U.S. patent application number 12/534681 was filed with the patent office on 2009-11-26 for fluidics devices.
This patent application is currently assigned to Osmetech Technology Inc.. Invention is credited to Charles E. Clemmens, Clark Foster, Gary R. Gust, Rudolph A. Montalvo, Robert Mucic, Gary T. Olsen, Thomas P. Robinson.
Application Number | 20090291507 12/534681 |
Document ID | / |
Family ID | 39690548 |
Filed Date | 2009-11-26 |
United States Patent
Application |
20090291507 |
Kind Code |
A1 |
Clemmens; Charles E. ; et
al. |
November 26, 2009 |
FLUIDICS DEVICES
Abstract
The invention relates to fluidics as used in medical and
diagnostic equipment and relates further to means for purifying,
abstracting, filtering, detecting and/or measuring analytes in
liquid samples.
Inventors: |
Clemmens; Charles E.;
(Encinitas, CA) ; Mucic; Robert; (Glendale,
CA) ; Montalvo; Rudolph A.; (Woodland Hills, CA)
; Foster; Clark; (Mission Viejo, CA) ; Gust; Gary
R.; (Huntington Beach, CA) ; Robinson; Thomas P.;
(Encinitas, CA) ; Olsen; Gary T.; (La Crescenta,
CA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS, LLP
ONE MARKET SPEAR STREET TOWER
SAN FRANCISCO
CA
94105
US
|
Assignee: |
Osmetech Technology Inc.
Pasadena
CA
|
Family ID: |
39690548 |
Appl. No.: |
12/534681 |
Filed: |
August 3, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12032356 |
Feb 15, 2008 |
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12534681 |
|
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60890180 |
Feb 15, 2007 |
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60953197 |
Jul 31, 2007 |
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Current U.S.
Class: |
436/501 ;
422/400; 422/68.1 |
Current CPC
Class: |
B01L 2400/0439 20130101;
B01L 2200/027 20130101; B01L 2300/0874 20130101; B01L 2300/0645
20130101; B01L 2300/0816 20130101; B01L 9/527 20130101; B01L
2300/088 20130101; B01L 2400/0478 20130101; B01L 2200/025 20130101;
B01L 2300/023 20130101; B01L 2400/0442 20130101; B01L 2400/0655
20130101; B01L 2300/1827 20130101; B01L 2400/0605 20130101; B01L
2200/10 20130101; B01L 3/502707 20130101; B01L 3/502738 20130101;
B01L 3/502715 20130101; B01L 2400/0481 20130101; B01L 2300/0887
20130101; B01L 2200/0684 20130101; B01L 3/502723 20130101; B01L
2300/123 20130101; B01L 2400/0487 20130101; B01L 7/52 20130101;
B01L 7/525 20130101; B01L 2300/024 20130101; G01N 27/27 20130101;
B01L 2300/0636 20130101; B01L 2300/0867 20130101 |
Class at
Publication: |
436/501 ; 422/99;
422/68.1 |
International
Class: |
G01N 33/53 20060101
G01N033/53; B01L 3/00 20060101 B01L003/00 |
Claims
1. A fluidics device comprising: a body, said body comprising; a
flow channel for transporting a fluid sample, said flow channel
comprising a situs where an operation is performed on one or more
components in said fluid sample; an inlet port for receiving said
fluid sample, said inlet port in fluid communication with said flow
channel and also in communication with an enclosed gaseous
environment; and an outlet port in fluid communication with said
inlet port and configured such that upon operation of said device
said fluid sample is recirculated through said flow channel to
release gaseous bubbles into said enclosed gaseous environment
while simultaneously allowing for facilitated diffusion and
performance of said operation on said one or more components in
said fluid sample.
2. The fluidics device of claim 1 further comprising at least one
working electrode in said flow channel.
3. The fluidics device of claim 2 further comprising one or more
auxiliary electrodes.
4. The fluidics device of claim 1 further comprising connectors for
interface with a detection device.
5. The fluidics device of claim 1 further comprising an EEPROM.
6. The fluidics device of claim 1 further comprising: one or more
check valves in fluid communication with said flow channel for
regulating the flow of said fluid sample therethrough; and a
diaphragm for interface with a pump, said diaphragm opearationally
coupled to said flow channel and said one or more check valves.
7. The fluidics device of claim 1 comprising one or more
immobilized biological binding partners or ligands in said flow
channel.
8. The fluidics device of claim 7 wherein said one or more
biological binding partners or ligands is immobilized using a self
assembling monolayer affixed to one or more electrode surfaces.
9. The fluidics device of claim 1 comprising an array of electrodes
bound to one or more biological binding partners or ligands.
10. The fluidics device of claim 9 wherein said array is located in
a serpentine channel.
11. The fluidics device of claim 1 wherein said channel comprises a
cross-sectional dimension comprising a greater width than
height.
12. The fluidics device of claim 1 wherein said device has a sample
capacity of from about 50 ul to about 200 ul.
13. A diagnostics kit comprising the device of fluidics device of
claim 1.
14. A method of determining analyte binding, comprising: providing
a fluidics device according to claim 9; adding to said device a
liquid sample suspected of containing one or more analytes specific
for said one or more biological binding partners or ligands;
circulating and recirculating said sample across said array; and
detecting binding of said one or more analytes to said one or more
biological binding partners or ligands.
15. The method of claim 14 wherein said circulating and
recirculating comprises flowing said liquid sample at a rate of
from about uL/sec to about 40 uL/sec.
16. The fluidics device of claim 1 wherein said device has a single
fluid sample capacity of no more than about 200 uL in said flow
channel and said flow channel has a length of from about 100 mm to
about 200 mm, a cross-sectional dimension ranging from about 0.75
mm to about 2.0 mm in width, and a height of from about 0.125 mm to
about 0.40 mm.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/032,356, filed Feb. 15, 2008, entitled the same, and
claims the benefit of U.S. Provisional Patent Applications Nos.
60/890,180, filed Feb. 15, 2007, and 60/953,197, filed Jul. 31,
2007, entitled the same, all of which are incorporated by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to fluidics as used in medical and
diagnostic equipment and relates further to means for purifying,
abstracting, filtering, detecting and/or measuring analytes in
liquid samples.
BACKGROUND OF THE INVENTION
[0003] The following documents are all incorporated herein by
reference in their entireties, although none is admitted to be
prior or relevant art. Collectively they reflect that there is a
current and long-felt need for, and past failure of success as
relates to, adequate microcassette fabrication and microfluidics,
not only from a cost and ease of fabrication and reproducibility
standpoint, but also from the standpoint that such systems are
typically restricted to laminar flow and feature inefficient mixing
compounded by gaseous bubble formation that obstructs or restricts
flow and diffusion. The present invention, depending on aspect and
embodiment, provides useful solutions to one or more of these
historic deficiencies.
[0004] Woolley et al. (1996) report functional integration of PCR
amplification and capillary electrophoresis in a microfabricated
DNA analysis device. Anal. Chem., 68:40814086. The chips are
generated by photolithography and etching of silicone wafers. The
prospects for electrophoretic valving and active microfabricated
valves made from polymer diaphragms are also discussed.
[0005] Martynova et al. (1997) report fabrication of plastic
(poly(methylmethacrylate)(PMMA)) microfluid channels by imprinting
methods for use in electrophoretic and chromatographic
applications. Anal. Chem., 69:4783-4789. Mechanical pumping is
referenced, as are the fabrication techniques of casting, molding,
laser ablating and machining plastic. Bubble entrapment is noted as
a problem that can be solved by heating.
[0006] Roberts et al. (1997) report micro channel construction
using UV laser machined polymer substrates (e.g., polystyrene,
polycarbonate, cellulose acetate, and poly(ethylene terephthalate)
(PET) for the development of microdiagnostic systems. Anal. Chem.,
69:2035-2042. The article also discusses laminate sealing. Pump and
valving are not addressed.
[0007] Burns et al. (1998) report an integrated nanoliter DNA
analysis device made of a glass and silicone substrate and
containing microfabricated channels, heaters, temperature sensors,
and fluorescence detectors. Science, vol. 282, pp. 484-487. The
device is made using photolithography and is reportedly capable of
measuring, mixing, amplifying and digesting DNA.
[0008] Kopp et al. (1998) report continuous-flow PCR on a glass
microchip. Science, vol. 280, pp. 1046-1048.
[0009] Waters et al. (1998) report a microchip device for cell
lysis, multiplex PCR amplification, and electrophoretic sizing and
fluorescence-based detection. Anal. Chem., 70:158-162.
Microchannels are polymer-etched and 50 um wide by 10.4 um deep.
Valving, pumps, and the identity of the polymer substrate are not
addressed.
[0010] Duffy et al. (1999) report microfabricated centrifugal
microfluidic systems having microscopic channels formed in a
plastic disk by casting molded PDMS or machining
pmethylmethylacrylate. Anal. Chem. 71:4669-4678. The channels are
reported to have diameters of 5 um-0.5 mm and depths of 16 um-3
mm.
[0011] Anderson, J. et al. (2000) report fabrication of
three-dimensional microfluidic systems in PDMS using "membrane
sandwiches", in which thin membranes having channel structures
molded on each face are fixed under pressure between two thicker,
flat slabs. Anal. Chem. 72, pp 3158-3164.
[0012] Anderson, R. et al. (2000) report a miniaturized integrated
polycarbonate device ("disposable cartridge") the size of a credit
card for automated multistep genetic assays. Nucl. Acid. Res., Vol.
28, No. 12, pp. i-vi. The device employs laminate valves made out
of a 0.01 mm thick mylar held in place by ultrasonic welding or
adhesives. Fluids are moved therethrough using a pneumatic
diaphragm valve and vacuum. Porous hydrophobic membranes are
reported that allow the passage of gas but not liquids.
[0013] Barker et al. (2000) report polystyrene, PDMS, polycarbonate
and polyethylene terephthalate glycol (PETG) plastic microfluidic
devices having surfaces modified with polyelectrolyte multilayers
(PEMs). Anal. Chem. 72: 4899-4903.
[0014] Beebe et al. (2000) report a PDMS microfluidics platform
that combines liquid-phase photopolymerization cartridges using
lithography, channels, pH-actuated hydrogel valving, and sensors.
PNAS, vol. 95, no. 25, pp. 13488-13493.
[0015] Liu et al. (2000) report chaotic advection passive mixing in
a three-dimensional serpentine microchannel having a C-shaped
repeating unit. J. Micro-electromech. Sys., Vol. 9 No. 2, pp.
190-197. The device is fabricated in a silicon wafer using a
double-sided KOH we-etching. Discussed are active mixing techniques
versus passive techniques, the relative sophistication and
difficulties presented by the former, and the need for at least one
such mechanism when small dimension (tens of micrometers) channels
are employed. Also discussed is the fluid dynamics principle of
Reynolds numbers, R.sub.e,=Q/A (flow rate over cross-sectional
area).times.D.sub.h/v (hydraulic diameter of channel over kinetic
viscosity of fluid. The repeating C-units emanate away from the
inlet and toward a distinct outlet.
[0016] Oleschuck et al. (2000) report trapping of bead-based
reagents within microfluidic systems and on-chip solid-phase
extraction and electrochromatography, coupled with
electrofluorescence detection. Anal. Chem., 72:585-590. The system
is made of etched glass and features continuous, valveless
flow.
[0017] Unger et al. (2000) report on monolithic microfabricated
valves and pumps for multilayer soft lithography. Science 288;
113-116. Soft lithography is described as an alternative to
silicon-based micromachining and uses replica molding of
nontraditional elastomeric materials to fabricate stamps and
microfluidic channels, with advantages afforded in terms of rapid
prototyping, ease of fabrication and biocompatibility. Systems
containing on-off valves, switching valves, and pumps made entirely
out of elastomer are described. Those systems include
microlectromechanical structures ("MEMS") that are either bulk or
surface micromachined from silica or other semiconductor-type
materials (e.g., polysilicon, metals, silicon nitride, silicon
dioxide, etc.), with the latter sequentially applied and patterned
in 3D structures, or else replication molding-based by patterned
curing of elastomeric material ("soft lithography"). The elastomer
used is a two-component addition-cured silicon rubber fused by
hermetic sealing and irreversible bonding. Up to seven (7)
independent layers are combined into one using this technique. Each
of the layers and resulting device is monolithic (i.e., all made
from the same material). The valves described are crossed-channel
in architecture, 100 um wide by 10 um high, mediated by polymer
membrane typically 30 um in thickness, and sealed with a glass
bottom layer. The flexibility and durability of the layers permits
the repeated opening and closing of valves upon pneumatic actuation
without appreciable fatigue. Tubular flow channels are urged as
opposed to rectangular or other shapes, and there is also
discussion of the problem of electrolytic bubble formation and
avoidance thereof. A peristaltic pump consisting of three valves
arranged in a single channel is also reported. The figures also
show unidirectional flow in which the inlets are remote from the
"waste" outlet points.
[0018] Xu et al. (2000) report a room-temperature imprinting method
for microchannel fabrication in PMMA. Anal. Chem., 72:1930-1933.
PDMS film is used to seal the channels, which are imprinted from a
micromachined silicone template. Pumping and valving are not per se
addressed.
[0019] Chabinyc et al. (2001) report an integrated fluorescence
detection system in combination with disposable PDMS microfluidic
implements. Anal. Chem., 73:44914498.
[0020] Gioradano et al. (2001) report use of the polymerase chain
reaction (PCR) in polyimide microchips using 1.7 ul volumes and
IR-mediated thermocycling. Anal. Biochem., 291:124-132.
[0021] Ismagilov et al. (2001) report multi-phase laminar fluid
flow and "switching" through a three-dimensional elastomeric
microstructure formed by two microfluidic channels, fabricated in
layers that contact one another face-to-face (typically at a 90
angle), with the fluid flows in tangential contact. Anal. Chem.
73:4682-4687. There is no discussion of valves or valving per se,
pressure is administered by syringe, polydimethylsiloxane (PDMS)
membranes of 4-5 mm in dimension are used in construction, and
channels of .about.25-200 .mu.m operative height, 100-400 .mu.m
operative width, and 2-4 cm operative length are used. Further, the
inlet and outlet ports are remote to another, the adhesion of the
individual layers is accomplished by oxidizing the mating surfaces
in an air plasma system for approximately 1 minute, and a glass
cover slip is also used.
[0022] Kamholz and Yager (2001) theoretically analyze molecular
diffusion in pressure-driven laminar flow in microfluidic channels.
Biophys. J., 80:155-160. The authors conclude there is reduced
diffusivity in microfluidic systems, including, e.g., systems
employing self-assembling monolayers (SAMS).
[0023] Lachner et al. (2001) report the advantages of planar
microchip capillary electrophoresis in conjunction with
electrochemistry, including miniaturization potential while
preserving sensitivity. Electrophoresis 22:2526-2536. Emphasis is
on the selective grounding of a detection reservoir relative to a
"separation channel" into which sample is first introduced. The
system also features sample waste and buffer reservoirs, as well as
a high-voltage source to effect separation. By definition, the
system depends on electric field establishment for sample migration
and there is no circulation or recirculation of liquid sample.
These systems feature glass or plastic chips, with the latter
fashioned from laser ablation or injection molding techniques, and
a variety of electrode surfaces, including carbon, platinum,
palladium, copper and gold. Applications discussed include those
for separation and detection of catecholes, amino acids, peptides,
carbohydrates, nitroaromatics, PCR products, organophosphates and
hydrazines. As expected for electrophoretic applications,
separation of PCR products is coordinated with restriction enzyme
digestion.
[0024] Whitesides et al. (2001) review soft lithograpy techniques
and the implications for microfabrication and biochip patterning
and configuration. Annu. Rev. Biomed., 3:335-73. Soft lithography,
as opposed to photolithography, is based on printing and molding
using elastomeric stamps with patterns of interest in bas-relief.
PDMS is a substrate of choice that is patterned with
self-assembling monolayers (SAMs) and microcontact printing (uCP).
Membrane-stacking is noted as a method of synthesizing and
configuring 3-dimensional microfluidic structures. Pumps (including
pneumatic) and soft PDMS membrane flap valving is also briefly
noted.
[0025] Yuen et al. (2001) report a microchip module of blood sample
preparation and nucleic acid amplification reactions. Genome Res.,
11:405-412. The module is a computer numerical control-machined
Plexiglas microchip. A syringe pump is used in tandem with
valving.
[0026] Auroux et al. (2002) review micro analysis systems for the
period 1997 to 2002. Anal. Chem. 74: 2637-2652.
[0027] Beebe et al. (2002) review microfluidics in general and the
fabrication of valves, mixers and pumps for the same as of 2002.
Annu. Rev. Biomed. Eng. 4:261-86. Micromatching, soft lithography,
embossing, in situ construction, injection molding and laser
ablation are discussed, as well as the advantages and disadvantages
attendant thereto.
[0028] Jeon et al. (2002). Report design and fabrication of
integrated passive valves and pumps for flexible polymer
3-dimensional microfluidic systems. Biomed Microdevices
4:117-121.
[0029] Johnson et al. (2002) report rapid microfluidic mixing in
preformed T-microchannel imprinted in a hot-imprinted polycarbonate
silicon stamped substrate and modified with a pulsed UV excimer
laser to create slanted wells at the junction. Anal. Chem.
74:45-51. PETG "lids" were sealed to the PC by heat-bonding.
[0030] Stroock et al. (2002) report chaotic mixers for
microchannels. Science 295: 647-651. The difference between laminar
and turbulent flow is discussed in terms of efficient mixing, with
the former (characteristic of systems having channels of dimension
.about.100 .mu.m or less) described as less efficient and
characteristic of microfluidic systems in general. Stroock et al.'s
solution is to employ textured relief structures deposited by
planar lithographic techniques inside PDMS microfluidic channels in
order to impart differential resistance across varied topographic
surfaces, thereby improving passive mixing in the process.
[0031] Klank et al. (2002) report CO.sub.2 laser micromachining and
back-end processing for rapid production of PMMA-based microfluidic
systems. Lab Chip, 2:242-246.
[0032] McDonald and Whitesides (2002) report poly(dimethylsiloxane)
(PDMS) as a useful material for fabricating microfluidic devices.
Accounts of Chemical Res., vol. 35, no. 7, pp. 491499. Silicone
adhesive tapes are noted for their ability to reversibly effect
water-tight binding between different PDMS components. 3-D
"membrane sandwich" fabrication by stacking multiple layers is also
discussed, as is the ability to configure the devices with chambers
that fit pipette tips.
[0033] Pugmire et al. (2002) report surface characterization of
laser-ablated polymers used for microfluidics. Anal. Chem.,
74:871-878. Electroosmotic flow comparisons are made between PMMA,
PETG, PVC and PC after ablation under different gaseous conditions.
Pumping and valving are not addressed.
[0034] Qi et al. (2002) report high-aspect-ration microstructures
(HARMS) in microfluidic devices fabricated from PMMA using
hot-embossing with integrated sampling capillary and fiber optics
for fluorescence detection. Lab Chip, 2:88-95. Aspect ratio is
described as the ratio of feature height to lateral dimension.
Pumping and valving are not addressed.
[0035] Reyes et al. (2002) briefly review the historical evolution
of micro total analysis systems ("uTAS"; synonymous with "lab on a
chip") theory and technology, including microfabrication, bonding,
surface modification, design, interfaces and connections,
microvalves and flow control, and micropumps. Anal. Chem.
74:2623-2636. Construction materials noted include, e.g., PDMS,
PMMA, PC, poly(ethyleneterephthalate) (PET), and
poly(tetrafluoroethylene)(Teflon.RTM.). Valving and pumping are
generally discussed on page 2631 et seq. Passive membraneous check
valves are noted, as well as flap, lever and duckbill
varieties.
[0036] Wang, J. (2002) reports on electrochemical detection in
microscale analytical systems and how such systems offer possible
advantages by way of miniaturization, portability and, more
tenuously, disposability. Talanta 56:223-231. Emphasis is again on
electrophoretic separations and sample reservoir to waste reservoir
directionality and flow, with the reservoirs made of PDMS/glass.
Included is discussion of capillary electrophoretic (CE) systems,
micromachining and ablation techniques, as well as discussion of
different electrode types/compositions and forms of electronic
detection, e.g., fixed-potential/current monitoring (amperometric)
and voltammetry. Detection of nucleic acids is discussed using
intercalating iron-phenanthroline redox markers.
[0037] Breadmore et al. (2003) report microchip-based purification
of DNA from biological samples. Anal. Chem. 75:1880-1886.
[0038] Fiorini et al. (2003) report fabrication of thermoset
polyester microfluidic devices and embossing masters using rapid
prototyped polydimethylsiloxane (PDMS) molds. Lab Chip,
3:158-163.
[0039] Glasgow and Aubry (2003) report enhanced microfluidic mixing
using time pulsing. Lab Chip, 3:114-120. Mixing is accomplished by
time varying and pulsing fluid flow using, e.g., variable channel
dimensions. Bubble entrapment is stated as a problem to be avoided.
The substrate used is not identified and there is no discussion of
venting or valving.
[0040] Jensen et al. (2003) report microstructure fabrication in
poly(methyl methylacrylate) (PMMA) with a CO.sub.2 laser system,
including raster scanning to produce cavities 50 um wide and 200 um
deep. Lab Chip, 3:302-307.
[0041] Koh et al. (2003) report integrated PCR, valving and
electrophoresis in a plastic device for bacterial detection. Anal.
Chem. 75:45914598. The device is made from cyclic polyolefin having
graphite ink electrodes and photopatterned gel domains that
function as passive valves. Detection is optical and accomplished
using laser-induced fluorescence of an interchalating dye. Volumes
used were 29-84 nL.
[0042] Kricka and Wilding (2003) review microchip PCR systems
bearing serpentine channels and fabricated from molded PDMS,
micromachined polycarbonate or assembled layers of ceramic tape,
held together, e.g., by use of adhesives. Anal. Bioanal. Chem.
377:820-825. A host of passivation agents are also discussed that
avoid adverse surface interactions, including, e.g., silicon oxide,
PDMS, polypropylene, BSA, and polyvinylpyrrolidone.
[0043] Landers (2003) reports inter alia on the potential for
performing single nucleotide polymorphism (SNP) diagnostics on
electrophoretic microchips, preferably using optical detection on
capillary based systems. Anal. Chem., 75:2919-2927. See, e.g., pp.
2922.
[0044] Liu et al. (2003) report sophisticated microfluidic PCR
systems devised of multilayer elastomeric PDMS formed using
photolithography and active pumping and valving schemes. Anal.
Chem. 75:4718-4723.
[0045] Wang et al. (2003) report low-density microarrays assembled
in microfluidic chips fabricated from hot-embossed PMMA for the
detection of low-abundant DNA mutations. Anal. Chem., 75:1130-1140.
Appropriate ligand linking chemistry is also addressed.
[0046] Buch et al. (2004) report DNA mutation detection in a
modular polycarbonate microfluidic network using temperature
gradient gel electrophoresis. Anal. Chem. 76:874-881. One module is
embossed with microchannels and the other contains a tapered
microheater lithographically patterned along with an array of
temperature sensors.
[0047] Gustafsson et al. (2004) report integrated peptide sample
preparation and MALDI Mass Spectometry on a Microfluidic Compact
Disk, in which sample fluid is pushed using centripetal force.
Anal. Chem. 76:345-350.
[0048] Hashimoto et al. (2004) report rapid PCR in a continuous
flow embossed polycarbonate device. Lab Chip, 4:638-645.
Microchannel dimensions used were 6 cm(L).times.50 um(W).times.150
um(H).
[0049] Howell et al. (2004) report on fluid dynamics principles and
the design and evalution of a Dean vortex-based micromixer on a
machined PMMA chip. Lab chip, 4:663-669. A double-sided adhesive
tape is used to fix the machined chip to a glass slide and bubble
avoidance is urged.
[0050] Lagally et al. (2004) report an integrated portable genetic
analysis microsystem for pathogen/infectious disease detection
using PCR, electrophoresis and laser-excited fluorescence
detection. Anal. Chem., 76:3162-3170. The system is said to be of
etched glass wafer design and contain active solenoid PDMS
"membrane valves", with one particular configuration possessing
three such valves in series to collectively form a "diaphragm"
pump. Sample volumes are 200 nL.
[0051] Lai et al. (2004) report a resin-gas injection packaging
technique for bonding and surface modification of polymer-based
microfluidic platforms such as glass, silicon, polyethylene,
polystyrene poly(methyl methacrylate) (PMMA), polyamide, and
polycarbonate. Anal. Chem., 76:1175-1183. Also noted are adhesive
layer techniques and the problem of bubble accumulation/obstruction
and the suggestion to use a vacuum to minimize such.
[0052] Laser and Santiago (2004) review micropump structures in J.
Micromech. Microeng., 14:R35-R64. The detrimental problem of
bubbles in microfluidic systems is noted repeatedly throughout, as
is the general dearth of effective pumping systems in microfluidics
systems. Despite this, reciprocating pneumatically-driven diaphragm
pumps flanked by passive check valves are discussed in the context
of multilayer constructions, see, e.g., FIG. 1 and .sctn. 2.1,
although diaphragms made out of soft polymer membranes are said to
be a "concern" because of stability. Etching, micromachining and
photolithography are also discussed as means of creating device
channels and chambers.
[0053] Noerholm et al. (2004) report a disposable polycarbonate
microfluid chip for online monitoring of microarray hybrizations.
Lab Chip, 4:28-37. The chip is 25.times.76.times.1.1 mm in
dimension and manufactured by micro injection molding. The chip is
said to contain an inlet, a 10 ul hybridization chamber, a waster
chamber and a vent to allow air to escape when sample is injected.
Its utility is demonstrated using hybridization buffer, wash
buffers, fluorescence-based detection and a computer controlled
syringe pump. The system would appear to be capillary-action
mediated, continued flow, non-recirculating and valveless. Use of
plastic polymers is said to endow advantages by way of milling,
laser ablation, hot embossing and injection molding. The use of
adhesive tape in fashioning microstructures is also noted. The
problem of bubble development is also noted but the vent used is
located remote to the inlet and proximal to a waste chamber.
[0054] Schonfeld et al. (2004) report an optimized
split-and-recombine (SAR) micro-mixer formed from milled PMMA and
featuring active, uniform "chaotic" mixing. Lab Chip, 4:65-69.
[0055] Vilkner et al. (2004) review various micro total analysis
systems (uTAS), including microfabrication, bonding techniques,
microvalves and flow control, and micropumps. Anal. Chem.,
76:3373-3386. Their review builds on that of Reyes (2002) and, in
addition to discussing PDMS, PMMA, PC, poly(ethyleneterephthalate)
(PET), and poly(tetrafluoroethylene) (Teflon.RTM.) as construction
materials, and general valving, further includes discussion, e.g.,
of thermoresponsive hydrogel plugs and valving.
[0056] Yaralioglu et al. (2004) report ultrasonic mixing in PDMS
microfluidic channels using integrated piezoelectric transducers.
Anal. Chem., 76:3694-3698.
[0057] Fiorini and Chiu (2005) review disposable microfluidic
device fabrication, function and application. BioTechniques vol.
38, no. 3, pp. 429-446. Methods of fabrication include replica and
injection molding, embossing, and laser ablation. Fluid pumping and
valving is also described, as is mixing and analyte separation and
detection. Deformable membrane pressure pumps and valves are
particularly discussed at pp. 434-5, as is the concept of pulsatile
flow. Strategies for mixing include use of 3-dimensional serpentine
channels. P. 435. Multilayer fabrication with plastics is also
mentioned, as are electrochemical detection schemes and advantages
attendant thereto, and nucleic acids as detectable analyte. pp.
438-9.
[0058] Howell et al. (2005) report a microfluidic mixer with
grooves placed on the top and bottom of milled PMDA channels. Lab
Chip, 5:524-530.
[0059] Klapperich et a. (2005) report hot-embossed fabrication of a
cyclic polyolefin microfluidic device for on-chip isolation of
nucleic acids onto silicon particles embedded in the device,
followed by elution. Proc. ICMM2005, 3.sup.rd Int. Conf. on
Microchannels and Minichannels. Toronto, CANADA.
[0060] Lee et al. (2005) report development of a passive
3-dimensional PDMS micromixer based on repeated fluid twisting and
flattening of the channels, and its application to DNA
purification. Anal. Bioanal. Chem., 383:776-782. Multi-layer
stacking and multi-step photolithography are noted as device
fabrication techniques. The system has discreet inlets and outlets
that are remote relative to one another.
[0061] Roper et al. (2005) report advances in polymerase chain
reaction (PCR) methodology on polycyclic olefin microfluidic chips
using hydraulic valves and pneumatic pumps. Anal. Chem.,
77:3887-3894. Reported reaction volumes are approximately 30
nl.
[0062] Skelley et al. (2005) report development and evaluation of a
sophisticated capillary electrophoresis microdevice made of glass
wafers and PDMS membranes for amino acid biomarker detection and
analysis use on Mars. PNAS, vol. 102, no. 4, pp. 1041-1046. The
device is vacuum driven and said to possess 34 individual membrane
valves and 8 pumps. The wafers are 10 cm in diameter with 20 um
deep.times.70 um wide.times.21.4 cm long channels.
[0063] Wang et al. (2005) report label-free detection of
small-molecule-protein interactions using nanowire nanosensors
(silicone; SiNW) and field effect transistors (FETs) on a surface
plasma resonance (SPR)-like chip. PNAS, vol. 102, no. 9, pp.
3208-3212.
[0064] Whitesides et al. (2005) report a technique for storing and
delivering a sequence of reagents to a microfluidic device.
Abstract, Anal. Chem., 77(1):64-71. The technique makes use of
cartridges of tubing filled by sequentially injecting plugs of
reagents separated by air spacers.
[0065] Liu et al. (2006) report integrated microfluidic biochips
for DNA microarray analysis by fluorescence imaging that contain
electromechanical pumps, low-cost check valves, fluid channels and
reagent storage containers. Expert Rev. Mol. Diagn., 6(2):253-261
(Abstract).
[0066] Soper et al. (2006) forecast point-of-care (POC) biosensor
systems for cancer diagnostics/prognostics. Biosensors and
Bioelectronics, 21:1932-1942. The article only generally speaks to
the future of the field and the need for mass-production, low cost
fabrication and specialized valving and pumping systems. Techniques
contemplated for construction of such devices include injection
molding, nanoprint lithography and hot-embossing.
[0067] U.S. Pat. Nos. 7,101,509 and 6,368,87,1 assigned to Cepheid
share a common specification and collectively report and claim
temperature controlled devices and methods for the manipulation of
materials in a fluid sample using a plurality of microstructures
bearing insulator films (selected from silicon dioxide, silicon
carbide, silicon nitride, and electrically insulating polymers).
The devices employ integrated loading chambers, reaction vessels,
and aspirators in connection with the insulator-film bearing
structures. Application of a voltage to the structures induces the
desired electrophoretic separation and attraction, followed by
washing and elution steps. U.S. Pat. Nos. 6,893,879, 6,664,104 and
6,403,037 assigned to Cepheid report and claim similar analyte
flow, capture and elution techniques and devices.
[0068] U.S. Pat. No. 6,818,185 reports and claims a cartridge for
conducting a chemical reaction that consists of a body having at
least first and second channels formed therein, a reaction vessel
extending from the body, a reaction chamber, an inlet port
connected to the reaction chamber via an inlet channel, and an
outlet port connected to the reaction chamber via an outlet
channel. The inlet port of the vessel is connected to the first
channel in the body, and the outlet port of the vessel is connected
to the second channel in the body. The walls of the reaction
chamber contain polymeric films, and vents for exhausting gas from
the second channel are also described. The system also employs
differential pressure sources for forcing sample through the
system, which can further include thermal surfaces, heating
elements, mixing and lysing chambers, and optically transmissive
walls.
[0069] U.S. Pat. No. 6,374,684 reports a fluid control and
processing system having a plurality of chambers and a valve body
that includes a fluid sample processing region coupled with a fluid
displacement region, the fluid displacement region depressurizable
to draw fluid into the fluid displacement region and pressurizable
to expel fluid from the fluid displacement region.
[0070] U.S. Pat. Nos. 6,830,936, 6,197,595, 6,043,080, 5,922,591,
and 5,856,174, each entitled "Integrated nucleic acid diagnostic
device" and assigned to Affymetrix, describe and/or claim diaphragm
or controllable valve actuated miniature fluid flow systems for
measuring and processing fluid samples. The systems described make
use of a plurality of different chambers and channels, as well as
inlet and vent ports. U.S. Pat. Nos. 6,733,977 and 6,168,948 are
similar to these in content.
[0071] U.S. Pat. Nos. 7,223,363 (entitled "Method and System for
Microfluidic Interfacing to Arrays") and 7,235,400 (entitled
"Laminated Microarray Interface Device"), each assigned to BioMicro
Systems Inc., report pump-driven multi-laminate microfluidics
systems having a gasket that defines the walls of a reaction
chamber (e.g., serpentine), integrated passive valving, diaphragm
and multiple bladder use to promote mixing, the possibility for
sample (re)circulation and bubble elimination using an external
pumping scheme, the relative positioning of multiple such devices
such that there is a pitch of 9 mm between devices for ease of
loading by a multipipetteman, and the merit of using construction
materials that permit visualization/optical assessment of the
system.
[0072] U.S. Pat. Nos. 5,063,081 ("Method of manufacturing a
plurality of uniform microfabricated sensing devices having an
immobilized ligand receptor"), 5,096,669 ("Disposable sensing
device for real time fluid analysis") and 5,124,661 ("Reusable test
unit for simulating electrochemical sensor signals for quality
assurance of portable blood analyzer instruments") to I-STAT
Corporation discuss inter alia use of a disposable cartridge system
that makes use of an internal bladder to manipulate liquid
sample.
[0073] Micronics, Inc. also holds numerous patents in the field of
microfluidics including, e.g., U.S. Pat. Nos. 7,223,371
("Microfluidic channel network device"), 6,743,399 ("Pumpless
microfluidics"), 6,742,661 ("Well-plate microfluidics"), 6,581,899
("Valve for use in microfluidic structures"), 6,557,427
("Capillaries for fluid movement within microfluidic channels") and
6,488,896 ("Microfluidic analysis cartridge").
[0074] Numerous patents and papers published by Paul Yager are also
germane to the topic of microfluidics and include, e.g., U.S. Pat.
Nos. 5,716,852 and 5,972,710 ("Microfabricated diffusion-based
chemical sensor"), 6,007,775 ("Multiple analyte diffusion-based
chemical sensor"), 6,039,897 ("Multiple patterned structures on a
single substrate fabricated by elastomeric micro-molding
techniques"), 6,110,354 and 6,790,341 ("Microband electrode
arrays"), 6,159,739 ("Device and method for 3-dimensional alignment
of particles in microfabricated flow channels"), 6,454,945
("Microfabricated devices and methods"), Sensors in Biomaterials
Science: An Introductory Text, Ratner, B. D. and Hoffman, A. S.,
Eds. Academic Press, Inc., Orlando, (1996), Low Reynolds number
micro-fluidic devices, Proceedings Hilton Head MEMS conference,
Solid-State Sensor and Actuator Workshop, 105-108, (1996),
Biotechnology at low Reynolds numbers, Biophysical Journal. 71 (6),
3430-3441, (1996), Integration of microelectrodes with etched
microchannels for in-stream electrochemical analysis, Micro Total
Analysis Systems, 105-108 (1998), Design of microfluidic sample
preconditioning systems for detection of biological agents in
environmental samples, SPIE Proceedings, 3515, 252-259 (1998),
Whole blood diagnostics in standard gravity and microgravity by use
of microfluidic structures (T-sensors), Mikrochimica Acta, 131,
75-83 (1999), A novel microfluidic mixer based on successive
lamination, Micro Total Analysis Systems, Mesa Monographs, 495-498
(2003), On the importance of quality control in microfluidic device
manufacturing, Micro Total Analysis Systems, Mesa Monographs,
1069-1072 (2003), Lab-on-a-chip and fluorescence sensing on the
microscale, Fluorescence Sensors and Biosensors, R. B. Thompson,
ed., ISBN 0-8247-2737-1, CRC Press, Boca Raton, Fla., c. 400 pp
(2005), Rapid, parallel-throughput, multiple analyte immunoassays
with on-board controls on an inexpensive, disposable microfluidic
device, Micro Total Analysis Systems, Vol. 2, Transducer Research
Foundation, Pubs., 1000-1002 (2005), Recirculating flow accelerates
DNA microarray hybridization in a microfluidic device, Lab on a
Chip, in press.
[0075] Microfluidic systems and function is also addressed in
patents and publications by Stanford's Stephen Quake, including
U.S. Pat. Nos. 7,232,109 ("Electrostatic valves for microfluidic
devices"), 7,216,671, 7,169,314, 7,144,616, 7,040,338, 6,929,030,
6,899,137, and 6,408,878 ("Microfabricated elastomeric valve and
pump systems"), 7,143,785 ("Microfluidic large scale integration"),
6,960,437 ("Nucleic acid Microfabricated elastomeric valve and pump
systems, 6,793,753 ("Method of making a microfabricated elastomeric
valve"), 6,767,706 ("Integrated active flux microfluidic devices
and methods"), and "A nanoliter-scale nucleic acid processor with
parallel architecture," Nat. Biotechnol, 22: 4: 435-9 (2004),
"Solving the "world-to-chip" interface problem with a microfluidic
matrix." Anal. Chem., 75: 18: 4718-23 (2003), "Microfluidics in
structural biology: smaller, faster em leader better." Curr. Opin.
Struct. Biol. 13: 5: 538-44 (2003), "Integrated nanoliter systems,"
Nat. Biotechnol., 21: 10: 1179-83 (2003), "Microfabricated fountain
pens for high-density DNA arrays," Genome Res., 13: 10: 2348-52
(2003), "Microfluidic memory and control devices," Science, 300:
5621: 955-8 (2003), "Microfluidic large-scale integration,"Science,
298: 5593: 580-4 (2002), "A nanoliter rotary device for polymerase
chain reaction," Electrophoresis, 23:10:1531-6 (2002), "Dynamic
pattern formation in a vesicle-generating microfluidic device."
Phys. Rev. Lett. 86: 18: 4163-6 (2001), "Monolithic microfabricated
valves and pumps by multilayer soft lithography." Science, 288:
5463: 113-6 (2000); "From micro- to nanofabrication with soft
materials," Science, 290: 5496: 1536-40 (2000), and "A
microfabricated device for sizing and sorting DNA molecules." Proc.
Natl. Acad. Sci. USA 96: 1: 11-3 (1999).
[0076] In addition to the foregoing work of others, commonly-owned
U.S. Pat. Nos. 7,172,897, 6,960,467, 6,875,619, 6,833,267,
6,761,816, 6,642,046, 6,592,696, 6,572,830, 6,544,734, 6,432,723,
and 6,361,958 also speak to microfluidics and microfluidics
operations, including integration of individual electronic
components and positionment into detection devices, including
electrochemical detection devices.
[0077] As will be become apparent, the configuration and function
of the above third party devices is different from aspects and
embodiments of the inventions described herein in one or more of
construction, valving, mixing, diaphragm positionment and function,
bubble elimination, pump interfacing and recirculation design.
These differences give rise to real advantages and prospects for
the inventions described herein.
BRIEF SUMMARY OF THE INVENTION
[0078] Accordingly, in one aspect the invention provides fluidics
devices comprising at least one membranous diaphragm having a first
and second side. The first side is for coupling to a pump, and the
second side is for fluidic coupling to a flow channel (which can
include a detection chamber as a flow channel) and a plurality of
check valves. The check valves each comprise a sealing surface
comprising a valve seat that allows for regulated flow of fluid
through the flow channel and a flexible sealing structure for
contacting the valve seat and occluding fluid flow therethrough
when in a first position, and for promoting fluid flow therethrough
when in a second position. Generally, in this aspect, the diaphragm
and the flexible sealing structure are integral to one or more
layers of a multilayer assembly (e.g. a multilaminate structure).
The plurality of check valves alternate opening and closing in
coordinated reciprocal fashion according to alternating positive
and negative forces exerted on the diaphragm. In an additional
aspect, the device comprises a body comprising a channel (which can
be the detection chamber) within for transporting a fluid sample,
the channel comprising a situs where an operation is performed on
one or more components in the fluid sample. The body also comprises
an inlet port for receiving the fluid sample, the inlet port in
fluid communication with the channel and also in communication with
an enclosed gaseous environment. The inlet port can be configured
to receive the fluid sample from a pipette tip, including
micropipette tips. The body also comprises an outlet port in fluid
communication with the inlet port such that upon operation of the
device the fluid sample is recirculated through the channel to
release gaseous bubbles into the enclosed gaseous environment while
simultaneously allowing for facilitated diffusion and performance
of the operation on the one or more components in said fluid
sample.
[0079] In a further aspect, the fluidics devices process no more
than about 1.5 mL of fluid at a time, with some aspects processing
no more than about 150 ul of fluid at a time. In some aspects, the
sample capacity of the device is from about 50 ul to about 200 ul.
The channels can comprise a cross-sectional dimension comprising a
greater width than height. In some aspects, the channels have a
cross-sectional dimension ranging from about 0.030'' to about
0.060'' in width and from about 0.006'' to about 0.014'' in
height.
[0080] In an additional aspect, the fluidics devices are made at
least in part from the stacking of multiple individual polymeric
laminate sheets, optionally held together by pressure sensitive
adhesive sheets. In some aspects, the devices are constructed from
alternating laminate and adhesive layers having individual
thicknesses in the range of from about 0.0005'' to about 0.030''.
In some aspects, the individual laminate layers are thicker than
the individual adhesive layers. In some aspects, the individual
laminate layers are from about 0.0005'' to about 0.010'' thick and
said individual adhesive layers are from about 0.001'' to about
0.003'' thick.
[0081] In a further aspect, one or more of the channels of the
fluidics devices are formed by grooves, cuts or recesses in one or
more of these individual plastic laminate sheets, which can be
produced by a die-stamp, laser, chemical etching, or molding.
[0082] In an additional aspect, the channels that serve as
detection chambers further comprise at least one working electrode
in the flow channel, optionally comprising capture binding ligands
and SAMs, and thus is optionally a diagnostics device. Thus,
aspects of the invention provide for immobilized biological binding
partners in the flow/detection channel. These biological binding
partners can be immobilized using a self assembling monolayer that
is affixed to a surface, optionally an electrode. The biological
binding partners can be polynucleotides or proteins (including
peptides and antibodies).
[0083] In a further aspect, the fluidics devices of the invention
further optionally comprise one or more auxiliary electrodes and/or
connectors for interface with a detection device. In some aspects,
the connectors are configured in a two-dimensional grid of contact
points. In some aspects, the connectors may be ZIF connectors
and/or side or edge connectors. The fluidics devices can also
optional comprise an EEPROM, and/or an internal unused space
designed for integration of one or more future functionalities.
[0084] In an additional aspect, the fluidics devices of the
invention can comprise a transparent or translucent plastic (e.g.
an optical window) that permits visualization of circulation and
recirculation of the fluid sample within.
[0085] In a further aspect, the fluidics devices can comprise one
or more valves in fluid communication with the channel for
regulating the flow of the fluid sample therethrough. The valves
can be check valves, passive valve and bridge valves. In some
aspects, or more of of the valve seats protrude from a planar
structure to promote sealing upon engagement with a corresponding
sealing structure. The magnitude of the protuberances relative to
the base surface is independently selected from about 0.001'' to
about 0.005''. Optionally, one of more of the valve seats are part
of a hollow boss having a plurality of ports adjacent and fluidly
coupled to one another in a nonlinear configuration, optionally a
"u-structure", and routing fluid from one fluidics plane in the
device to one or more other fluidics planes in the device.
[0086] In an additional aspect, the fluidics devices can further
comprise a means for pumping (e.g. a pump) the fluid sample through
the channel. Thus, the devices can comprise a plurality of bridge
or check valves actuated by a pneumatic, electromagnetic, or
hydraulic pump and a diaphragm. The pneumatic pump can be
electrically driven.
[0087] In an additional aspect, the devices can further comprise
caps for sealing the device following the addition of a sample, and
optionally a filter, adsorbent and/or absorbent for reducing or
eliminating solutes or analytes in the fluid sample.
[0088] In a further aspect, the fluidics devices of the invention
further comprise a detector that detects binding events between
binding partners and complementary binding partners in a fluid
sample, which can be a colorimetric detector or an electronic
detector that detects electronic properties of the binding events.
In some cases, electrochemiluminescent detection is not preferred.
In some cases, fluorescent detection is not preferred.
[0089] In an additional aspect, the fluidics devices of the
invention are micofluidics devices, filtration devices, or
purification or abstraction devices.
[0090] In some aspects, particularly when bubble removal is
desired, the fluidics devices receives the fluid sample through the
inlet port in an upright position of from 15.degree.-90.degree.
relative to horizontal and processes the fluid sample in a
15.degree.-90.degree. position relative to horizontal, and wherein
the receiving upright position and the processing upright position
are not necessarily the same.
[0091] In a further aspect, the invention provides racks for
carrying a plurality of fluidics devices of the invention, wherein
the rack is designed to position said devices relative to one
another having a pitch of about 9 mm between successive sample
reservoirs of said devices.
[0092] In an additional aspect, the invention provides devices that
are diaphragm-mediated two-stroke circulation and recirculation
devices mediated by passive valving.
[0093] In a further aspect, the invention provides diagnostic kits
comprising the devices of the invention, and optionally
reagents.
[0094] In an additional aspect, the invention provides methods of
determining the presence, absence and/or amount of analyte in a
sample, or analyte binding. The methods comprise providing a
fluidics device as outlined herein, adding a sample (usually
liquid) suspected of containing one or more of the analytes,
circulating and recirculating the sample across the array; and
detecting binding of the analytes to the ligands.
BRIEF DESCRIPTION OF THE DRAWINGS
[0095] FIG. 1A is a perspective view of a fully assembled fluidics
device (cartridge) according to one embodiment of the invention,
showing integral handle and cover, integral fill reservoir, pump
interface, and cap. Also shown is one of two detents to help align
cartridge upon insertion into a cartridge module and provide
tactile/audible feedback. FIG. 1B is an exploded view of the
individual components within the cartridge.
[0096] FIG. 2 is another exploded view of FIG. 1 showing PCB
(printed circuit board), valve/diaphragm laminate assembly, plate,
cover and cap, with visible sample addition fill reservoir,
serpentine channel, pump interface, bridge valves, and serpentine
electrode array on PCB board configured to interface with
serpentine channel in laminate assembly to thereby create one or
more fluid channels.
[0097] FIG. 3 is a perspective view of a partially assembled
cartridge embodiment (less cover) containing pump interface, sample
fill chamber/reservoir (with cap), alignment holes, and EEPROM.
[0098] FIG. 4 is a bottom perspective view of a cartridge plate
assembly embodiment (less cover, cap and PCB), including alignment
holes, void for receiving EEPROM, sample fill reservoir, sample
inlet port, and sample outlet port. Also shown are two valve seats,
one at the fill reservoir outlet and the second below the ports of
the oval recess (above and to the right of diaphragm), a bubble
stripping/fill reservoir, and diaphragm for interface with
pump.
[0099] FIG. 5 is a perspective view of a partially assembled
cartridge that contains the laminate assembly overlaying the PCB
board and that better shows the operational relation between bridge
valves, diaphragm, and channel.
[0100] FIG. 6 is a perspective view of a PCB board configured with
reference, auxiliary and working electrodes in serpentine
configuration, alignment holes and EEPROM. Not visible are the
traces and vias linking the individual electrodes to a
two-dimensional array of gold contact points on the underside of
the PCB board.
[0101] FIG. 7 shows the underside view of FIG. 6, including
connector pads and rectangular area showing the thermal interface
location for receipt of a temperature-controlled metal plate
thereover. There are also alignment features (cut-outs or "cookie
bites") shown to facilitate proper connection to the connector. The
connector pads are clamped into a detection device preferably using
a zero-insertion force (ZIF) mechanism. In another embodiment, the
connector pads may be concentrated in mass on a side facing ("edge"
connectors).
[0102] FIG. 8 shows individual modular laminate components of the
laminate assembly, each having features cooperative with and
complementary to each other when in operative use. The top piece is
an adhesive layer with voids for functional overlay over the middle
laminate which contains bridge valve slits and orifices that
cooperate with those on the lower laminate/adhesive to form
functional diaphragm, valves and channels. The lower piece is a
laminate covered on both sides with an adhesive layer, the bottom
of which mates with the PCB board.
[0103] FIG. 9 is a top perspective view of the plate, which houses
the sample fill reservoir, pump interface, void for receipt of an
EEPROM and oval recess over which is sealed a jumper laminate. Also
shown are raised bosses that facilitate engagement of plate to
cover.
[0104] FIG. 10 is a perspective view of a rack containing multiple
cartridges vertically aligned and stacked, with sample fill
reservoirs facing upward for receipt of liquid samples from a
liquid delivery system, such as a multi-pipetteman.
[0105] FIG. 11 is a sectional view of a fully assembled cassette
embodiment, showing cooperativity of the PCB board, laminate
assembly, plate, and cover to afford fluidic operation. Shown are
the channels, valving, diaphragm and pneumatic pump interface.
[0106] FIG. 12 is similar to FIG. 8, showing an exploded view of
the individual laminate and adhesive layers comprising the laminate
assembly.
[0107] FIG. 13A shows an assembled cartridge module and FIGS. 13B
and C respectively show the bottom and top hemispheres of the
module, including internal components and design. FIG. 13D shows
the buckle beam assembly, with one bracket displaced showing buckle
beams. FIG. 13E is a perspective view of the top of the module
showing connection to pump.
[0108] FIG. 14 shows an embodiment of a detection device for
housing multiple cartridges according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0109] Ease of use and cost considerations are driving systems into
a paradigm where inexpensive, disposable consumables are used
together with capital equipment. This is especially true in
analytic and diagnostic applications where concern over possible
contamination of one sample with another leads consumers to prefer
single-use devices where feasible. While the inventions discussed
here apply more generally to any system configuration and to
applications beyond diagnostics, this description will focus on the
use of a disposable device--a cartridge--that manipulates
sample-specific reagents in concert with separate, reused
instrumentation for diagnostic purposes.
[0110] Accordingly, in one embodiment the present invention is
directed to a fluidics device designed to analyze a plurality of
target analytes. In one embodiment the fluidics device of the
present invention includes a membranous diaphragm, one side of
which, in one embodiment, is coupled to a pump. The second side is
coupled to a flow channel, and a plurality of check valves for
fluidic coupling. The valves include a sealing surface that include
a valve seat allowing for for regulated flow of fluid through the
flow channel, and a flexible sealing structure for contacting the
valve seat and occluding fluid flow through the sealing structure
when in a first position and for promoting fluid flow through the
seal when in a second position. In one embodiment the diaphragm and
flexible sealing structure are integral to one or more layers of a
multilayer assembly. The plurality of check valves alternate
opening an closing in coordinated reciprocal fashion according to
alternating positive and negative forces exerted ion the
diaphragm.
[0111] In another embodiment the fluidics device includes a body.
The body includes a channel within it for transporting a fluid
sample. The channel includes a situs where an operation is
performed on one or more components in the fluid sample. In
addition the body includes an inlet port for receiving the fluid
sample. The inlet port is in fluid communication with the channel
and also in communication with an enclosed gaseous environment. The
body also includes an outlet port in fluid communication with the
inlet port such that upon operation of the device the fluid sample
is recirculated through the channel to release gaseous bubbles into
the enclosed gaseous environment while simultaneously allowing for
facilitated diffusion and performance of the operation on the one
or more components in the fluid sample.
[0112] Thus, the present disclosure provides compositions and
methods for detecting the presence or absence of target analytes in
samples. As will be appreciated by those in the art, the sample
solution may comprise any number of things, including, but not
limited to, bodily fluids (including, but not limited to, blood,
urine, serum, lymph, saliva, anal and vaginal secretions,
perspiration and semen, of virtually any organism, with mammalian
samples being preferred and human samples being particularly
preferred); environmental samples (including, but not limited to,
air, agricultural, water and soil samples); biological warfare
agent samples; research samples (i.e. in the case of nucleic acids,
the sample may be the products of an amplification reaction,
including both target and signal amplification as is generally
described in PCT/US99/01705, such as PCR amplification reaction);
purified samples, such as purified genomic DNA, RNA, proteins,
etc.; raw samples (bacteria, virus, genomic DNA, etc.); as will be
appreciated by those in the art, virtually any experimental
manipulation may have been done on the sample.
[0113] The methods are directed to the detection of target
analytes. By "target analyte" or "analyte" or grammatical
equivalents herein is meant any molecule or compound to be detected
and that can bind to a binding species, defined below. Suitable
analytes include, but are not limited to, small chemical molecules
such as environmental or clinical chemical or pollutant or
biomolecule, including, but not limited to, pesticides,
insecticides, toxins, therapeutic and abused drugs, hormones,
antibiotics, antibodies, organic materials, etc. Suitable
biomolecules include, but are not limited to, proteins (including
enzymes, immunoglobulins and glycoproteins), nucleic acids, lipids,
lectins, carbohydrates, hormones, whole cells (including
procaryotic (such as pathogenic bacteria) and eucaryotic cells,
including mammalian tumor cells), viruses, spores, etc.
Particularly preferred analytes are proteins including enzymes;
drugs, cells; antibodies; antigens; cellular membrane antigens and
receptors (neural, hormonal, nutrient, and cell surface receptors)
or their ligands.
[0114] In one embodiment, the target analyte is a protein. As will
be appreciated by those in the art, there are a large number of
possible proteinaceous target analytes that may be detected using
the present invention. By "proteins" or grammatical equivalents
herein is meant proteins, oligopeptides and peptides, derivatives
and analogs, including proteins containing non-naturally occurring
amino acids and amino acid analogs, and peptidomimetic structures.
The side chains may be in either the (R) or the (S) configuration.
In one embodiment, the amino acids are in the (S) or
L-configuration.
[0115] As discussed below, when the protein is used as a binding
ligand, it may be desirable to utilize protein analogs to retard
degradation by sample contaminants. Suitable protein target
analytes include, but are not limited to, (1) immunoglobulins,
particularly IgEs, IgGs and IgMs, and particularly therapeutically
or diagnostically relevant antibodies, including but not limited
to, for example, antibodies to human albumin, apolipoproteins
(including apolipoprotein E), human chorionic gonadotropin,
cortisol, a-fetoprotein, thyroxin, thyroid stimulating hormone
(TSH), antithrombin, antibodies to pharmaceuticals (including
antieptileptic drugs (phenytoin, primidone, carbariezepin,
ethosuximide, valproic acid, and phenobarbitol), cardioactive drugs
(digoxin, lidocaine, procainamide, and disopyramide),
bronchodilators (theophylline), antibiotics (chloramphenicol,
sulfonamides), antidepressants, immunosuppresants, abused drugs
(amphetamine, methamphetamine, cannabinoids, cocaine and opiates)
and antibodies to any number of viruses or bacteria outlined
below.
[0116] As will be appreciated by those in the art, a large number
of analytes may be detected using the present methods; basically,
any target analyte for which a binding ligand, described below, may
be made may be detected using the methods of the invention.
[0117] In one embodiment, the target analytes are nucleic acids. By
"nucleic acid" or "oligonucleotide" or grammatical equivalents
herein means at least two nucleotides covalently linked together. A
nucleic acid of the present invention will generally contain
phosphodiester bonds, although in some cases, as outlined below,
nucleic acid analogs are included that may have alternate
backbones, comprising, for example, phosphoramide (Beaucage et al.,
Tetrahedron 49(10):1925 (1993) and references therein; Letsinger,
J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem.
81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986);
Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem.
Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141
91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437
(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et
al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press), and peptide nucleic acid
backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895
(1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen,
Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all
of which are incorporated by reference). Other analog nucleic acids
include those with bicyclic structures including locked nucleic
acids, Koshkin et al., J. Am. Chem. Soc. 120:13252-3 (1998);
positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA
92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023,
5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al.,
Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J.
Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside &
Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series
580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S.
Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic &
Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular
NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose
backbones, including those described in U.S. Pat. Nos. 5,235,033
and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,
"Carbohydrate Modifications in Antisense Research", Ed. Y. S.
Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp
69-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of ETMs, or to increase the stability and half-life of
such molecules in physiological environments.
[0118] As will be appreciated by those in the art, all of these
nucleic acid analogs may find use in the present invention. In
addition, mixtures of naturally occurring nucleic acids and analogs
can be made; for example, at the site of conductive oligomer or ETM
attachment, an analog structure may be used. Alternatively,
mixtures of different nucleic acid analogs, and mixtures of
naturally occuring nucleic acids and analogs may be made.
[0119] The nucleic acids may be single stranded or double stranded,
as specified, or contain portions, of both double stranded or
single stranded sequence. The nucleic acid may be DNA, both genomic
and cDNA, RNA or a hybrid, where the nucleic acid contains any
combination of deoxyribo- and ribo-nucleotides, and any combination
of bases, including uracil, adenine, thymine, cytosine, guanine,
inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc. One
embodiment utilizes isocytosine and isoguanine in nucleic acids
designed to be complementary to other probes, rather than target
sequences, as this reduces non-specific hybridization, as is
generally described in U.S. Pat. No. 5,681,702. As used herein, the
term "nucleoside" includes nucleotides as well as nucleoside and
nucleotide analogs, and modified nucleosides such as amino modified
nucleosides. In addition, "nucleoside" includes non-naturally
occurring analog structures. Thus for example the individual units
of a peptide nucleic acid, each containing a base, are referred to
herein as a nucleoside.
[0120] Thus, in one embodiment, the target analyte is a target
sequence. The term "target sequence" or "target nucleic acid" or
grammatical equivalents herein means a nucleic acid sequence on a
single strand of nucleic acid. The target sequence may be a portion
of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including
mRNA and rRNA, or others. As is outlined herein, the target
sequence may be a target sequence from a sample, or a secondary
target such as a product of an amplification reaction, etc. It may
be any length, with the understanding that longer sequences are
more specific. As will be appreciated by those in the art, the
complementary target sequence may take many forms. For example, it
may be contained within a larger nucleic acid sequence, i.e. all or
part of a gene or mRNA, a restriction fragment of a plasmid or
genomic DNA, among others. As is outlined more fully below, probes
are made to hybridize to target sequences to determine the presence
or absence of the target sequence in a sample. Generally speaking,
this term will be understood by those skilled in the art. The
target sequence may also be comprised of different target domains;
for example, a first target domain of the sample target sequence
may hybridize to a capture probe or a portion of capture extender
probe, a second target domain may hybridize to a portion of an
amplifier probe, a label probe, or a different capture or capture
extender probe, etc. The target domains may be adjacent or
separated as indicated. Unless specified, the terms "first" and
"second" are not meant to confer an orientation of the sequences
with respect to the 5'-3' orientation of the target sequence. For
example, assuming a 5'-3' orientation of the complementary target
sequence, the first target domain may be located either 5' to the
second domain, or 3' to the second domain.
[0121] Suitable target analytes include biomolecules associated
with: (1) viruses, including but not limited to, orthomyxoviruses,
(e.g. influenza virus), paramyxoviruses (e.g respiratory syncytial
virus, mumps virus, measles virus), adenoviruses, rhinoviruses,
coronaviruses, reoviruses, togaviruses (e.g. rubella virus),
parvoviruses, poxviruses (e.g. variola virus, vaccinia virus),
enteroviruses (e.g. poliovirus, coxsackievirus), hepatitis viruses
(including A, B and C), herpesviruses (e.g. Herpes simplex virus,
varicella-zoster virus, cytomegalovirus, Epstein-Barr virus),
rotaviruses, Norwalk viruses, hantavirus, arenavirus, rhabdovirus
(e.g. rabies virus), retroviruses (including HIV, HTLV-1 and -11),
papovaviruses (e.g. papillomavirus), polyomaviruses, and
picomaviruses, and the like; and (2) bacteria, including but not
limited to, a wide variety of pathogenic and non-pathogenic
prokaryotes of interest including Bacillus; Vibrio, e.g. V.
cholerae; Escherichia, e.g. Enterotoxigenic E. coli, Shigella, e.g.
S. dysenteriae; Salmonella, e.g. S. typhi; Mycobacterium e.g. M.
tuberculosis, M. leprae; Clostridium, e.g. C. botuliniin, C.
tetani, C. difficile, C. perfringens; Cornyebacterium, e.g. C.
diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae;
Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae;
Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. G.
lamblia Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida;
Chlamydia, e.g. C. trachonmatis; Bordetella, e.g. B. pertussis;
Treponema, e.g. T. palladium; and the like.
[0122] Other suitable target analytes include, but are not limited
to, (1) enzymes (and other proteins), including but not limited to,
enzymes used as indicators of or treatment for heart disease,
including creatine kinase, lactate dehydrogenase, aspartate amino
transferase, troponin T, myoglobin, fibrinogen, cholesterol,
triglycerides, thrombin, tissue plasminogen activator (tPA);
pancreatic disease indicators including amylase, lipase,
chymotrypsin and trypsin; liver function enzymes and proteins
including cholinesterase, bilirubin, and alkaline phosphotase;
aldolase, prostatic acid phosphatase, terminal deoxynucleotidyl
transferase, and bacterial and viral enzymes such as HIV protease;
(2) hormones and cytokines (many of which serve as ligands for
cellular receptors) such as erythropoietin (EPO), thrombopoietin
(TPO), the interleukins (including IL-1 through IL-17), insulin,
insulin-like growth factors (including IGF-1 and -2), epidermal
growth factor (EGF), transforming growth factors (including TGF-n
and TGF-P), human growth hormone, transferrin, epidermal growth
factor (EGF), low density lipoprotein, high density lipoprotein,
leptin, VEGF, PDGF, ciliary neurotrophic factor, prolactin,
adrenocorticotropic hormone (ACTH), calcitonin, human chorionic
gonadotropin, cotrisol, estradiol, follicle stimulating hormone
(FSH), thyroid-stimulating hormone (TSH), leutinzing hormone (LH),
progeterone and testosterone; and (3) other proteins (including
(Y-fetoprotein, carcinoembryonic antigen CEA, cancer markers,
etc.).
[0123] Suitable target analytes include carbohydrates, including
but not limited to, markers for breast cancer (CA15-3, CA 549, CA
27.29), mucin-like carcinoma associated antigen (MCA), ovarian
cancer (CA125), pancreatic cancer (DE-PAN-2), prostate cancer
(PSA), CEA, and colorectal and pancreatic cancer (CA 19, CA 50,
CA242).
[0124] Other suitable target analytes include metal ions,
particularly heavy and/or toxic metals, including but not limited
to, aluminum, arsenic, cadmium, selenium, cobalt, copper, chromium,
lead, silver and nickel.
[0125] In one embodiment, the methods of the invention are used to
detect pathogens such as bacteria. In this embodiment, target
sequences include rRNA, as is generally described in U.S. Pat. Nos.
4,851,330; 5,288,611; 5,723,597; 6,641,632; 5,738,987; 5,830,654;
5,763,163; 5,738,989; 5,738,988; 5,723,597; 5,714,324; 5,582,975;
5,747,252; 5,567,587; 5,558,990; 5,622,827; 5,514,551; 5,501,951;
5,656,427; 5.352.579; 5,683,870; 5,374,718; 5,292,874; 5,780,219;
5,030,557; and 5,541,308, all of which are expressly incorporated
by reference.
[0126] In one embodiment nucleic acid sequencing methods are used.
Sequencing methods are described in U.S. Ser. Nos. 09/626,096,
filed Jul. 26, 2000, 09/847,113, filed May 1, 2001, 10/137,710,
filed Apr. 30, 2002, 10/336,255, filed Jan. 2, 2003 and 10/823,502,
filed Apr. 12, 2004, all of which are expressly incorporated herein
by reference.
[0127] As will be appreciated by those in the art, a large number
of analytes may be detected using the present methods; basically,
any target analyte for which a binding ligand, described below, may
be made may be detected using the methods of the invention. While
many of the techniques described below exemplify nucleic acids as
the target analyte, those of skill in the art will recognize that
other target analytes can be detected using the same systems.
[0128] If required, the target analyte is prepared using known
techniques. For example, the sample may be treated to lyse the
cells, using known lysis buffers, electroporation, etc., with
purification and/or amplification as needed, as will be appreciated
by those in the art. When the target analyte is a nucleic acid, the
target sequence may be amplified as required; suitable
amplification techniques are outlined in PCT US99/01705, hereby
expressly incorporated by reference. In addition, techniques to
increase the amount or rate of hybridization can also be used; see
for example WO 99/67425 and U.S. Ser. Nos. 09/440,371 and
60/171,981, all of which are hereby incorporated by reference.
[0129] The samples comprising the target analytes can be added to
the fluidics devices described herein. By "fluidics device" is
meant device comprising a substrate, at least one channel, inlet
ports and outlet ports as well as valves. The fluidics device of
this disclosure can take on numerous configurations.
[0130] By "cartridge" herein is meant a casing or housing for the
biochip. As outlined herein, and as will be appreciated by those in
the art, the cartridge can take on a number of configurations and
can be made of a variety of materials. Suitable materials include,
but are not limited to, fiberglass, teflon, ceramics, glass,
silicon, mica, plastic (including acrylics, polystyrene and
copolymers of styrene and other materials, polypropylene,
polyethylene, polybutylene, polycarbonate, polyurethanes,
Teflon.TM., and derivatives thereof, etc.), etc. Particularly
preferred cartridge materials are plastic (including polycarbonate
and polyproplylene) and glass.
[0131] As will be appreciated by those in the art, the cartridge
can comprise a number of components, including reaction chambers,
inlet and outlet ports, heating elements including thermoelectric
components, RF antennae, electromagnetic components, memory chips,
sealing components such as gaskets, electronic components including
interconnects, multiplexers, processors, etc.
[0132] In one embodiment, the cartridge comprises a reaction
chamber. Generally, the reaction chamber comprises a space or
volume that allows the contacting of the sample to the biochip
array. The volume of the reaction chamber can vary depending on the
size of the array and the assay being done. In general, reaction
chamber ranges from 1 nL to about 1 mL, with from about 1 to about
250 .mu.l being preferred and from about 10 to about 100 .mu.l
being especially preferred. In some embodiments, to avoid the
introduction of air bubbles into the reaction chamber (which can be
disruptive to detection), the reaction chamber is less than the
size of the sample to be introduced, to allow a slight overflow and
thus ensure that the reaction chamber contains little or no
air.
[0133] In one embodiment, the biochip cartridge can be configured
to include additional chambers that can used for any number of
different reactions, such as sample preparation, cell lysis, rare
target capture/concentration, sample clean-up, nucleic acid
amplification, including PCR, post-amplification clean-up, sample
concentration, reagent storage, mixing baffles/devices, etc. In
other embodiments, the reaction chamber may be configured for other
types of reactions as generally described below.
[0134] In one embodiment, the biochip cartridge reaction chamber is
configured to include at least one nucleic acid amplification
chamber. However, multiple amplification chambers may be used. That
is, a cartridge may comprise from about 1 to about 10 or more
chambers, with 2, 3, 4, 5, 6, 7, 8 or 9 also being preferred.
[0135] In one embodiment, the biochip cartridge reaction chamber is
configured to include at least one PCR chamber. However, multiple
PCR chambers may be used. That is, a cartridge may comprise from
about 1 to about 10 or more chambers, with 2, 3, 4, 5, 6, 7, 8 or 9
also being preferred.
[0136] In one embodiment, the chamber of the cartridge should be
made from biocompatible materials. In particular, materials that
provide a surface that retards the non-specific binding of
biomolecules, e.g. a "non sticky" surface, are preferred. For
example, when the reaction chamber is used for PCR or amplification
reactions a "non sticky" surface prevents enzymatic components of
the reaction mixture from sticking to the surface and being
unavailable in the reaction. In addition, the biocompatible
properties of the chamber may be improved by minimizing the surface
area.
[0137] Biocompatible materials include, but are not limited to,
plastic (including acrylics, polystyrene and copolymers of styrene
and other materials, polypropylene, polyethylene, polybutylene,
polyimide, polycarbonate, polyurethanes, Teflon..TM.., and
derivatives thereof, etc.) Other configurations include
combinations of plastic and printed circuit board (PCB; defined
below). For example at least one side of the chamber is printed
circuit board, while one or more sides of the chamber are made from
plastic. In one embodiment, three sides of the chamber are made
from plastic and one side is made from printed circuit board. In
addition, the chambers, channels, valves, pumps, etc. of the
systems described herein may be coated with a variety of materials
to reduce non-specific binding. These include proteins such as
caseins and albumins (bovine serum albumin, human serum albumin,
etc.), parylene, other polymers, etc.
[0138] In one embodiment, the reaction chamber of the cartridge
comprises an inlet port for the introduction of the sample to be
analyzed. Depending on the reaction being run, multiple inlet ports
may be used, that may feed from a variety of storage chambers or
from the outside of the chamber. The inlet port may optionally
comprise a seal to prevent or reduce the evaporation of the sample
or reagents from the reaction chamber. In one embodiment, the seal
comprises a gasket, or valve through which a pipette or syringe can
be pushed. The gasket or valve can be rubber or silicone or other
suitable materials, such as materials containing cellulose.
[0139] The reaction chamber can be configured in a variety of ways.
In one embodiment, the reaction chamber is configured to minimize
the introduction or retention of air bubbles or other sample
impurities. Thus for example, assuming that the cartridge is held
in an upright angle, the inlet port allows the flow of fluid sample
into the "bottom" of the reaction chamber, to allow the escape of
air or fluid through the "top" of the reaction chamber, for example
through an outlet port. Thus the fluid sample flows up into the
reaction chamber and contacts the array. Thus, in one embodiment,
the reaction chamber further comprises an outlet port to allow air
or excess sample to exit the reaction chamber. In some embodiments,
the outlet port vents to either a waste storage well, as is further
described below, to an external surface of the chip or cartridge,
or, in one embodiment, back into the inlet port. Thus for example
one embodiment utilizes a system wherein the exit port vents to the
inlet port, preferably above the point of loading. For example,
when a pipette is used to load the cartridge, the tip of the
pipette extends below the exit port, such that air from the exit
port is not introduced into the reaction chamber. In addition, the
materials of the cartridge housing and biochip can be chosen to be
similar in hydrophobicity or hydrophilicity, to avoid the creation
of air bubbles.
[0140] In one embodiment, an anti-siphon vent is used to prevent
liquid from being sucked back into a chamber as a result of the
negative pressure generated when an air pump heater is turned off.
For example, a anti-siphon vent comprising a paraffin valve and an
open port can be constructed between the reaction chamber and an
air pump.
[0141] In addition, in one embodiment, the reaction chamber/inlet
and/or outlet ports optionally include the use of valves. For
example, a semi-permeable membrane or filter may be used, that
preferentially allows the escape of gas but retains the sample
fluid in the chamber. For example, porous teflons such as
Gortex.TM. allow air but not fluids to penetrate.
[0142] In one embodiment, a reaction chamber in the biochip
cartridge (such as a PCR chamber) has one or more valves
controlling the flow of fluids into and out of the chamber. The
number of valves in the cartridge depends on the number of channels
and chambers. Alternatively, the biochip cartridge is designed to
include one or more loading ports or valves that can be closed off
or sealed after the sample is loaded. It is also possible to have
multiple loading ports into a single chamber; for example, a first
port is used to load sample and a second port is used to add
reagents. In these embodiments, the biochip cartridge may have a
vent. The vent can be configured in a variety of ways. In some
embodiments, the vent can be a separate port, optionally with a
valve, that leads out of the reaction chamber. Alternatively, the
vent may be a loop structure that vents liquid and/or air back into
the inlet port.
[0143] As will be appreciated by those in the art, a variety of
different valves may be used. Microvalves can be categorized into
two major types: passive microvalves (without actuation) and active
microvalves (with an actuation). Generally, active microvalves
couple a flexible diaphragm to a thermopneumatic, piezoelectric,
electrostatic, electromagnetic, bimetallic actuator. Additional
valves find use in the invention and are described in more detail
in US Pub. No. 2007/0098600 which is expresslly incorporated herein
by reference for disclosure describing valves.
[0144] Accordingly, in a first aspect, the invention features a
fluidics device, preferably multilaminate, having a diaphragm and a
plurality of check valves contiguous with said diaphragm through
one or more flow channels. The diaphragm is acted upon by a pump,
which in one embodiment is extraneous to the device itself but
nevertheless engageably interfaceable therewith. In one embodiment
the check valves are passive "bridge" valves as described herein,
which essentially comprise more or less parallel slits in a
flexible laminate sheet which can allow displacement of the section
therebetween upon application of force, thereby routing fluid flow
along a new path, in one embodiment, in a direction more or less
parallel to the laminate portion flanking the slits. In one
embodiment the system is pump or vacuum-driven.
[0145] By "multilaminate" is meant prepared from multiple, e.g.
more than one, layers. Polyethyleneterepthalate (PET) and etched
polytetrafluoroethylene (PTFE) find particular use as construction
materials, but other materials as discussed herein can also be
used, with the overall multilaminate comprising a single material
or multiple different materials. In general, as described herein,
at least 2, 3, 4, 5 or more layers are used, as is shown in the
figures. Generally, one or more of the layers have vias, e.g.
shaped holes therein, such that when sandwiched between two
additional layers (e.g. a "top" and a "bottom") they form the
channels of the chips as described herein. Similarly, one of the
layers generally includes the array of electrodes (which as
outlined herein can be within a detection chamber as a rectilinear
array, within a detection chamber comprising a serpentine (or other
geometries) channel, etc.), which is laminated to a layer defining
a channel. As described herein, the layers may be attached in a
wide variety of ways, including adhesives (pressure sensitive, heat
sensitive, etc.). In addition, as outlined herein, the individual
layers can also contain features for attachment, like posts or pegs
that couple to corresponding holes in another layer.
[0146] By "diaphragm" is meant a flexible seal that flexes in a
positive and negative fashion. In one emboidment the diaphragm is
positioned in a first chamber or separates multiple chambers. In
one embodiment the diaphragm is within an enclosed system. Thus,
upon movement of the diaphragm ni a positive or negative direction,
either air or materials within the enclosed system move in response
to the movement of the diaphragm. In one embodiment the diaphragm
includes a magnet. This allows for movement of the diaphragm by
controlling movement of the magnet, which can be accomplished
electrically.
[0147] By "flow channels" is meant a channel through which a liquid
flows in the cartridge of this disclosure. The dimensions of the
flow channel are significant in that the linear speed of the fluid
over the electrodes is dependent on the channel cross section.
Additionally, the channel height and width must be great enough to
allow any bubbles to freely flow through the channel and eventually
be trapped/cleared in the reservoir. Therefore, it is important to
design a channel that is small enough to provide sufficient linear
velocity but not too restrictive with respect to bubble movement.
Another consideration is that the lower channel dimensions will
utilize less analyte solution, often a desired characteristic.
Channel widths of may be from 0.020-0.100'', more preferably,
0.025-0.080'' and more preferably from 0.030-0.060''. Channel
heights may be from 0.002-0.020'', more preferably from
0.004-0.015'' and more preferbly from 0.006-0.015''. For example,
channel widths 0.030-0.060'' and heights of 0.006''-0.014'' were
tested as compatible ranges, with the preferred dimensions being a
width of 0.040'' and height of 0.010''.
[0148] Valves are described above. In one embodiment the check
valve is a "bridge valve" as decribed herein. In some embodiments,
the bridge valves are contained in the same laminar piece or
layered composite as the diaphragm.
[0149] In one embodment the diaphragm is in a first chamber such as
a reaction chamber and a secone chamber such as a detection chamber
may be separate. In some embodiments channels connect the first and
second chambers as described herein. Alternatively, the channel
itself is used for detection.1 In this embodiment the detection
chamber or the channel, when it is used for detection, includes
ligands for binding target analytes as described herein. Also as
described herein the ligands may form an array on a substrate in
the detecion chamber or channel.
[0150] The channels are preferably defined by two or more laminate
layers and preferably elongated, most preferably serpentine.
Preferably, inside the channel are surfaces containing affixed
ligand(s), which in turn abstract or bind specific complement,
anti-ligand or analyte from a fluid sample that is pumped through
the channel and across those surfaces. Preferably the abstracting
surfaces are configured in a dimension that does not waste surface
area space, the effect of which is to conserve volume and
sensitivity per sample volume, and thereby allow for or facilitate
miniaturization.
[0151] There are two primary means by which fluid can be moved in
the biochip cartridge. These are: (1) through the use of a pump
that pushes the fluid in or out; or, (2) by suction that pulls
fluid in or out of the chamber.
[0152] Generally, a device such as a moving piston is used to
create suction, however cooling of gases, vacuum chambers and gas
consuming reactions can be used. When suction is used to move
liquid in or out of the chamber, a vacuum may be created elsewhere
in the system.
[0153] In some embodiment the pump can be on the chip or off the
chip. By "on chip" is meant that the pump is integral to the
cartridge itself. by "off chip" is meant that the chip is separate
from and not integral to the chip. Basically, two major groups of
pumps, classified based on different pump mechanisms (i.e.,
actuation), can be use in the present invention: membrane actuated
(i.e., mechanical) and non-membrane actuated pumps. Membrane
actuated pumps can be further divided into three types:
piezoelectric, electrostatic, and thermopneumatic. Non-membrane
pumping principles include electrohydrodynamic, electroosmotic,
traveling wave, diffuser, bubble, surface wetting, rotary, etc.
[0154] In one embodiment, an "air pump" is used to move the liquid
out of the PCR chamber. In this embodiment, a chamber of air is
incorporated in the chip with an "on chip" heater. When the heater
is turned on, the air in the chamber expands according to PV=nRT.
In some embodiments, the air pump is incorporated into the
cartridge.
[0155] Preferably, heaters (as are also described below) are
incorporated into the middle of the chip. In some embodiments, more
than one heater is incorporated in a chip to create "heater zones".
Air chambers or pockets are located over the heater zones. The air
chambers are connected to the reaction chamber via a channel that
runs up to the top of the reaction chamber with a valve or a plug
blocking it off. When the air is heated, it expands. The resulting
build up in pressure forces the valve or plug to move out of the
way, thereby forcing the liquid out of the chamber via an outlet
port.
[0156] Other ways of moving liquid out of the reaction chamber or
reaction chamber include using a low boiling liquid in place of
air. In this embodiment, the low boiling liquid expands when heated
and displaces the liquid contained in the reaction chamber.
Alternatively, a chemical reaction may be used to move liquid out
of the reaction chamber. For example, the chemical reaction used to
expand car air bags may be used to move liquid out of the reaction
chamber, or other reactions in which gases are generated.
[0157] Other types of pumps that can be used include syringe driven
pumps. These pumps can be actuated either by expanding air behind
the syringe or by mechanical means. For example, TiNi alloys,
nitinol wire, or "shape memory metals" can be used to mechanically
actuate a syringe driven pump. By "TiNi alloys", "nitinol wire" or
"shape memory metals" herein is meant materials that when heated
above a certain transition temperature contract (i.e., usually up
to 3 to 5% over the original length of the metal), thereby changing
shape. Other materials that change shape upon heating include shape
memory plastics.
[0158] Pumps also may be created using spring loaded pistons. In
this embodiment, a spring that can be released is compressed or
restrained within the body of the cartridge. For example, wax may
be used to hold a spring in its compressed state. Upon heating, the
wax is melted, and the spring is released, thereby generating
sufficient force to move a piston and displace liquid. Other
versions include incorporating materials that change from solids to
liquids at a given transition temperature, or moving a mechanical
blockade from the spring's pathway.
[0159] Pumps that utilize PZT driven actuations are also known and
may be incorporated int this invention. By "PZT" herein is meant a
material comprised of lead, zirconium and titanium which upon
application of a voltage undergoes a rearrangement of the crystal
lattice and generates a force and a displacement. This so called
piezoelectric effect can be used to constrict and expand a pump
chamber and result in a net movement of liquid. Other materials
like shape memory alloys that under a change in shape upon
application of a current such that the temperature of the metal is
raised above a certain transition temperature can also be used.
[0160] In addition, commercially available micro pumps may be used
in to move liquid from one location to another in the cartridge.
Examples of commercially available pumps include, moulded plastic
micro pumps available from IMM (see liganews@imm.uni-mainz.de),
thin film shape alloy microactuators (TiNi Alloy Company, San
Leandro, Calif.), silicon micro pumps (see M. Richter & J.
Kruckow, aktorik/paper/2000jahresbericht/Paper2, 16.11.00).
[0161] In addition, based on the geometry of the chamber, air can
be used to push liquid out of the reaction chamber or mix liquids
within the reaction chamber. Whether the air pumps the fluid or
bubbles through to generate a mixing effect is determine by the
relative size of the bubble, the geometry of the chamber/channel
and the surface tension of the liquid. Larger air-liquid interfaces
tend to favor mixing over pumping. Mixing of liquid within the
biochip cartridge can occur by pumping the liquid back and forth in
the biochip cartridge.
[0162] In one embodiment, flow-induced mixing is used to induce
convectional flow. Preferably, this is used in a vertical system,
such that fluid gravity may be used to induce convectional flow.
The convectional flow results in bulk fluid mixing between two
liquid solutions. In addition, meniscus recirculation mixing can be
used to induce circulation flow (Anderson, et al., (1998)
Solid-State Sensor and Actuator Workship, Hilton Head Island, June
9-11, pp 7-10; incorporated herein by reference in its
entirety).
[0163] In one embodiment, mixing is accomplished by creating a
thermal gradient across a chip. For example, a thermal gradient may
be created by heating the bottom of the chip to 65.degree. C. and
cooling the top of the cartridge cover to 10.degree. C. This can be
accomplished by placing the chip between two peltier heaters, or by
using an imbedded heater and a single peltier or other
thermoelectric cooling devices.
[0164] In one embodiment, mixing is accomplished by recirculating
liquid in a given chamber using an on chip or "off chip" pump
attached to a chip.
[0165] In alternative embodiments, mixing is accomplished by
recirculating liquid using a micro disk-pump, such as a plastic
disk embedded with a magnetic steel bar. Rotation of the disk pump
may be achieved using an external magnetic filed provided by a
standard stirrer or custom built with multiple fields. See also
U.S. Ser. No. 60/308,169, filed Jul. 26, 2001 and a provisional
application by Gallagher, et al., entitled "System and methods for
mixing within a microfluidic chamber", filed Jul. 11, 2002; both of
which are incorporated by reference in their entirety.
[0166] In other embodiments, biochannel based mixing can be used to
enhance hybridization rates. In this embodiment, a bubble is
intentionally introduced into one corner of the chip. By
alternately expanding and contracting the bubble volume via the
application of heat from either an in chip or off chip heat source,
mixing occurs as a result of the pressure flow created by changing
the volume of the bubble within the chip. Alternatively, resonance
induced mixing of bubbles can be done using PZT devices as
well.
[0167] In some embodiments, mixing may be accomplished using
non-contact mixing technologies like that describe by Covaris,
Inc.
[0168] In one embodiment, heaters are incorporated onto or into the
chip, to allow "on chip" heating (in addition, as described below,
"off chip" thermocontrollers within the device may also be used).
In this embodiment, the reaction chamber is designed to maximize
thermal conductivity between the chamber and the heater or
themocontroller. Generally, designs that minimize thermal mass
(i.e., making the surface of the chamber in contact with the heat
source as thin as possible), impose certain geometric constraints
to ensure the complete removal of liquid from the chamber,
incorporate materials that are good thermal conductors (i.e.,
metals), and thermally isolate the chamber from the rest of the
chip are preferred. Often one makes a trade off between minimizing
surface to volume ratios to reduce surface area for the
non-specific binding of biological components and maximizing
surface-to-volume ratis in order to obtain rapid heat transfer
rates for heating and cooling.
[0169] In one embodiment, air pockets or vents are used to
thermally isolate the amplificationchamber from the rest of the
chip. That is, the there is a break in the continuity of the
cartridge around the amplification chamber.
[0170] In one embodiment, thermally conductive materials are
incorporated into or below the reaction chamber, forming hybrid
chambers. For example, by using "layers" of different materials,
effective heaters are constructed. Thus for example, one embodiment
utilizes one or more resistive heaters in the form of resistive
metallic inks can be applied to a first layer of PC board. These
heaters are powered by interconnects. In one embodiment, a thin
sheet of a thermally conductive material, preferably a metal such
as copper, is applied, to allow even heat distribution. In one
embodiment, the copper layer is then coated with a thin layer of
biocompatible material, such as plastic. See FIG. 10A.
[0171] The total thickness of the hybrid chamber may vary from a
few microns to millimeter dimension. A preferred thickness is
approximately 200 microns.
[0172] In one embodiment, multiple thermal heaters are incorporated
into the device to allow for the creation of multiple thermal
zones. The temperature in the respective zones is maintained via
either active or passive control. Frequently, thethermal
connectivity of the cartridge materials are taken into account
during the design. In one embodiment, a chip may contain a thermal
heater in the detection chamber of the cartridge in order to
maintain the temperature of the detection chamber as well as
constructing unique temperature zones in another part of device. In
one embodiments, these temperature zones may be maintained to allow
an enzymatic reaction to run efficiently. In another embodiment,
multiple temperature zones may be maintained to simulate the
temperatures normally used during PCR heat cycling. In order to
effect the necessary temperature, the liquid can be maintained
stationary and the temperature of the amplification chamber cycled
(i.e. 95-55-72), alternatively, the liquid can be pumped over
different temperature zones in order to obtain heat cycling. This
embodiment can be realized in different material substrates such as
glass, plastic, ceramic and PCB.
[0173] Similarly, there may be portions of the substrate that
require heating, and those that do not. Thus more than one heater
may be incorporated into the substrate. Similarly, these thermal
zones may or may not be thermally isolated from other parts of the
substrate. For example, PC board is significantly thermally
insulative, and thus just putting distance between the heaters and
thermal zones and the areas of the substrate that do not require
heating may be sufficient. In other embodiments, thermally
insulative materials may be incorporated. For example, when the
substrate is a ceramic material, thermal isolation may be
accomplished by cutting out sections of the ceramic substrate such
that solid regions of ceramic are separated from one another by a
"cut out".
[0174] Other embodiments include the incorporation of temperature
sensors into the substrate such that the temperature throughout the
board can be monitored. In one embodiment, temperature sensors are
created using resistive devices, including silicon diodes. Other
embodiments include the use of capillary thermostats and limiters,
such as those offered by Thermodisc.
[0175] As will be appreciated by those in the art, there are a
variety of reaction chamber geometries which can be used in this
way. Generally having the intersection of the inlet port and the
reaction chamber be at the "bottom" of the cartridge, with a small
aperture, with the reaction chamber widening, is preferred. In
addition, the "top" of the reaction chamber may narrow, as well.
Thus, preferred embodiments for the size and shape of the reaction
chamber allow for smooth loading of the reaction chamber. Preferred
embodiments utilize reaction chamber geometries that avoid the use
of sharp corners or other components that serve as points for
bubble formation.
[0176] In addition, in some embodiments, the reaction chamber can
be configured to allow mixing of the sample. For example, when a
sample and a reagent are introduced simultaneously or separately
into the chamber, the inlet port and/or the reaction chamber can
comprise weirs, channels or other components to maximize the mixing
of the sample and reagent. In addition, as is outlined below, the
reaction may utilize magnetic beads for mixing and/or
separation.
[0177] In one embodiment, the cartridge comprises a sealing and/or
venting mechanism to prevent the cartridge from exploding due to a
build up in pressure during a reaction, or to prevent leakage of
the sample or reagents onto other parts of the substrate,
particularly (in the case of electronic detection) onto electronic
interconnects. As will be appreciated by those in the art, this may
take on a variety of different forms. In one embodiment, there is a
gasket between the biochip substrate comprising the array and the
cartridge, comprising sheets, tubes or strips. Alternatively, there
may be a rubber or silicone strip or tube used; for example, the
housing may comprise an indentation or channel into which the
gasket fits, and then the housing, gasket and chip are clamped
together. Furthermore, adhesives can be used to attach the gasket
to the cartridge, for example, a double sided adhesive can be used;
for example, silicone, acrylic and combination adhesives can be
used to attach the gasket to the biochip, which is then clamped
into the cartridge as described herein.
[0178] In embodiments where the surfaces are electrodes, each
electrode surface preferably occupies a majority of the channel
width and is positioned in series relative to other electrodes,
with a channel height thereover that allows fluid to flow
thereacross through the channel.
[0179] In some embodiments, the individual laminate pieces are
substantially planar and "stack" to thereby form the multilaminate
device and internal features in operable form. In other
embodiments, the individual laminate pieces are substantially
nonplanar or modular, and still cooperatively stack or interface,
e.g., like Ruffles.RTM. or Pringles.RTM. potato chips.
[0180] In a second aspect, the invention features a method of
constructing a multilaminate fluidics device by mating/conjoining
the individual laminate pieces noted above. This is preferably
accomplished by stacking and fusing or otherwise sealing the
individual pieces together to thereby form a device having one or
more functional chambers, channels, diaphrams and/or valves, etc.
therein.
[0181] In yet a third aspect, the invention features a method of
using a multilaminate fluidics device as above by adding a fluid
sample thereto and interfacing with a pump mechanism (e.g., syringe
pneumatic, hydrolic, thermal, or electromechanical) and magnetic,
electronic, or other detection device (e.g., colorimetric,
electrochemical, isotopic, densitometric, etc.), and prectifying
flow through the device so that the sample can be acted upon or
analyzed, e.g., by a laser or detector in one or more downstream
channels or chambers.
[0182] In some embodiments, flow is preferably of a two-stroke
design wherein the diaphragm is periodically moved back and forth
or up and down to actuate/rectify directional fluid flow across
cooperating tandem valves having the diaphragm fluidly coupled
therebetween. In operation, one valve is substantially closed when
the other is substantially open, depending on stroke, and
vice-versa.
[0183] In some embodiments the device is a disposable one, and
reversibly engageable with a pump and/or electronic stimulation or
detection component that can be used over and over again, e.g., a
laser and/or an optical, voltametric, amperometric, and/or thermal
reader.
[0184] In some embodiments, the device is used for in vitro
diagnostics.
[0185] In some embodiments, the device is used with or has
integrated a filtration, purification, separation, and/or mixing
means as known in the art.
[0186] In a fourth aspect the invention features a single laminar
sheet bearing one or more of a diaphragm, valving, and/or channels.
This sheet can be a component of the first aspect. In embodiments,
the sheet is formed of either PET, polypropylene, ultra high
molecular weight polyethylene, low density polyethylene, high
density polyethylene, linear low density polyethylene and/or
Teflon.RTM., and is preferably 5 mm or less in thickness, more
preferably 2 mm or less in thickness, and most preferably 1 mm or
less in thickness. These sheets may be present as part of a large
roll of individual sheets of identical or complementary dimension.
The sheets may be individually machined, molded, stamped,
chemically-etched and/or laser ablated to carry the individual
features noted. The individual sheets may also be perforated or
otherwise rendered separable from the roll to be thereafter
incorporated into a larger multilaminate fluidics device according
to the invention. One can readily envision an automated or
semi-automated procedure whereby a uniform roll of laminate
undergoes stamping, machining, molding, etching, welding and/or
ablation to endow microfluidics features in a volume batching
format using standard methodologies and capabilities known in the
art.
[0187] In a fifth aspect the invention features a device having
inlet and outlet ports that are joined to effectively re-circulate
sample while simultaneously facilitating gas removal and thereby
facilitating molecular diffusion and efficiency of the microfluidic
system. This aspect can be combined with any of the preceding
aspects and embodiments as appropriate, e.g., by suitable injection
molding, machining, stamping, etching, ablation, and mated sealing
of individual laminates using adhesives, gaskets, clamps, solvent
bonding, ultrasonic welding, etc. Preferably, gas removal is
accomplished by circulating the fluid sample past a bubble trap or
enclosed gaseous environment located above the fluid flow path.
[0188] In some embodiments, the multilaminate device processes (or
are designed to process) no more than 1 mL of fluid/analyte sample
at a time. In some other embodiments, no more than 150 uL of fluid
is processed at a time. In still others, preferably no more than
about 100 uL or less is processed at a time.
[0189] In multilaminate embodiments, the individual pieces may
optionally be held together and sealed by pressure sealing adhesive
sheets, welding, and/or using conformable gasket-like material such
as silicone or silicone sheeting.
[0190] In some embodiments, one or more channels or chambers are
formed by grooves, cuts or recesses in one or more of the
cooperating laminate sheets, e.g., as provided by a die-stamp,
chemical etching or laser ablation technique, as those techniques
are commonly understood in the art.
[0191] In some embodiments, biological binding partners such as
proteins, peptides, antibodies, nucleic acid and
polynucleotides/oligonucleotides are preferably attached to
surfaces in the system, e.g., on electrodes, which are preferably
contained in some of the channels or chambers that receive the
liquid samples upon flow through the device. Preferably those
samples are recirculated, which improves binding efficiency and
result by simultaneously augmenting diffusion and effecting mixing,
washing and shear strain to overcome problems associated with
laminar flow.
[0192] A continuous flow rate of about 10-40 uL/sec in connection
with the dimensions used herein has been found to be optimal, but
pulsed flow is also envisoned to work. These principles need not be
tied to multilaminate devices alone, but can be adopted for any
device and with like effect.
[0193] Preferred attachment means for the binding partners are by
adsorption or self assembling monolayer derivatization and
addition/spotting to/of the surfaces on which they are affixed,
e.g., electrode(s).
[0194] In some embodiments, the devices are preferably made of a
transparent or translucent material that permits visualization of
the circulation and recirculation of fluid sample.
[0195] In some embodiments, syringes or pneumatic pumps or other
means may drive the system, which may or may not be
electromechanical in nature.
[0196] In some embodiments, the individual cartridges are "stacked"
and oriented such that a set pitch exists between individual inlet
sample ports in neighboring cartridges. This allows for convenient
use of multi-well pipetting devices and the like to load samples. A
common pitch in the industry for this is .about.9 mm.
[0197] In some embodiments, channels are wider than tall. In some
embodiments, channel dimensions range from about 0.030'' to about
0.060'' in width and 0.006'' to about 0.014'' in height.
[0198] In some alternating laminate and adhesive layer embodiments,
individual layer thicknesses range of from about 0.0005'' to about
0.010''. In some preferred embodiments, laminate layers are thicker
than adhesive layers. In some embodiments, laminate layers range
from about 0.0005'' to about 0.030'' thickness and adhesive layers
from about 0.001'' to about 0.003'' thickness.
[0199] By "substantially parallel" is meant not perpendicular to
one another.
[0200] Other aspects and embodiments will be apparent to one of
ordinary skill in the art from the background documents, drawings,
detailed description, and claims to follow.
[0201] The individual aspects and embodiments of the invention can
be combined as appropriate in any combination. Advantages from the
combinations include, as appropriate for a given aspect/embodiment:
lower cost, ease of fabrication and mass fabrication, ease of
reproducibility, improved flow, improved mixing, and elimination or
minimization of gaseous bubble formation that would otherwise
obstruct sample flow, and facilitated analyte diffusion and
electrolyte conductivity.
Ease of Use
[0202] As the diagnostic testing environment becomes more
decentralized (from large central labs to smaller hospital labs, to
patient bedsides, and into the field), highly-trained and
specialized operators become more scarce. Ease of use is a key
criterion for successful implementation in such an environment.
This invention includes several features that address this
need.
[0203] One way to make a system easier to use is to incorporate
more "intelligence", which is most effective when software is
provided with useful information. Incorporating inexpensive
information storage into the cartridge in a format that can be read
from and written to is quite advantageous. Barcodes are an example
of media that provide read-only storage that may transfer
information from the cartridge manufacturer to the instrument, e.g.
identification of what test protocol should be run or what the
cartridge expiration date is. Read-only systems used in conjunction
with instrument databases further allow the association of fixed
identification information (e.g. a cartridge serial number) with
data unknowable at manufacture (such as the specific locale and
instrument used for testing, the testing status, and the reported
results). Media such as EEPROMs and some RFID formats have the
added ability to write to the cartridge, which allows independence
from a database and permits any instrument to retrieve the
information without requiring networked access to a central server.
(An "EEPROM" is short for Electronically Erasable Programmable
Read-Only Memory, which is a non-volatile storage chip used in
computers and other devices to store small amounts of volatile
(configuration) data. EEPROMs come in a range of capacities from a
few bytes to over 128 kilobytes and are typically used to store
configuration parameters, and in modern computers they replace the
hitherto common CMOS nonvolatile BIOS memory).
[0204] Another way to drive ease of use is to make a system fully
compatible with preexisting technologies for sample handling. For
example, liquid samples in labs are commonly transferred using
volumetric pipettes. While there is some degree of standardization,
variations in available tip geometries could make compatibility
with a custom interface difficult. However, given that these
pipettes have been designed to transfer fluids into microcentrifuge
tubes and microtiter plates, any cartridge with a sample input
reservoir that mimics these other systems will be at an
advantage.
[0205] Further advantage can be gained through compatibility with
standard parallel processing and automation equipment. Manual
multipipettes and robotic pipetting systems are generally designed
to interface with a standard 96-well microtiter plate format, i.e.
with tips separated by a 9 mm pitch and in arrangements of
8.times.12 (or a subset of this array). This makes cartridges that
can nest together at a 9 mm pitch especially advantageous.
[0206] Yet further ease of use can be achieved by making customer
interaction with the physical cartridge simple and obvious,
especially when troubleshooting. For example, transparency is a
valuable though often overlooked feature. A transparent sample
input reservoir allows the operator to confirm that the sample has
been added to the cartridge, and a graduated reservoir allows
confirmation that the necessary volume has been added. A
transparent fluid path allows operators to see if there has been a
problem with fluid handling or with bubbles.
Analyte Capture
[0207] While ease of use is important for broad acceptance by
consumers, the keystone of a diagnostic system is its detection
technology. In one broad class of such technologies, the specific
binding of an analyte to an immobilized partner (commonly affixed
to a surface) is the prelude to an observable signal. The rate at
which such a capture event occurs is generally proportional to the
concentration of analyte present at that surface.
[0208] As the analyte molecules nearest to the surface bind, the
local analyte concentration becomes depleted. This can slow the
capture of subsequent molecules, reducing the sensitivity of the
system and the time-to-answer, so efforts to replenish the analyte
can reap rewards. For example, quickly flowing the
analyte-containing fluid over the capture site shrinks the boundary
layer that bulk analyte molecules must diffuse across in order to
be captured. In cases of extreme depletion, such fluid motion also
carries new, "un-accessed" fractions of the sample nearer to the
capture region.
[0209] There are many possible configurations for flowing a fluid
across a surface. While not the only workable geometry, one
convenient arrangement is to flow the analyte fluid through a
channel over the capture site. In this layout the constricted cross
section increases the flow velocity for a given volume transferred,
and several capture surfaces can be placed sequentially along the
channel to experience a more or less equivalent flow profile.
[0210] Actuating the motion of fluid through this channel can also
be done in many different ways. For a rectified flow, one
configuration comprises inserting a pump at one end of the system,
or one in the middle with a check valve on each side. Oscillating
flow likely needs no valving but might require a compression
chamber if in a linear rather than cyclical configuration.
[0211] One advantage to rectified flow that could justify its
additional complexity can be seen when considering bubbles, an
additional impediment to analyte capture. If a capture surface is
stored dry, it is possible to generate bubbles of trapped air as
the sample is introduced. Outgassing from the cartridge materials
is also possible, and outgassing from the sample is virtually
guaranteed given that the mixing techniques commonly used to
prepare samples will also aerate them. In each of these scenarios,
bubbles might reduce how effectively an analyte is captured.
However, the ability to strip bubbles from the system can mitigate
the issue. In a system with rectified flow, a bubble-stripping
chamber can be inserted into the fluid path.
[0212] For example, consider a design that could make use of the
bubbles' buoyancy to extract them. Create a holding reservoir with
an inlet that adds fluid near the top and an outlet that removes it
from the bottom. (Note that "top" and "bottom" do have a critical
meaning in this context because buoyancy is only meaningful in
gravity or a similar mass acceleration field.) Some reserve of
standing fluid is present in the reservoir. When new fluid with
bubbles enters from the top, the bubbles float on the top of this
reserve until they collapse, while the new fluid mingles with the
other liquid. Fluid is pulled from the bottom of this standing
liquid to refresh the system.
[0213] It is important to design the system so that there is always
standing liquid at the bottom of the reservoir, otherwise air or
un-collapsed bubbles will get pulled from the chamber into the rest
of the system and defeat the purpose. (For example, there needs to
be at least enough liquid to accommodate any pulsing nature of the
fluid propulsion.) However, there does not necessarily need to be
any dead air space at the top of the reservoir for this
bubble-stripping chamber to work. The chamber just needs to have a
geometry such that the force due to the fluid flow pulling the
liquid into the drain is less than the buoyancy force propelling
the bubbles away from the drain.
Electrochemical Detection
[0214] After an analyte has been captured, this binding event must
be converted into a signal that is observable by a detection
instrument. One way is to use electrochemistry to convert the
chemical binding event into an electrical current. In this
embodiment, the capture surface must also be an electrode.
[0215] Electrochemical detection can have several advantages over
alternative methods, but it has its own special requirements.
Electrochemical techniques call for the creation of an
electrochemical cell, often a three-electrode cell where there is a
working electrode (the sensor surface) linked through a conductive
electrolyte solution to an auxiliary electrode (a current
source/sink) and a reference electrode (a voltage reference). Any
disruption in this conductive link can impact the electrochemical
scan, so the bubble stripping described for analyte capture is just
as important for electrochemical detection. The use of redundant
auxiliary electrodes is an additional way to reduce the risk of a
break in conductivity: the path to each auxiliary electrode would
have to be blocked in order to compromise the electrochemical
circuit.
[0216] Of course, electrical connectivity with the instrument is
just as important as electrical connectivity through the
electrolyte solution. Given the paradigm that many disposable
cartridges will be interfacing with a non-disposable piece of
equipment, that equipment's connector must be robust to many
cycles. Furthermore that connector should be able to create many
connections in order to enable the analysis of many different
working electrodes from one cartridge. A zero insertion force (ZIF)
connector with a two-dimensional grid of contacts is one good
solution.
[0217] The following commonly owned or controlled patents describe
electrochemical detection principles and methodologies in more
detail: U.S. Pat. Nos. 5,591,578, 5,824,473, 6,177,250, 6,277,576,
6,268,149, 6,268,150, 6,180,352, 6,200,761 6,238,870, 6,258,545,
6,528,266, 5,770,369, 6,096,273, 7,014,992, 6,221,583, 6,090,933,
7,045,285, 6,479,240, 6,977,151, 7,125,668, 6,265,155, 6,291,188,
7,033,760, 6,232,062, 6,495,323, 7,056,669, 6,013,459, 6,013,170,
6,248,229, 7,018,523, 6,740,518, 6,063,573, 6,600,026, 7,160,678,
6,290,839, 6,264,825, 6,761,816, 7,087,148, 6,541,617, 6,942,771,
6,432,723, 6,833,267, 7,090,804, 6,686,150, 5,620,850, 6,197,515,
6,322,979, 6,306,584, 7,172,897, 6,753,143, 6,518,024, 6,544,734,
6,642,046, 6,592,696, 6,572,830, 6,361,958, 6,960,467, 6,602,400,
6,824,669, 6,596,483, and 6,875,619, all of which are incorporated
herein by reference for their disclosure related to detection
principles and methodologies.
[0218] Specific ligand attachment chemistries, including
self-assembling monolayer technology, is also discussed in detail
in U.S. Pat. Nos. 6,306,584, 5,620,850, 6,472,148, 6,197,515,
6,322,979, 6,809,196, 5,620,850, 6,197,515, 6,322,979, and
6,306,584, all of which are expressly incorporated herein by
reference for disclosure related to ligand attachment chemistries,
including self-assembling monolayer technology (for example when
the electrodes of the invention comprise self-assembled monolayers
(SAMs)). The compositions of these SAMs will vary with the
detection method used. In general, there are two basic detection
mechanisms. In one embodiment, detection of an ETM is based on
electron transfer through the stacked ni-orbitals of double
stranded nucleic acid. This basic mechanism is described in U.S.
Pat. Nos. 5,591,578, 5,770,369, 5,705,348, and PCT US97/20014 and
is termed "mechanism-1" herein. Briefly, previous work has shown
that electron transfer can proceed rapidly through the stacked
n-orbitals of double stranded nucleic acid, and significantly more
slowly through single-stranded nucleic acid. Accordingly, this can
serve as the basis of an assay. Thus, by adding ETMs (either
covalently to one of the strands or non-covalently to the
hybridization complex through the use of hybridization indicators,
described below) to a nucleic acid that is attached to a detection
electrode via a conductive oligomer, electron transfer between the
ETM and the electrode, through the nucleic acid and conductive
oligomer, may be detected.
[0219] Alternatively, the ETM can be detected, not necessarily via
electron transfer through nucleic acid, but rather can be directly
detected on an electrode comprising a SAM; that is, the electrons
from the ETMs need not travel through the stacked n orbitals in
order to generate a signal. As above, in this embodiment, the
detection electrode preferably comprises a self-assembled monolayer
(SAM) that serves to shield the electrode from redox-active species
in the sample. In this embodiment, the presence of ETMs on the
surface of a SAM, that has been formulated to comprise slight
"defects" (sometimes referred to herein as "microconduits",
"nanoconduits" or "electroconduits") can be directly detected. This
basic idea is termed "mechanism-2" herein. Essentially, the
electroconduits allow particular ETMs access to the surface.
Without being bound by theory, it should be noted that the
configuration of the electroconduit depends in part on the ETM
chosen. For example, the use of relatively hydrophobic ETMs allows
the use of hydrophobic electroconduit forming species, which
effectively exclude hydrophilic or charged ETMs. Similarly, the use
of more hydrophilic or charged species in the SAM may serve to
exclude hydrophobic ETMs.
[0220] The person of ordinary skill in the art will appreciate that
electrochemical detection may be accomplished in a variety of ways
to detect a variety of different analytes. Osmetech's eSensor.RTM.
DNA Detection Technology is illustrative of one way of detecting
nucleic acid sequences.
[0221] The eSensor.RTM. microarray is composed of a printed circuit
board (PCB) consisting of an array of gold electrodes that are each
modified with a multi-component, self-assembled monolayer (SAM)
that includes pre-synthesized oligonucleotide capture probes.
Nucleic acid detection is based on a sandwich assay principle.
Signal and capture probes are designed with sequences complementary
to immediately adjacent regions on the corresponding target DNA
sequence. A three-member complex is formed between capture probe,
target, and signal probe based on sequence-specific hybridization,
which brings the 5'-end of the signal probe containing
electrochemically-active ferrocene labels into close proximity with
the electrode surface. The ferrous ion within each ferrocene group
undergoes cyclic oxidation and reduction, leading to loss or gain
of an electron, which is measured as current at the electrode
surface using alternating current voltammetry (ACV) and
higher-order harmonic signal analysis. The ferrocene labels are
only detected when the signal probe is captured at the surface of
the electrode by sequence-specific hybridization. In the absence of
target, no specific signal is detected.
[0222] Osmetech's current system is adapted to specifically detect
genetic mutations and polymorphisms by employing allele-specific
signal probes containing ferrocene labels with distinguishable
redox potentials. The signal probe matching the wild-type sequence
contains a ferrocene label of one electrochemical potential, and a
second signal probe matching the mutant sequence contains a second,
distinguishable ferrocene label. Both the wild-type and mutant
targets bind to the capture probe at a site adjacent to the
mutation. The wild-type and mutant signal probes then compete for
binding to their complementary sequences. The probe with the
perfect match to the target is bound with a high degree of
preference. The genotype is then determined by the ratio of signals
generated by the bound wild-type and mutant signal probes.
Genotyping boundaries are established based on statistical analysis
of data from a large number of samples, and subsequent
identification of unknown samples requires no further calibration
of the instrument or cartridge lot. This approach can be used to
discriminate single- or multiple-base changes, insertions and
deletions. A mutation site with multiple alleles, or two adjacent
mutation sites, can be genotyped using additional ferrocene
labels.
Superior Cartridge/Instrument Interface
[0223] Regardless of the chosen analytical method, the interface
between cartridge and instrument must be designed for reliability
and ease-of-use. For example, appropriate alignment between the
cartridge and the fluid flow actuation must be ensured, keying
features should be added to prevent an operator from inserting a
cartridge in the wrong orientation, and protective features may
need to be added to prevent damage to the instrument if an
inappropriate cartridge interface is forced. In addition, there
should be tactile (and/or perhaps audible) feedback to the operator
to communicate to them that a cartridge has been inserted
correctly. Common detent methods such as spring-loaded balls or
compressible lever latches will serve this purpose.
[0224] It is also important to be forward-looking when designing
the cartridge/instrument interface. Under the "disposable cartridge
with reusable instrument" paradigm, new cartridges are frequently
shipped to customers to be used with a device already installed in
the field. Design choices can be made with that instrument's
cartridge interface such that it will be possible to upgrade
cartridge design without triggering modification of the instruments
themselves. For example, in one embodiment a pressure plate may
press against the cartridge in intimate physical contact (to clamp
that cartridge into place). However, if the pressure plate were to
be designed to conform perfectly with the cartridge's natural
design, then no other physical changes to the cartridge shape will
be possible in the future without retrofit. However, if a
mechanical adaptor is integrated into the cartridge so that a more
generic surface is presented to the pressure plate, then future
cartridge designs are free to change internal shape with a
corresponding adjustment to the adaptor space within, the outside
of the cartridge remaining substantially the same.
[0225] It should be noted that one or more of the above-noted
preferred features may not apply for a given application or
embodiment, as the person of ordinary skill in the art will
appreciate.
EXAMPLES
Example 1
Cartridge Design & Manufacture
[0226] With reference to FIG. 6, we have chosen a printed circuit
board (PCB) (1) to provide the gold surfaces for a 3-electrode
electrochemical cell controlled by a potentiostat circuit. It has
dual auxiliary electrodes (3) shorted to one another, a
multiplicity of working electrodes (4), and a reference electrode
(5). The reference electrode is coated with the same electrically
conductive silver material (epoxy) that is used to attach an EEPROM
(6), and this silver on the reference is electrochemically oxidized
immediately prior to analysis to generate the necessary Ag/AgCl
redox couple. (While a more standard solder could have been used to
attach the EEPROM, this would have introduced additional chemical
compounds and manufacturing steps). The PCB geometry lays the
working electrodes in 3D wells, which simplifies the process of
applying differing chemical treatments to each electrode to create
the desired capture surfaces.
[0227] In embodiment, the cartridge device consists of a PCB chip,
a cover, and a microfluidic component. The microfluidic component
is composed of a plate and a multilayer laminate. The PCB chip
includes 72 gold-plated working electrodes (that is twice density
of the CFCD chip), a silver/silver chloride reference electrode,
and two gold-plated auxiliary electrodes. Each working electrode
has its own connector contact pad on the opposite side of the chip
to allow electrical connection to an instrument. The entire surface
of PCB is coated with an insulating solder mask, leaving only the
center (250 mm diameter) of the electrodes exposed. The PCB chip
also contains an EEPROM (Electrically Erasable-Programmable
Read-Only Memory) component, a memory device that stores
information related to the cartridge, such as assay protocol,
cartridge lot number, and expiration date.
[0228] With reference to FIGS. 1B, 2, 3, 4 and 5, a laminate
assembly (2) is affixed on top of the PCB (1), which assembly
combines individual layers of pressure-sensitive adhesive (7a-c)
and thin plastic laminate layers (8a,b). On top of the laminate
assembly is a molded polycarbonate plate (9). The laminate layers
define the fluid channel (10) and provide a diaphragm (11) (for
interface with a pump) and two check valves. The polycarbonate
plate (9) provides the rigid chimney (14), the extreme end of which
constitutes the pump interface (15), and also contains a fill
reservoir/sample chamber (16) that in this embodiment serves double
duty as a bubble-stripping chamber, but that in other embodiments
may be a separate chamber/port. All of this is created in a thin
enough arrangement to allow cartridges to be held side-by-side in a
loading rack (17) with their reservoir openings (18) offset by a 9
mm standard multipipette tip pitch or distance between individual
cartridge chambers. For loading rack, see FIG. 10.
[0229] With reference to FIGS. 1B, 4 and 5, the walls of the fluid
channel (10) are defined by one or more layers (7, 8) of the
laminate assembly (2), and are sealed to the PCB (1) by an adhesive
layer. While clamping the parts together might be sufficient in
some systems, the adhesive provides a more reliable seal,
especially for low viscosity solutions. (Although the conductive
electrolyte solution necessary for electrochemical detection is
usually water-based, the specific additional salts, detergents, or
non-aqueous solvents required by the individual chemistries of
diagnostic technologies can have a significant impact on the
fluid's surface tension and capillary behavior.)
[0230] An adhesive layer can also seal the walls of the channel to
its ceiling, which can be flat, beveled or otherwise. In one
embodiment, the ceiling is formed from a plastic layer comprising a
facing of the laminate or conforming channel within the laminate.
This allows the laminate part to hold its shape during manufacture.
Cutting the channel entirely through to the polycarbonate plate
could make the laminate prone to stretching, twisting, or
distortion when handled, depending on the geometry of the channel
path. For this reason, in certain embodiments, certain of the
laminate layers are only partially cut into or sculpted, and not
completely so, with other functionalities or complementing features
provided by other layers so that no one layer is too weak for
practical manufacturing. In other embodiments, the ceiling is
provided by the polycarbonate plate/cover.
[0231] With reference to FIGS. 2, 4, 5, and 11, rectified fluid
flow through the channel (10) is actuated by a diaphragm (11)
flanked by two bridge beams (13). The diaphragm (11) is formed from
the same layer of plastic laminate that creates the channel
ceiling, covering an area where the channel widens into a circular
region. However, while the channel ceiling is attached to the
polycarbonate plate (9) with a layer of adhesive, the diaphragm
(11) is free to oscillate. A local absence of adhesive and a
recess/beveled underside (29) in the plate gives the diaphragm (11)
freedom to flex toward and away from the PCB (1) surface and form a
diaphragm chamber (22). See FIG. 11.
[0232] In one embodiment, the cartridge diaphragm (11) is driven by
a pneumatic pump (12), preferably one integrated into or alongside
a detection instrument/device (23). See FIGS. 13C, E. The use of
alternating positive and negative pressure air pulses allows a full
push-and-pull cycle, rather than the push-and-relax characteristic
of other driver sources. Furthermore, the pump interface (15)
between the pneumatic pump (12) and cartridge (24) is quite simple.
A flexible tube (25) carries the pressure wave, diverting it around
any internal obstacles, and delivers it to an outlet (26)
configured for interface with a compressible grommet (27). Rigid
chimney (14) molded into the polycarbonate plate (9) mates with
this opening and transfers the pulse through a narrowed baffle (28)
and into beveled underside (29), which in turn mates in conforming
fashion with cartridge diaphragm (11) upon supply of a negative
pressure/vacuum pulse from pump (12). See FIGS. 4, 11.
[0233] With reference to FIGS. 4, 5, 8, and 11, rectified flow
requires diaphragm pump zone to be communicatively flanked by a
plurality of cooperating check valves, here two. One embodiment
incorporates what is referred to as a "bridge" valves. Similar in
concept to the common reed valve, a bridge valve functions by
seating a flat, flexible bridge beam (13) across a port (31) and
valve seat (30) in a rigid or substantially rigid substrate. When a
pressure differential pulls the fluid through port (31), then
flexible membrane (11) flexes away and allows fluid to pass around
it. When the direction of the pressure differential reverses, the
membrane gets pulled flat against the rigid valve seat and seals
the port (31) against backwash. In one embodiment, the flexible
bridge beam (13) is formed from the same laminate layer (8a) that
creates the channel ceiling and diaphragm (11). The valve seats
(30) are preferably molded into the polycarbonate plate (9) and
have slightly raised annular bosses relative to membrane (11) that
facilitate sealing when in closed position. The bridge valve has a
simple construction that does not require small floating parts or
separate small pieces, which leads to easier manufacture and
greater reliability. The bridge valve is more manufacturable even
than its cousin, the reed valve. The "bridge" is constrained on
both sides--on both "shores" if you will--whereas the reed would be
attached on one side and free on the other (like a diving board or
flap), open to being bent or folded over during assembly (and thus
subject to fatigue/weakening). The bridge valve has another
advantage over the reed valve in that it can be biased open,
neutral, or closed. This is controlled by the relative placement of
the flexible membrane and the rigid valve seat. If the flat,
relaxed bridge is exactly flush against the seat, the bias is
neutral; if the relaxed bridge is separated from the seat, the bias
is open; if the valve seat protrudes so that the relaxed bridge is
stretched and pressed against it, the bias is closed. The common
reed valve cannot be biased closed.]
[0234] One distinct feature of the cartridge over the CFCD
cartridge is the utilization of microfluidic technology to
introduce fluidic circulation in order to accelerate hybridization
and decrease the hybridization time. The microfluidic component
consists of a plastic plate and a multi-layer laminate sandwich,
which form functional microfluidic components such as a micropump,
two check valves, and a hybridization channel. The laminate
consists of multiple layers of silicone adhesive, Teflon, and a PET
(polyethylene terephthalate) layer. The PET layer embodies two thin
membranes in a bridge configuration that function as check valves
and a diaphragm which is part of a pneumatically driven pump. A
serpentine channel (275 um deep and 1 mm wide) within the laminate
assembly forms the hybridization chamber above the working
electrode array. The pneumatic pump is connected to a pneumatic
source from the instrument. The pneumatic pump provides
unidirectional pumping of the analyte through the serpentine
channel past all the electrodes during the hybridization. Previous
studies have shown that flow circulation in the hybridization
channel brought a large number of target molecules per time unit to
pass by individual electrodes and allowed continuously replenishing
the area around the electrode that has been depleted of
complementary targets. Moreover, since hybridization is a rate
limiting process that relies on diffusion of target molecules
across the diffusion boundary layer to their binding sites, the
rapid fluid movements can enhance the transport of target within
the diffusion boundary layer by reducing the thickness of the
diffusion boundary layer. As a result, the hybridization kinetics
is greatly improved and hybridization time is reduced from 2 hr to
30 min."
[0235] In operation, and with particular reference to FIGS. 2, 3, 4
and 11, fluid is introduced to the cartridge (24) by way of a
filling reservoir (16) molded into the polycarbonate plate (9). The
underside of the outlet from this reservoir provides the valve seat
(30) that together with bridge beam (13) forms the first bridge
valve. Fluid flows out of the reservoir (16), across this valve and
into a diaphragm chamber (22). From the diaphragm chamber (22)
fluid is diverted back up through the polycarbonate plate (9) and
across an overpass (34) so it can return to the second bridge
valve, exiting through a second valve seat (30) and traversing a
second bridge beam (13) to enter the primary channel (10). Fluid
flows through this channel (10) over the electrodes (4) until it is
released back into the filling/sample reservoir/chamber (16) where
it is naturally stripped of bubbles before it begins another
circuit. A jumper laminate (35) seals the oval recess (36) in plate
(9) associated with overpass (34) and second valve seat (30) and is
planar and proximate to the ends of two substantially parallel
ports (72), which act to re-direct fluid from one plane to another
in cartridge (24) and facilitate rectified valve-actuated flow by
taking advantage of gravity. Jumper laminate (35) conforms
substantially to the dimension of the oval overpass recess (36) and
is affixed by a jumper laminate pressure sensitive adhesive layer
(33) of conforming dimension. See FIGS. 2, 3. Any dimension other
than oval can be used, e.g., square, rectangular, circular. A
separate molded polypropylene cap (38) with a living hinge (39)
provides a seal for the filling reservoir. A rigid cover (40), also
made from molded polycarbonate, fits over the top of the
polycarbonate plate (9). It incorporates detents (41) to hold the
PCB alignment features against their mating partners within the
module (42) (discussed below) and to provide tactile and/or audible
feedback. Cover (40) also includes a handle (43) for easy
manipulation in and out of module (42), keying features to ensure
appropriate orientation before clamping the cartridge into the
instrument, and clearance hole/recess (45) for receipt of chimney
(14) and for easy viewing of the filling/sample reservoir (16). A
large, flat area (47) on the cover's surface interfaces with the
module clamping mechanism discussed below, and internal ribs
(underside of cover; not shown) transfer the pressure evenly across
the polycarbonate plate (9). Space spanned by these ribs affords
adaptability for future inclusion of one or more additional
functionalities. Cover (40) is made of polycarbonate
(injection-molded) and has a recess (49) to accommodate the fill
reservoir/chamber (16) of plate (9). The combined plate/assembly is
attached to cover (40) by way of two bosses (67) on plate that have
reciprocal engagement recesses in the underside of cover. See FIGS.
1A, B and 2. Each of laminate assembly (2), plate (9) and
individual laminate layers (8) and adhesive layers (7) also have an
EEPROM recess/void (70) for allowing access to the EEPROM (6).
[0236] The laminate assembly (2) and rigid plate (9) are both
preferably made of polyethyleneterepthalate (PET) and etched
polytetrafluoroethylene (PTFE), and one or more of the individual
laminate layers and PCB board are held together by pressure
sensitive adhesive (PSA) membranous layers, preferably
silicone-based. Other laminates that can be used include but are
not limited to, e.g., polycarbonate, polyethylene, ultra high
molecular weight polyethylene (UHMW PE) and polypropylene. Criteria
for an acceptable laminate include one or more of flexibility
(tensile modulus), conformability (form by cold flow), pliability,
ability to cut/machine or ablate (laser), ability to slice/make
thin (form into a film), durability, heat stability, and chemical
inertness relative to the fluidic components and assay chemistry
therein. In addition, in place of one or more laminates, silicone
layers can be used that have resilience/pliability and
compressibility to facilitate sealing. The materials used for
construction are readily available commercially: Adhesive layers of
thicknesses 0.001-0.003'' from silicone pressure sensitive
adhesives, for example, Tran-Sil NT1001 pressure sensitive adhesive
of thickness 0.002'' (Dielectric Polymers INC, Holyoke Mass.).
Flexible materials such as skived Teflon.RTM. (PTFE) film of
thickness 0.002-0.010'' come from Fralock, Valencia Calif., and
polycarbonate, polypropylene, and PET films 0.0005''-0.005'' thick
come from Now Plastics, INC, East Longmeadow Mass. Pump specs:
Micro Diaphragm, KNF Neuberger GmbH (Freiburg, DE),
PU1947-NMP09-1.07, 6 Volts. These and other parts and materials are
generic and commercially available or known in the art, or else
readily produced and assembled by the person of ordinary skill in
the art with the guidance of the present disclosure.
[0237] The dimensions of the flow channel are significant in that
the linear speed of the fluid over the electrodes is dependent on
the channel cross section. Additionally, the channel height and
width must be great enough to allow any bubbles to freely flow
through the channel and eventually be trapped/cleared in the
reservoir. Therefore, it is important to design a channel that is
small enough to provide sufficient linear velocity but not too
restrictive with respect to bubble movement. Another consideration
is that the lower channel dimensions will utilize less analyte
solution, often a desired characteristic. Channel widths of
0.030-0.060'' and heights of 0.006''-0.0014'' were tested as
compatible ranges, with the preferred dimensions being a width of
0.040'' and height of 0.010''.
[0238] The overall channel dimensions directly impact the minimum
amount of fluid required to flow without lapses in analyte fluid
(large bubbles/gaps) in the system. However, amounts in excess of
the channel volume can be cycled through the reservoir. The
preferred system as described above allows for analyte volumes of
about 1100-200 uL, preferably about 130 uL or less. This range can
be tuned easily by increasing or decreasing the channel height via
the thickness of laminate assembly and contributing layers.
[0239] The dimensional aspects require exacting manufacturing
methods such as the use of laser cutting machinery instead of die
cutting, as die cutting cannot form the thin channels (width of
0.030-0.040'') preferred. However, die-cutting could be used for
laminates with features at the upper ends of the suggested ranges,
e.g., 0.060''.
[0240] Material compatibility was thoroughly tested for Tran-Sil
adhesives, with PET and PTFE Teflon.RTM. membrane components. The
preferred laminate assembly consists of 0.003'' Transil.RTM.
adhesive applied to the one side of 0.006'' Teflon.RTM. (the side
to be adhered to the PCB), another Tran-Sil.RTM. layer of 0.002''
thickness between the other side of the Teflon.RTM. the 0.001'' PET
middle laminate containing the bridge valves. The final adhesive
layer of 0.002'' Transil.RTM. is applied to the PET laminate and
eventually bonds the polycarbonate cover to the laminate and
PCB.
Example 2
Cartridge Module Design & Manufacture
[0241] With reference to FIGS. 13A-E, the interface between
detection device (23) and cartridge (24) is mediated by a cartridge
module (42), which consists of a base (50) and top (51), both of
which are made of molded polyetherimide plastic (glass-filled
Ultem.RTM., General Electric), and both of which contain aligned
embedded metal screw castings (52) for fastening to each other.
Module top (51) contains a connector lever (53) and engagement
plate (54) for slideably engaging and disengaging cartridge firmly
against base (50) and buckle beam assembly (73) electrical
connection points therein. Top (51) also contains a conduit (55)
for mating with the cartridge chimney (14) and feeding and
withdrawing air to and from said cartridge. Base (50) has a heater
area (56) for receiving and mating with a gold/nickel plated copper
thermal plate (57) and buckle beam assembly (73) that consists of
an array of gold/nickel plated beryllium copper pins parallel and
proximate to said heater area (56) for electrically engaging
contact pads (58) beneath PCB (1) (see FIG. 7), and two
spring-loaded stainless steel metal detent locating pins (59) and
radii (60) to aid final functional positioning of cartridge upon
initial positioning into module (42). Thermal plate (57) overlays
heater area (56) and presses against the flat underside of the PCB
(1) directly beneath cartridge fluid channel (10) to act as a
modulable heat source for reactions taking place therein.
[0242] The electrical interface between device and cartridge relies
on reciprocally cooperating features possessed by both. See FIGS.
7, 13A-D. In the PCB (1), traces (not shown) are routed from the
electrodes (2-5) through vias (not shown) to the underside of the
PCB (1), where the two-dimensional array of gold contact pads (58)
are exposed to said buckle beam assembly (73), individual pads
mating with individual pins thereon. Module (42) is a clamping
zero-insertion-force (ZIF) connector wherein the cartridge is
slotted in loose and then firmly engaged within module (42) upon
positionment against detent pins (59) and radii (60) and clamping
with slideable lever (53) that forces engagement plate (54) down
against top of cartridge (24), thereby securing bottom of cartridge
(24), including pads (58), firmly against buckle beam assembly
(73). Clamping motion also forcibly mates compressible outlet (63)
of pneumatic conduit (55) with cartridge chimney (14). Alignment
features (64) cut into the PCB ensure that the array of pads (58)
on underside of the PCB (1) and the array of pins from buckle beam
assembly (73) line up.
[0243] Buckle beam contacts can be substituted by pogo pins,
anisotropic electrically conductive tapes, films, elastomers, and
or other connectors known in the art. Elastomeric parts for
pneumatic interface, including compressible outlet, are made of
molded polyurethane, but silicone and like compositions are also
considered to be an adequate alternative.
Example 3
Detection Device
[0244] In typical use, and with reference to FIG. 14, the cartridge
and cartridge module are coupled to a detection device (23) and/or
computer, which parts may be combined into one, as is known and
readily implementable in the art. FIG. 14 shows an embodiment of a
detection device (23) for housing multiple cartridges (24). This
embodiment contains three vertical towers (65) each possessing
eight cartridges (24), each of which fits into its own module (42).
To the left is a computer screen (66) for programming detection
parameters and evaluating results. In this particular embodiment
the screen is a touch-pad screen from which a user may conveniently
select from various programming options merely by touching the
screen. The fill reservoir/chambers (16) of the cartridges (24)
face out at an approximate 15.degree. angle relative to horizontal
to take advantage of gravity and 3-dimensional space to
economize/optimize onlooker or technician view and facilitate
loading/clamping of cartridges into cartridge modules. For maximum
utility, the detection device is programmed to perform either a
random-access or batch mode operation, which allows for one or more
different tests to be run simultaneously.
[0245] The preceding is useful for any detection device. Specifics
of electrochemical detection and other detection methods are as
described previously and in the above-cited documents.
[0246] All articles and documents referenced herein, as well as all
the citations cited therein, are incorporated by reference for an
understanding of the invention and are indicative of what the
person of ordinary skill requires to know to make the invention
operable using no more than routine experimentation, as well as
appreciate the advantages of the invention.
[0247] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The methods and compositions described illustrate
preferred embodiments, are exemplary, and are not intended as
limitations on the scope of the invention. Certain modifications
and other uses will be apparent to those skilled in the art, and
are encompassed within the spirit of the invention as defined by
the scope of the claims.
[0248] The terms and expressions which have been employed are used
as terms of description and not of limitation, and there is no
intention in the use of such terms and expressions of excluding any
equivalents of the features shown and described, or portions
thereof. It is recognized that various modifications are possible
within the scope of the invention claimed. Thus, it should be
understood that although the present invention has been
specifically disclosed by preferred embodiments, optional features,
modifications and variations of the concepts herein disclosed may
be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention as defined by the description and the appended
claims.
[0249] In addition, where features or aspects of the invention are
described in terms of ranges or Markush groups or other grouping of
alternatives, e.g., genuses, those skilled in the art will
recognize that the invention is also thereby described in terms of
any individual measurement, member or subgroup of members of the
range, Markush group or subgenus, and exclusions of individual
members as appropriate, e.g., by proviso.
* * * * *