U.S. patent application number 12/650479 was filed with the patent office on 2010-04-29 for miniaturized fluid delivery and analysis system.
Invention is credited to Ping Chang, Chi-Chen Chen, Rong-I Hong, Shaw-Tzuv Wang, James Russell Webster.
Application Number | 20100105065 12/650479 |
Document ID | / |
Family ID | 32028401 |
Filed Date | 2010-04-29 |
United States Patent
Application |
20100105065 |
Kind Code |
A1 |
Webster; James Russell ; et
al. |
April 29, 2010 |
Miniaturized Fluid Delivery and Analysis System
Abstract
The present invention provides a method for combining a fluid
delivery system with an analysis system for performing
immunological or other chemical of biological assays. The method
comprises a miniature plastic fluidic cartridge containing a
reaction chamber with a plurality of immobilized species, a
capillary channel, and a pump structure along with an external
linear actuator corresponding to the pump structure to provide
force for the fluid delivery. The plastic fluidic cartridge can be
configured in a variety of ways to affect the performance and
complexity of the assay performed.
Inventors: |
Webster; James Russell;
(Hsinchu, TW) ; Chang; Ping; (Hsinchu, TW)
; Wang; Shaw-Tzuv; (Hsinchu, TW) ; Chen;
Chi-Chen; (Hsinchu, TW) ; Hong; Rong-I;
(Hsinchu, TW) |
Correspondence
Address: |
AGNEW INTERNATIONAL;PATENT & TRADEMARK LAW FIRM
7700 Irvine Center Drive, SUITE 800
Irvine
CA
92618
US
|
Family ID: |
32028401 |
Appl. No.: |
12/650479 |
Filed: |
December 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11504303 |
Aug 15, 2006 |
7666687 |
|
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12650479 |
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|
10437046 |
May 14, 2003 |
7241421 |
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11504303 |
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Current U.S.
Class: |
435/6.13 ;
435/7.9 |
Current CPC
Class: |
B01L 2300/0816 20130101;
B01L 2400/0481 20130101; B01L 2400/0605 20130101; B01L 3/50273
20130101; Y10T 436/2575 20150115; B01L 2300/0883 20130101; B01L
2200/10 20130101; B01L 2300/0867 20130101; F04B 43/043 20130101;
B01L 2300/0887 20130101; B01L 2400/0638 20130101; B01L 3/502738
20130101 |
Class at
Publication: |
435/6 ;
435/7.9 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/542 20060101 G01N033/542 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 27, 2002 |
TW |
91122431 |
Claims
1-11. (canceled)
12. A method of performing immunological assay of a fluid sample,
wherein the method comprises the steps of: (a) pumping said fluid
sample from a fluid reservoir, where said fluid sample is placed
therein, to a reaction chamber, wherein said fluid reservoir and
said reaction chamber are defined in a fluidic cartridge and said
reaction chamber comprises therein a plurality of immobilized
species; (b) allowing said fluid sample to react with said
plurality of immobilized species for a predetermined reaction time;
and (c) excluding said fluid sample from said reaction chamber
through an exit port.
13. The method, as recited in claim 12, further comprising the
steps of: (d) placing an antibody solution containing a specific
secondary antibody conjugated with a detectable molecule into a
fluid reservoir; (e) pumping said antibody solution from said fluid
reservoir to said reaction chamber; (f) pumping said antibody
solution out through an exit port after a predetermined reaction
time; and (g) providing a detectable signal.
14. The method as recited in claim 12, further comprising at least
a washing step of pumping a wash buffer placed in a fluid reservoir
through said reaction chamber and out an exit port.
15. The method as recited in claim 13, after step (c) and step (f),
each further comprising a washing step of pumping a wash buffer
placed in a fluid reservoir through said reaction chamber and out
said exit port.
16. The method, as recited in claim 12 or 14, wherein said fluid
sample contains a plurality of different antibodies.
17. The method, as recited in claim 12, 13 or 15, wherein said
detectable molecule is selected from the group consisting of
peroxidase enzyme, alkaline phosphatase enzyme and fluorescent
tag.
18. The method, as recited in claim 17, after step (c), further
comprising a step of pumping a substrate buffer placed in said
fluid reservoir to said reaction chamber to allow a substrate in
said substrate buffer to react with any enzyme captured in step (b)
with said immobilized species providing a detectable signal.
19. The method, as recited in claim 17, after step (f), further
comprising a step of pumping a substrate buffer placed in said
fluid reservoir to said reaction chamber to allow a substrate in
said substrate buffer to react with any enzyme captured in step
(e).
20. The method, as recited in claim 12 or 14, wherein said fluid
reservoir, said reaction chamber and said exit port are connected
by one or more channels of capillary dimensions, wherein said
fluidic cartridge includes a first substrate, a second substrate
and an flexible intermediate interlayer sealedly interfaced between
said first substrate and said second substrate to form therein said
fluid reservoir, said one or more channels, said reaction chamber,
and said exit port, and wherein said fluidic cartridge further
provides a fluid flow controlling structure therein to restrict a
flow of said fluid sample through said reaction chamber via said
one or more channels in one direction only.
21. The method, as recited in claim 13 or 15, wherein said fluid
reservoir, said reaction chamber and said exit port are connected
by one or more channels of capillary dimensions, wherein said
fluidic cartridge includes a first substrate, a second substrate
and an flexible intermediate interlayer sealedly interfaced between
said first substrate and said second substrate to form therein said
fluid reservoir, said one or more channels, said reaction chamber,
and said exit port, and wherein said fluidic cartridge further
provides a fluid flow controlling structure therein to restrict a
flow of said fluid sample and said antibody solution through said
reaction chamber via said one or more channels in one direction
only.
22. The method as recited in claim 12, wherein said fluid sample
comprises a plurality of bio-molecules at unknown concentrations or
an analyte conjugated with a detectable molecule.
23. The method as recited in claim 12, wherein said immobilized
species comprises a protein, an antibody, a nucleic acid, a DNA, a
compound, or a combination thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. patent application
Ser. No. 10/437,046, filed on May 14, 2003. This application is a
continuation of U.S. patent application Ser. No. 11/505,303 filed
on Aug. 15, 2006, which is a divisional of U.S. patent application
Ser. No. 10/437,046, filed on May 14, 2003, which are hereby
incorporated by reference herein in their entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] This invention relates to a system comprising a fluid
delivery and analysis cartridge and an external linear actuator.
More particularly, the invention relates to a system for carrying
out various processes, including screening, immunological
diagnostics, DNA diagnostics, in a miniature fluid delivery and
analysis cartridge.
[0003] Recently, highly parallel processes have been developed for
the analysis of biological substances such as, for example,
proteins and DNA. Large numbers of different binding moieties can
be immobilized on solid surfaces and interactions between such
moieties and other compounds can be measured in a highly parallel
fashion. While the sizes of the solid surfaces have been remarkably
reduced over recent years and the density of immobilized species
has also dramatically increased, typically such assays require a
number of liquid handling steps that can be difficult to automate
without liquid handling robots or similar apparatuses.
[0004] A number of microfluidic platforms have recently been
developed to solve such problems in liquid handling, reduce reagent
consumptions, and to increase the speed of such processes. Examples
of such platforms are described in U.S. Pat. Nos. 5,856,174 and
5,922,591. Such a device was later shown to perform nucleic acid
extraction, amplification and hybridization on HIV viral samples as
described by Anderson et al, "Microfluidic Biochemical Analysis
System", Proceeding of the 1997 International Conference on
Solid-State Sensors and Actuators, Tranducers '97, 1997, pp.
477-480. Through the use of pneumatically controlled valves,
hydrophobic vents, and differential pressure sources, fluid
reagents were manipulated in a miniature fluidic cartridge to
perform nucleic acid analysis.
[0005] Another example of such a microfluidic platform is described
in U.S. Pat. No. 6,063,589 where the use of centripetal force is
used to pump liquid samples through a capillary network contained
on compact-disc liquid fluidic cartridge. Passive burst valves are
used to control fluid motion according to the disc spin speed. Such
a platform has been used to perform biological assays as described
by Kellog et al, "Centrifugal Microfluidics: Applications," Micro
Total Analysis System 2000, Proceedings of the uTas 2000 Symposium,
2000, pp. 239-242. The further use of passive surfaces in such
miniature and microfluidic devices has been described in U.S. Pat.
No. 6,296,020 for the control of fluid in micro-scale devices.
[0006] An alternative to pressure driven liquid handling devices is
through the use of electric fields to control liquid and molecule
motion. Much work in miniaturized fluid delivery and analysis has
been done using these electro-kinetic methods for pumping reagents
through a liquid medium and using electrophoretic methods for
separating and perform specific assays in such systems. Devices
using such methods have been described in U.S. Pat. No. 4,908,112,
U.S. Pat. No. 6,033,544, and U.S. Pat. No. 5,858,804.
[0007] Other miniaturized liquid handling devices have also been
described using electrostatic valve arrays (U.S. Pat. No.
6,240,944), Ferrofluid micropumps (U.S. Pat. No. 6,318,970), and a
Fluid Flow regulator (U.S. Pat. No. 5,839,467).
[0008] The use of such miniaturized liquid handling devices has the
potential to increase assay throughput, reduce reagent consumption,
simplify diagnostic instrumentation, and reduce assay costs.
SUMMARY OF THE INVENTION
[0009] The system of the invention comprises a plastic fluidic
device having at least one reaction chamber connected to pumping
structures through capillary channels and external linear
actuators. The device comprises two plastic substrates, a top
substrate and a bottom substrate containing capillary channel(s),
reaction chamber(s), and pump/valve chamber(s)--and a flexible
intermediate interlayer between the top and bottom substrate which
provides providing a sealing interface for the fluidic structures
as well as valve and pump diaphragms. Passive check valve
structures are formed in the three layer device by providing a
means for a gas or liquid to flow from a channel in the lower
substrate to a channel in the upper substrate by the bending of the
interlayer diaphragm. Furthermore flow in the opposite direction is
controlled by restricting the diaphragm bending motion with the
lower substrate. Alternatively check valve structures can be
constructed to allow flow from the top substrate to the bottom
substrate by flipping the device structure. Pump structures are
formed in the device by combining a pump chamber with two check
valve structures operating in the same direction. A hole is also
constructed in the lower substrate corresponding to the pump
chamber. A linear actuator--external to the plastic fluidic
device--can then be placed in the hole to bend the pump interlayer
diaphragm and therefore provide pumping action to fluids within the
device. Such pumping structures are inherently unidirectional.
[0010] In one embodiment the above system can be used to perform
immunoassays by pumping various reagents from an inlet reservoir,
through a reaction chamber containing a plurality of immobilized
antibodies or antigens, and finally to an outlet port. In another
embodiment the system can be used to perform assays for DNA
analysis such as hybridization to DNA probes immobilized in the
reaction chamber. In still another embodiment the device can be
used to synthesize a series of oligonucleotides within the reaction
chamber. While the system of the invention is well suited to
perform solid-phase reactions within the reaction chamber and
provide the means of distributing various reagents to and from the
reaction chamber, it is not intended to be limited to performing
solid-phase reactions only.
[0011] The system of the invention is also well suited for
disposable diagnostic applications. The use of the system can
reduce the consumables to only the plastic fluidic cartridge and
eliminate any cross contamination issues of using fixed-tipped
robotic pipettes common in high-throughput applications.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A is a top view of a pump structure within the plastic
fluidic device of the invention.
[0013] FIG. 1B is a cross section view of the pump structure within
the plastic fluidic device of the invention.
[0014] FIG. 2 is a top view of a plastic fluidic device of the
invention configured as a single-fluid delivery and analysis
device.
[0015] FIG. 3 is a top view of a plastic fluidic device of the
invention configured as a 5-fluid delivery and analysis device.
[0016] FIG. 4 is a top view of a plastic fluidic device of the
invention configured as a re-circulating 3-fluid delivery and
analysis device.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The system of the invention comprises a plastic fluidic
cartridge and a linear actuator system external to the fluidic
cartridge. FIG. 1A shows a cross-sectional view of a pump structure
formed within the fluidic cartridge of the invention. The plastic
fluidic cartridge comprises three primary layers: an upper
substrate 21, a lower substrate 22, and a flexible intermediate
interlayer 23, as shown in FIG. 1B. The three layers can be
assembled by various plastic assembly methods such as, for example,
screw assembly, heat staking, ultrasonic bonding, clamping, or
suitable reactive/adhesive bonding methods. The upper and lower
substrates, depicted as 21 and 22 in FIG. 1B, both contain a
variety of features that define channels of capillary dimensions as
well as pump chambers, valve chambers, reaction chambers,
reservoirs, and inlet/outlet ports within the cartridge. FIG. 1B
shows a top view of the pump structure of FIG. 1A. The pump is
defined by a pump chamber 14 and two passive check valves 15 that
provide a high resistance to flow in one direction only. Passive
check valves 15 comprise a lower substrate channel 13 and an upper
substrate channel 11 separated by interlayer 23 such that holes
through interlayer 23, depicted as holes 12 in FIG. 1B, are
contained within upper substrate channel 11 but not within lower
substrate channel 13. Such check valve structures provide a low
resistance to a gas/liquid flowing from lower substrate channel 13
to upper substrate channel 11 and likewise provide a high
resistance to a gas/liquid flowing from upper substrate channel 11
to lower substrate channel 13. Pump chamber 14 comprises an upper
substrate chamber and a hole 141 in lower substrate 22 to free
interlayer 23 to act as a diaphragm 25, as depicted in FIG. 1B. A
linear actuator 24 external to the fluidic cartridge can then be
placed in the hole 131 to bend diaphragm 25 and therefore provide
the necessary force to deform the diaphragm.
[0018] FIG. 2 shows a top view of a plastic fluidic cartridge of
the invention configured as a single-fluid delivery and analysis
device. Fluid is first placed into the reservoir 31 manually or
automated using a pipette or similar apparatus. A pump structure 32
similar to that of FIG. 1B is contained within the device. By
repeatedly actuating an external linear actuator, fluid in
reservoir 31 is pumped through the pump structure 32, the capillary
channel 33 and into the reaction chamber 34. Reaction chamber 34
contains a plurality of immobilized bio-molecules 35 for specific
solid-phase reactions with said fluid. After a specified reaction
time, the fluid is pumped through reaction chamber 34 and out the
exit port 36.
[0019] Upper substrate 21 and lower substrate 22 of the plastic
fluidic cartridge of the invention can be constructed using a
variety of plastic materials such as, for example,
polymethyl-methacrylate (PMMA), polystyrene (PS), polycarbonate
(PC), Polypropylene (PP), polyvinylchloride (PVC). In the case of
optical characterization of reaction results within a reaction
chamber, upper substrate 21 is preferably constructed out of a
transparent plastic material. Capillaries, reaction chambers, and
pump chambers can be formed in upper substrate 21 and lower
substrate 22 using methods such as injection molding, compression
molding, hot embossing, or machining. Thicknesses of upper
substrate 21 and lower substrate 22 are suitably in, but not
limited to, the range of 1 millimeter to 3 millimeter in thickness.
Flexible interlayer 23 can be formed by a variety of polymer and
rubber materials such as latex, silicone elastomers,
polyvinylchloride (PVC), or fluoroelastomers. Methods for forming
the features in interlayer 23 include die cutting, rotary die
cutting, laser etching, injection molding, and reaction injection
molding.
[0020] Linear actuator 24 of the present invention, as depicted in
FIG. 1B, is preferred to be, but not limited to, an electromagnetic
solenoid. Other suitable linear actuators include a
motor/cam/piston configuration, a piezoelectric linear actuator, or
motor/linear gear configuration.
[0021] The invention will further be described in a series of
examples that describe different configurations for performing
different analyses using the plastic fluidic cartridge and external
linear actuator of this invention.
Example 1
Immunological Assay
[0022] The plastic fluidic cartridge, as shown in FIG. 2, can be
utilized to perform immunological assays within reaction chamber 34
by immobilizing a plurality of bio-molecules such as different
antibodies 35. In one exemplary embodiment, a sample containing an
unknown concentration of a plurality of antigens or antibodies is
first placed within reservoir 31. The external linear actuator is
then repeatedly actuated to pump the sample from reservoir 31 to
reaction chamber 34. The sample is then allowed to react with the
immobilized antibodies 35 for a set reaction time. At the end of
the set reaction time, the sample is then excluded from reaction
chamber 34 through exit port 36. A wash buffer is then placed in
reservoir 31 and the external linear actuator is repeatedly
actuated to pump the wash buffer through reaction chamber 34 and
out the exit port 36. Such wash steps can be repeated as necessary.
A solution containing a specific secondary antibody conjugated with
a detectable molecule such as a peroxidase enzyme, alkaline
phosphatase enzyme, or fluorescent tag is placed into reservoir 31.
The secondary antibody solution is then pumped into reaction
chamber 34 by repeatedly actuating the linear actuator. After a
predetermined reaction time, the solution is pumped out through
exit port 36. Reaction chamber 34 is then washed in a similar
manner as previously describe. In the case of an enzyme conjugate,
a substrate solution is placed into reservoir 31 and pumped into
reaction chamber 34. The substrate will then react with any enzyme
captured by the previous reactions with the immobilized antibodies
35 providing a detectable signal. For improved assay performance,
reaction chamber 34 can be maintained at a constant 37.degree.
C.
[0023] According to the present invention, the plastic fluidic
cartridge need not be configured as a single-fluid delivery and
analysis device. FIG. 3 shows a plastic cartridge configured as a
five fluid delivery and analysis device. Such a device can perform
immunological assays, such as competitive immunoassay,
immunosorbent immunoassay, immunometric immunoassay, sandwich
immunoassay and indirect immunoassay, by providing immobilized
antibodies in reaction chamber 46. Here reaction chamber 46 is not
configured as a wide rectangular area, but a serpentine channel of
dimensions similar to capillary dimension. This configuration
provides more uniform flow through the reaction chamber at the
expense of wasted space. For example, during immunoassays, a sample
containing unknown concentrations of a plurality of antigens or
antibodies is placed in reservoir 41. A wash buffer is placed in
reservoir 42. Reservoir 43 remains empty to provide air purging. A
substrate solution specific to the secondary antibody conjugate is
placed in reservoir 44. The secondary antibody conjugate is placed
in reservoir 45. Each reservoir is connected to a pump structure 1'
similar to that of FIG. 1. Pump structures 1' provide pumping from
reservoirs 41, 42, 43, 44, and 45 through reaction chamber 46 to a
waste reservoir 49. A secondary reaction chamber 47 is provided for
negative control and is isolated from the sample of reservoir 41 by
check valve 48. The protocol for performing immunoassays in this
device is equivalent to that described previously for the
single-fluid configuration with the distinct difference that each
separated reagent is contained in a separate reservoir and pumped
with a separate pump structure using a separate external linear
actuator. First, an external linear actuator corresponding to a
pump connected to reservoir 41 is repeatedly actuated until a
sample fluid fills reaction chamber 46. After a predetermined
reaction time, the sample fluid is pumped to waste reservoir 49
using either a pump connected to sample reservoir 41 or a pump
connected to air purge reservoir 43. Next the wash buffer is pumped
into reaction chamber 46 by repeatedly actuating the external
actuator corresponding to a pump structure connected to wash
reservoir 42. The wash and/or air purge cycle can be repeated as
necessary. A secondary antibody solution is then pumped into
reaction chamber 46 by repeatedly actuating the external linear
actuator corresponding to a pump structure connected to reservoir
45. After a predetermined reaction time, the secondary antibody
solution is excluded from reaction chamber 46 either by a pump
connected to reservoir 45 or a pump connected to air purge
reservoir 43. Reaction chamber 46 is then washed as before. The
substrate is pumped into reaction chamber 46 by repeatedly
actuating a linear actuator corresponding to a pump connected to
reservoir 44. After a predetermined reaction time, the substrate is
excluded from reaction chamber 46 and replaced with wash buffer
from reservoir 42. Results of the immunoassay can then be confirmed
by optical measurements through upper substrate 21.
[0024] Furthermore, the reactions performed with the plastic
fluidic cartridge of the invention need not be limited to reactions
performed in stationary liquids. FIG. 4 shows a plastic fluidic
cartridge according to the invention, configured to provide
continuous fluid motion through reaction chamber 55. In this
configuration, reservoirs 51, 52, and 53 are connected to separate
pump structures similar to those of the five fluid configuration of
FIG. 3, but in this case the pump structures are connected to an
intermediate circulation reservoir 56. For example, pump structure
57 is connected to circulation reservoir 56 to provide continuous
circulation of fluid from circulation reservoir 56 through reaction
chamber 55 and returning to circulation reservoir 56. In this
manner, a fluid can be circulated through reaction chamber 55
without stopping. Such a fluid motion can provide better mixing,
faster reactions times, and complete sample reaction with
immobilized species in reaction chamber 55. Pump structure 58 is
connected such that it provides pumping of fluids from circulation
reservoir 56 to waste reservoir 54. Immunological assays similar to
those described above can be performed in this device by
immobilizing antibodies in reaction chamber 55 placing the sample
containing unknown concentrations of antigens or antibodies in the
circulation reservoir 56, placing a solution of secondary antibody
conjugate in reservoir 52, placing a substrate solution in
reservoir 53, and placing a wash buffer in reservoir 51. The
remaining protocol is identical to the above method with the
addition of transferring fluids to and from the circulation
reservoir 56 and continuously circulating during all reaction
times.
Example 2
DNA Hybridization
[0025] The system of the present invention can also be used to
perform DNA hybridization analysis. Using the plastic cartridge of
FIG. 4, a plurality of DNA probes are immobilized in reaction
chamber 55. A sample containing one or more populations of
fluorescently tagged, amplified DNA of unknown sequence is placed
in reservoir 52. A first stringency wash buffer is placed in
reservoir 51. A second stringency wash buffer is placed in
reservoir 53. Reaction chamber 55 is maintained at a constant
temperature of 52.degree. C. The sample is transferred to
circulation reservoir 56 by repeatedly actuating a linear actuator
corresponding to a pump structure connected to reservoir 52. The
sample is then circulated through reaction chamber 55 by repeatedly
actuating a linear actuator corresponding to pump structure 57. The
sample is circulated continuously for a predetermined hybridization
time typically from 30 minutes to 2 hours. The sample is then
excluded from the circulation reservoir 56 and reaction chamber 55
by actuating pump structures 57 and 58 in opposing fashion. The
first stringency wash buffer is then transferred to circulation
reservoir 56 by repeatedly actuating the linear actuator
corresponding to the pump structure connected to reservoir 51. The
first stringency wash buffer is then circulated through reaction
chamber 55 in the same manner described above. After a
predetermined wash time, the first stringency wash buffer is
excluded from reaction chamber 55 and circulation reservoir 56 as
described above. A second stringency wash buffer is then
transferred to circulation reservoir 56 and circulated through
reaction chamber 55 in a manner similar to that previously
described. After the second wash buffer is excluded, the DNA
hybridization results can be read by fluorescent imaging.
[0026] The invention being thus described, it will be obvious that
the-invention may be varied in many ways. Such variations are not
to be regarded as a departure from the spirit and scope of the
invention, and all such modifications as would be obvious to one
skilled in the art are intended to be included within the scope of
the following claims.
* * * * *