use of external pressure relief valves and a
pump in the analysis device. As shown, cube 700 was constructed of
stacked layers of, for example, a thermoplastic such as
poly(methylmethacrylate) for layers 704, 709 but optionally having
a lower surface of layer 702 made of a compressible material such
as neoprene, for pressure based sealing of the cube 700 to, for
example, an assay flow cell as described in M. J. Feldstein et al.,
Array Biosensor: Optical and Fluidics Systems, Biomedical
Microdevices, 1, (2), 1999. Likewise, a lower surface of layer 709
and/or an upper surface of layer 708 can optionally be made from a
compressible material, for pressure based sealing of layer 709 to
an upper surface of layer 708. When aligned, using, for example
alignment holes 711 and joined together into cube 700, the
essentially two-dimensional features of each layer provide the
fluid circuit required for the present invention. Layers 704, 708
were stacked and then fused into cube under moderate pressure and
heating to just above the glass transition temperature so that cube
was made fluid-tight. Other methods of joining the layers together,
such as adhesives or applied pressure and compressible seals, could
also be used in place of or in combination with the thermal fusing
process. The top layer, layer 709, can be attached to the cube
using bolts in bolt holes 713 (with corresponding receptacles for
the bolts in at least one of layers 702-708) or a bolt receptacles
positioned below layer 702, that seals the cube using a gasket
arranged between the layer 702 and the rest of cube. Sample fluids
and reagent fluids can be placed into the cube before top layer 702
is attached thereto, or if desired, a dried reagent or sample can
be placed into reagent reservoirs 786 before sealing for use when
fluid is later added after sealing.
[0104] As shown in FIG. 7, cube 700 includes holes in each layer
forming sample reservoirs 716, reagent reservoirs 782, outlet
channels 780 (connected to the analyzing channels and the source of
negative pressure), first ducts 786, and second ducts 786. In layer
704, suitable fluid connections 715 are made at the bottoms of
sample reservoirs 716 to the bottoms of first ducts 718, and
similarly suitable fluid connections 717 are made at the bottoms of
reagent reservoirs 782 to the bottoms of second ducts 786. In layer
708 the tops of outlet channels 780 therebeneath are connected by
fluid connections 719 to the tops of first ducts 718 and similarly
by fluid connections 721 to the tops of second ducts 786. Finally,
as shown in layer 708, a network 723 of fluid connections can
connect the tops of sample reservoirs 716 with an expanded vent
cavity 725 whose top is then connected to the sample pressure
relief valve (provided in the analysis device) though small vent
hole 727; while a network 729 of fluid connections connects the
tops of reagent reservoirs 782 with an expended vent cavity 731
whose top is then connected to the reagent pressure relief valve
(also provided in the analysis device) though small vent hole 733.
Conveniently, all of the holes, channels, and ducts are formed in
cube by the simple drilling or machining.
[0105] With the fluid circuit embodied in cube 700, six selected
sample fluids are conveniently inserted into respective sample
reservoirs 716 while six selected reagent fluids are similarly
inserted into reagent reservoirs 782. Thereafter, the cube is
inserted as a modular unit into an analysis device adapted to
receive the cube. The analysis device is then actuated in a first
operation to draw the sample fluids from each sample reservoir 716
from the bottom thereof, through first ducts 718, and into outlet
channels 780 for analysis in corresponding analyzing channels of
the analysis device. During this first operation, it will be
appreciated that the pump (or alternatively pumps) is actuated. At
the same time that the sample pressure relief valve is opened so
that air flows through each vent hole 727 to each sample reservoir
716. During this first operation, the reagent pressure relief valve
is closed, so that no reagent fluid is drawn from reagent
reservoirs 782. After suitable analysis of the sample fluids (or
portions thereof), the sample pressure relief valve is closed and
the reagent pressure relief valve is opened, switching the flow
through outlet channels 780 from the associated sample fluids to
the associated reagent fluids.
[0106] It is anticipated that a standard cube would have reservoirs
716, 782 each sufficient to hold about 0.4 ml of fluid. However,
with a modular design, the reservoir volume could be increased or
decreased as desired, prior to annealing or assembling of cube, by
simply adding or subtracting layers 706. Layers 706 could thus be
designed to add or subtract to the volume of reservoirs 716 and 782
in 0.2 ml increments as desired. In addition, if the presence of
residual sample fluid in outlet channels 780 causes analysis
problems after switching is made, separate sample and reagent
outlet channels could be easily provided instead of the common
outlet channels 780.
[0107] The cube is designed to operate with currently available
miniature peristaltic pumps. Even if six such pumps were used, all
six pumps would be expected to add only about 120 cm.sup.3 to any
analysis device and would draw minimal current (50-75 mA max per
pump). This makes such pumps and cube ideal for extended battery
operation contemplated for portable bio/chemical analysis
systems.
[0108] While the fluidics system as described above has been
depicted as having two, three or six sets of reservoirs, it can be
appreciated by those of ordinary skill in the art that there is
really no limit to the number of reservoirs that can be used either
in a series or in a parallel arrangement, or combinations thereof.
In addition, while the fluidics systems have been disclosed as
being used to draw fluids out of different reservoirs, the present
invention is also applicable to controlling fluids being
selectively pumped into a reservoir. Further, while reservoirs of
glass or plastic are typical, the present invention is applicable
to reservoirs of almost any material, such as metal or ceramic, so
long as the reservoir can be effectively sealed from the
atmosphere. Still further, any suitable pressure relief valve,
whether manual or automatic, can be used, including physical and
chemical vent valves where the swelling and contracting of a
polymer could function as a vent.
[0109] FIG. 8 shows a simplified three-dimensional perspective view
of two sample reservoirs, two-reagent reservoirs fluidics system
that is similar to the six sample, six reagent system of FIG.
7.
EXAMPLE V
[0110] A fluidics cube, substantially as described in Example IV,
was used with a patterned multimode waveguide to perform
bio-chemical analysis on several samples. Staphylococcal
enterotoxin B (SEB) and anti-SEB antibodies were obtained from
Toxin Technologies (Sarasota, Fla.). To generate capture
antibodies, a long-chain derivative of biotin,
N-hydroxysuccinimidyl ester (EZ-Link NHS-LC-Biotin; Pierce,
Rockford, Ill.) was attached to the anti-SEB at a 10:1
biotin:protein ratio as recommended by the manufacturer. Labeled
protein was separated from unincorporated biotin using a Bio-Gel
P10 column, (Bio-Rad, Hercules, Calif.). Fluorescent tracer
antibodies were prepared by labeling anti-SEB antibodies with Cy5
bisfunctional reactive dye (.lambda..sub.ex=649 nm,
.lambda..sub.em=670 nm, Amersham Life Science Products, Arlington
Heights, Ill.) according to the manufacturer=s instructions. Dye to
protein ratios ranged from 2.5 to 4.0.
[0111] Silver-clad slides (Opticoat Associates, Protected Silver)
(Feldstein, M. J., Biomed. Microdevices, 1999, 1:2, pp. 139-153
(the disclosure of which is incorporated herein in its entirety by
reference)) were cleaned in a potassium hydroxide (KOH) solution
(10 grams KOH in 100 ml isopropanol) for 30 minutes at room
temperature in a Coplin jar. The slides were rinsed thoroughly with
de-ionized water and dried using a stream of nitrogen.
[0112] NeutrAvidinJ (Pierce, Rockford, Ill.) was immobilized on the
silvered side of the slides essentially according to the method of
Bhatia et al., (Bhatia, S. K. et al., Anal. Biochem., 1989, 178,
pp. 408-413 (the disclosure of which is incorporated herein in its
entirely by reference)) and modified to prevent removal of the
silver cladding. The cleaned slides were incubated for 1 hour in a
2% silane solution (1 ml 3-mercaptopropyl triethoxysilane in 50 ml
anhydrous toluene) in a glove bag under nitrogen. The slides were
washed three times in anhydrous toluene and air-dried briefly on a
lint-free cloth, silver side up. The silanized slides were
incubated for 30 minutes at room temperature in GMBS solution (12.5
mg B[g-maleimidobutyryloxy]-succinimide ester in 0.25 ml dimethyl
sulfoxide to which 43 ml absolute ethanol were added), then washed
three times in de-ionized water and placed in a fresh Coplin jar.
Finally, the slides were incubated in a NeutrAvidin solution (100
Fg/ml in 10 mM sodium phosphate buffer, pH 7.4) for 2 hours at room
temperature, and then rinsed three times in 10 mM sodium phosphate
buffer, pH 7.4, prior to storing them in the same buffer.
[0113] Physically isolated patterning, PIP, (Rowe, C. A., Anal.
Chem., 1999, 71, pp. 433-439 (the disclosure of which is
incorporated herein in its entirely by reference)) was used to form
an array of recognition elements on a planar waveguide. Briefly, a
patterning multi-channel flow cell was placed on the surface of a
waveguide that had been coated with NeutrAvidin. Biotinylated
anti-SEB antibodies were introduced into the channels of the flow
cell (each channel can contain a separate recognition molecule) and
incubated overnight at 4.degree. C., producing columns of the
capture antibody patterned on the waveguide surface, perpendicular
to its length. When used in combination with a multi-channel flow
cell aligned orthogonal to the patterned capture antibody, the
sensing surfaces present a 2-dimensional array of rectangular
recognition elements.
[0114] The PIP method used custom designed and molded flow cells,
which consisted of six parallel channels fabricated in widths from
0.75 to 1.5 mm. These flow cells were made from MED-6015 silicone
elastomer, polydimethylsiloxane, PDMS (NuSil Silicone Technology),
an elastomer known for its ability to mold and reproduce
three-dimensional structures. PDMS, once cured, is highly inert,
i.e., antibodies and antigens are not degraded by exposure to PDMS.
In addition, the elasticity and hydrophobicity of PDMS enables
temporary, fluid-tight seals to be made using only moderate
pressure. The PDMS patterning and assay flow cells were molded from
a polymethyl-methacrylate (PMMA) master mold created using a CNC
mill (CNC Software Inc., Tolland, Conn.). The PDMS flow cells were
reusable. They were cleaned and used to prepare dozens of patterned
substrates.
[0115] Cube layers were designed using MasterCam 8.0 software (CNC
Software Inc., Tolland, Conn.) and were manufactured from 0.25 inch
clear cast acrylic (AtoHaas North America, Inc., Philadelphia, Pa.)
using a 3-axis servo router (Techno-Isel, Hyde Park, N.Y.). Each
layer of acrylic was milled to contain a hole or groove or both.
When the layers were aligned, the holes and grooves combined to
form a three-dimensional network of channels and reservoirs. The
cube was designed to contain a bank of sample reservoirs on one
side and reagent reservoirs on the other with channels between the
reservoirs. Other features that were milled into the layers formed
holes for alignment of the pins and holes that were used to attach
the cube to the flow manifold. To form a solid cube, the layers
were secured with stainless steel pins then lightly clamped in a
vise and heated to 140.degree. C. for 3 hours. After cooling to
room temperature, stainless steel tubing was inserted into the
twelve exit holes to create exit ports. The tubing was secured with
a small amount of 5 Minute.RTM. Epoxy (Devcon, Inc., Danvers,
Mass.). After the epoxy had set, the tubing was cut to the desired
length using a variable-speed rotary tool equipped with a cut-off
wheel (Dremel, Inc., Racine, Wis.). Alternatively, the cube was
created by applying Weld-On 3, an acrylic solvent cement, (IPS
Corporation, Gardena, Calif.) to a layer then carefully placing the
next layer on top of it, with light manual pressure and allowing
the cement to dry. Layers were built up in this manner until the
entire cube was created. After cementing the layers into a cube, it
was placed in a vise under light pressure and heated to 140.degree.
C. for 3 hours. Each cube was tested for proper fluid flow and also
checked for leaks between reservoirs and channels or to the
exterior.
[0116] A flow manifold containing six channels and entry/exit holes
for fluid passage was designed using MasterCam 8.0 software (CNC
Software Inc., Tolland, Conn.). The flow manifold was manufactured
from 0.25" clear cast acrylic (AtoHaas North America, Inc.,
Philadelphia, Pa.) or black Lucite7.TM. cast acrylic (IC Acrylics,
Wilmington, Del.) using a 3-axis servo router (Techno-Isel, Hyde
Park, N.Y.). In the case of the manifold containing the PDMS gasket
(Leatzow et al., submitted), the flow channels were 2.74 mm
wide.times.38.1 mm long and 2.54 mm deep. The PDMS barrier
separation between each channel measured approximately 1.1 mm.
[0117] The flow manifold with the PDMS gasket was attached to the
glass waveguide through compression in a cassette assembly. The
assembly included the acrylic flow manifold with integrated PDMS
gasket, the glass waveguide, a bottom aluminum mounting bracket,
and nylon mounting screws. The waveguide was held in place between
the flow manifold and the mounting bracket by tightening the
mounting screws. The cube was attached to the top of the flow
manifold by a pair of nylon mounting screws. The screws extended
above the top surface of the manifold and entered into the cube
from below.
[0118] Following component assembly, the assay module (cube, flow
manifold, and waveguide) was placed on the detector. To verify the
system=s integrity and block nonspecific binding, the cube=s
reservoirs were filled with phosphate buffer saline containing
0.05% Triton.RTM. and 1 mg/ml bovine serum albumin, PB STB, which
was drawn through the flow manifold with negative pressure from a
downstream peristaltic pump. During the PBSTB flow-through, images
of the waveguide were captured to check for flow and leaks. To
assess nonspecific binding, 200 .mu.l of 10 .mu.g/ml Cy5-anti-SEB
antibody solutions were loaded into one bank of reservoirs and
drawn through the system. The system was flushed with 250 .mu.l of
PBSTB per reservoir and an image was captured. The image showed
negligible binding of Cy5-anti-SEB antibody to the waveguide or to
the edges of the flow manifold touching the waveguide.
[0119] Once the system checks were completed, 250 .mu.l of each
dilution of SEB (0-50 ng/ml) were loaded into one bank of sample
reservoirs. The other bank of reservoirs was loaded with 250 .mu.l
Cy5-anti-SEB, fluorescent tracer antibody, at 10 .mu.g/ml. By
opening and closing the appropriate air vents, the reservoirs
containing the tracer antibodies could be closed and the reservoirs
containing the samples could be opened, allowing only the sample
reservoirs to flow. An off-board peristaltic pump at a flow rate of
approximately 0.35 ml/min was used to create negative pressure
downstream of the assay module. After five minutes, the sample
reservoirs had been drained and the vents were then closed. The
tracer antibody reservoirs were then opened, and flow was confirmed
by capturing an image during the flow-through. After five minutes,
the antibody reservoirs had been drained. The antibody reservoirs
were filled with PBSTB and the buffer flushed through the flow
manifold. A final image, demonstrating detection of various
concentrations of SEB was captured and analyzed.
[0120] Digitized images were analyzed using Scion Image (Scion
Corporation, Frederick, Md.). To quantitate a region of interest
("spot"), a small rectangular selection was outlined around it and
the program calculated the average intensity of the pixels within
the spot. Using the same size rectangle, background readings were
taken on either side of the spot and the mean fluorescence of the
background was subtracted from the value determined for the spot.
There were six spots per channel (per SEB concentration) and their
mean and standard deviations were reported.
[0121] Premature mixing of sample and reagent upstream of the
waveguide surface could be minimized by configuring the flow path
so that neither the sample nor the reagent flowed through the
common channel, i.e., by separating the fluids with an extended
barrier.
[0122] Dilutions of SEB were loaded into the reservoirs of the cube
and assayed in our detector system. FIG. 9 shows the pattern of
signals captured by the imaging system. The fluorescent signals of
the spots were determined by subtracting the mean fluorescent
intensity of the adjacent regions with no capture antibody
(non-specific binding) from the fluorescent intensity of the region
including the capture antibody. The net fluorescence for each
capture antibody spot was plotted as a function of SEB
concentration. As shown in FIG. 10, the system was able to detect
concentrations of SEB from 5 to 50 ng/ml in a 200 .mu.l sample,
i.e., 1 to 10 ng (36-360 fmoles) of SEB. Six samples were analyzed
simultaneously with six assay replicates of each sample (i.e., six
separate assay spots) in under 20 minutes.
[0123] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
* * * * *