U.S. patent application number 10/447691 was filed with the patent office on 2004-01-22 for ultra high throughput sampling and analysis systems and methods.
This patent application is currently assigned to Caliper Technologies Corp.. Invention is credited to Biondi, Sherri Ann, Jensen, Morten J., Kopf-Sill, Anne R., Parce, J. Wallace, Wolk, Jeffrey A..
Application Number | 20040014239 10/447691 |
Document ID | / |
Family ID | 26870649 |
Filed Date | 2004-01-22 |
United States Patent
Application |
20040014239 |
Kind Code |
A1 |
Wolk, Jeffrey A. ; et
al. |
January 22, 2004 |
Ultra high throughput sampling and analysis systems and methods
Abstract
Ultra-high throughput systems and methods are used for sampling
large numbers of different materials from surfaces of substantially
planar library storage components. The systems and methods
typically employ: microfluidic devices having integrated capillary
elements for carrying out the analysis of the sampled materials;
library storage components, e.g., planar solid substrates, capable
of retaining thousands, tens of thousands and hundreds of thousands
of different materials in small areas; sensing systems for allowing
rapid and accurate sampling of the materials by the microfluidic
devices, and associated instrumentation for control and analysis of
the overall operation of these systems.
Inventors: |
Wolk, Jeffrey A.; (Half Moon
Bay, CA) ; Biondi, Sherri Ann; (Menlo Park, CA)
; Parce, J. Wallace; (Palo Alto, CA) ; Jensen,
Morten J.; (San Francisco, CA) ; Kopf-Sill, Anne
R.; (Portola Valley, CA) |
Correspondence
Address: |
CALIPER TECHNOLOGIES CORP
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043
US
|
Assignee: |
Caliper Technologies Corp.
Mountain View
CA
|
Family ID: |
26870649 |
Appl. No.: |
10/447691 |
Filed: |
May 29, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10447691 |
May 29, 2003 |
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09750450 |
Dec 28, 2000 |
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6620625 |
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60196468 |
Apr 11, 2000 |
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60174902 |
Jan 6, 2000 |
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Current U.S.
Class: |
436/180 ;
422/400 |
Current CPC
Class: |
B01J 2219/00626
20130101; G01N 2035/1034 20130101; B01J 2219/00585 20130101; B01L
3/02 20130101; B01L 2200/143 20130101; G01N 2001/028 20130101; B01J
2219/00621 20130101; B01J 2219/00369 20130101; B01L 2200/10
20130101; B01J 2219/00596 20130101; B01L 2400/0406 20130101; B01J
2219/00612 20130101; G01N 35/10 20130101; B01J 2219/00619 20130101;
C12Q 2565/629 20130101; G01N 2035/1037 20130101; G01N 2035/1039
20130101; B01J 2219/00527 20130101; B01L 2200/027 20130101; B01J
2219/00367 20130101; C40B 60/14 20130101; B01J 2219/00707 20130101;
Y10T 436/2575 20150115; B01J 2219/00641 20130101; B01J 2219/00628
20130101; B01L 2300/0819 20130101; B01J 2219/00587 20130101; B01J
19/0046 20130101; B01L 3/5027 20130101; B01L 3/50273 20130101; B01J
2219/00722 20130101; C40B 40/06 20130101; B01J 2219/00497 20130101;
B01J 2219/0061 20130101; B01J 2219/00659 20130101; G01N 21/6452
20130101; B01J 2219/00351 20130101; B01J 2219/00637 20130101; B01J
2219/00605 20130101; B01L 2300/0861 20130101; B01L 2400/022
20130101; B01L 3/502715 20130101; Y10T 436/25 20150115; B01L
2200/025 20130101 |
Class at
Publication: |
436/180 ;
422/58 |
International
Class: |
G01N 001/10 |
Goverment Interests
[0002] The present invention was made with government funding from
the United States National Institute of Standards and Technology
(NIST), through the Advanced Technology Program (ATP) under Grant
No. 70NANB8H4000, and the United States government has certain
rights in the invention.
Claims
What is claimed is:
1. A sample substrate array, comprising: a substrate having a first
surface; and at least 100 separate test compound spots dried onto
the first surface of the substrate, each test compound spot
comprising a test compound and at least one excipient agent.
2. The substrate array of claim 1, wherein the at least 100
separate compounds spots are present at a density of at least about
100 compounds/cm.sup.2 of substrate surface.
3. The substrate array of claim 1, wherein the at least 100 test
compounds spots comprise at least 500 test compounds which are
present at a density of at least about 500 compounds/cm.sup.2 of
substrate surface.
4. The substrate array of claim 1, wherein the at least 100 test
compounds spots comprise at least 1000 test compounds which are
present at a density of at least about 1000 compounds/cm.sup.2 of
substrate surface.
5. The substrate array of claim 1, wherein the first surface of the
substrate has a surface area of at least 1 cm.sup.2.
6. The substrate array of claim 1, wherein the first surface of the
substrate comprises at least 200 different compounds spots
reversibly immobilized thereon in discrete regions.
7. The substrate array of claim 1, wherein the first surface of the
substrate comprises at least 1000 different compound spots
reversibly immobilized thereon in discrete regions.
8. The substrate array of claim 1, wherein the surface of the
substrate comprises at least 10,000 different compound spots
reversibly immobilized thereon in discrete regions.
9. The substrate array of claim 1, wherein the first surface of the
substrate comprises a metal.
10. The substrate array of claim 1, wherein the substrate comprises
glass or quartz.
11. The substrate array of claim 1, wherein the first surface of
the substrate is nonconductive.
12. The substrate array of claim 1, wherein the first surface of
the substrate is selected from a metal oxide, SiO.sub.2,
Si.sub.3N.sub.4, siliconoxynitride and a polymeric material.
13. The substrate array of claim 1, wherein the first surface of
the substrate is a polymeric material.
14. The substrate array of claim 13, wherein the polymeric material
is selected from nitrocellulose, acrylic, polystyrene, parylene,
polyvinylidine difluoride (PVDF), polysulfone, polyvinyl chloride,
spun polypropylene, polytetrafluoroethylene (PTFE), and
polycarbonate.
15. The substrate array of claim 1, wherein the at least one
excipient agent is selected from a starch, dextran, glycol,
polyethylene oxide, polyvinylpyrrolidone, a detergent, sucrose,
fructose, maltose, and trehelose.
16. A method of fabricating a sample substrate array, comprising:
providing a substrate having a first surface; depositing at least
100 separate test compounds on the first surface of the substrate;
and freeze drying each of the at least 100 separate test compounds
on the first surface.
17. The method of claim 16, wherein the at least 100 separate
compounds are present at a density of at least about 100
compounds/cm.sup.2 of substrate surface.
18. The method of claim 16, wherein the at least 100 test compounds
comprise at least 500 test compounds which are present at a density
of at least about 500 compounds/cm.sup.2 of substrate surface.
19. The method of claim 16, wherein the at least 100 test compounds
comprise at least 1000 test compounds which are present at a
density of at least about 1000 compounds/cm.sup.2 of substrate
surface.
20. The method of claim 16, wherein the first surface of the
substrate has a surface area of at least 1 cm.sup.2.
21. The method of claim 16, wherein the first surface of the
substrate comprises at least 200 different compounds reversibly
immobilized thereon in discrete regions.
22. The method of claim 16, wherein the first surface of the
substrate comprises at least 1000 different compounds reversibly
immobilized thereon in discrete regions.
23. The method of claim 16, wherein the first surface of the
substrate comprises at least 10,000 different compounds reversibly
immobilized thereon in discrete regions.
24. The method of claim 16, wherein the first surface of the
substrate comprises a metal.
25. The method of claim 16, wherein the substrate comprises glass
or quartz.
26. The method of claim 16, wherein the first surface of the
substrate is nonconductive.
27. The method of claim 16, wherein the first surface of the
substrate is selected from a metal oxide, SiO.sub.2,
Si.sub.3N.sub.4, siliconoxynitride and a polymeric material.
28. The method of claim 16, wherein the first surface of the
substrate is a polymeric material.
29. The method of claim 28, wherein the polymeric material is
selected from nitrocellulose, acrylic, polystyrene, parylene,
polyvinylidine difluoride (PVDF), polysulfone, polyvinyl chloride,
spun polypropylene, polytetrafluoroethylene (PTFE), and
polycarbonate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 09/750,450, filed Dec. 28, 2000, which claims priority to
Provisional U.S. Patent Application No. 60/174,902, filed Jan. 6,
2000, and No. 60/196,468, filed Apr. 11, 2000, each of which is
hereby incorporated herein by reference in its entirety for all
purposes.
BACKGROUND OF THE INVENTION
[0003] The science of drug discovery has greatly benefited in
recent years from dramatic advances made in scientific research and
development. For example, on one end of the drug discovery
spectrum, the concerted international effort to sequence the human
genome has led to the discovery of large numbers of genes and gene
products that are potential targets for pharmaceutical agents in
the treatment of disease. On the other side of the equation,
numerous approaches to combinatorial chemical synthesis have led to
the generation of extremely large numbers of different chemical
compounds that can be screened for effects on those targets.
[0004] Linking the two technologies are a number of advances in
technology for screening the large libraries of compounds against
large collections of targets. For example, large automated robotic
systems have been developed to sample and mix reagents from
libraries in multiwell plate formats, performing thousands of
different screening reactions in a single day. These systems employ
a brute force approach to screening potential pharmaceutical
compounds by automating the fluid handling components of the assay
process. While these systems are widely used, they provide only a
first incremental increase in efficiency over the lone experimenter
working at his or her taken long for this incremental increase in
efficiency to be surpassed by the screening demand.
[0005] Microfluidic technology is one of the most recent
technologies to be applied in screening pharmaceutical libraries
(see, e.g., U.S. Pat. No. 5,942,443). These microfluidic
technologies provide benefits in terms of reagent consumption,
speed, reproducibility and automatability. Specifically, when
performed in the microscale format in fluid volumes on the order of
nanoliters or less, reagents mix more quickly, and assays require
much smaller quantities of expensive reagents. Further, the
integrated nature of microfluidic systems allows for precise
computer control of material flow, mixing, data acquisition and
analysis allowing for ease of use and improved reproducibility.
[0006] Microfluidic systems have also been developed to interface
with the traditional library storage format, namely the multiwell
plate. In particular, pipettor chips have been developed that
include an external sample accessing capillary, see, e.g., U.S.
Pat. No. 5,779,868. While such systems provide the advantages of
smaller reagent requirements in screening, conventional library
storage systems still utilize large reagent volumes, effectively
eliminating some of the advantages otherwise provided by
microfluidic technology.
[0007] While all of the foregoing advances in screening technology
have provided significant benefits to the pharmaceutical industry,
it would generally be desirable to be able to take advantage of all
of the advantages of microfluidic technology in terms of the
reagent storage and accessibility. The present invention meets
these and a variety of other needs.
SUMMARY OF THE INVENTION
[0008] The present invention is generally directed to improved
methods, devices and systems for use in high-throughput and even
ultra high-throughput assays. Generally, the methods, devices and
systems take advantage of novel automation, miniaturization and
integration techniques to achieve these goals.
[0009] For example, in a first aspect, the present invention
provides a method of sampling compounds into a microfluidic
channel. In these methods, a plurality of different compounds are
provided reversibly immobilized on a first surface of a substrate.
A capillary element is also provided having a capillary channel
disposed therethrough, where the capillary element has at least one
open end, and a volume of solubilizing fluid present at the open
end of the capillary element. In accordance with these methods, the
solubilizing fluid at the open end of the capillary element is
moved into contact with a first compound on the surface of the
substrate by sensing when the solubilizing fluid contacts the
surface of the substrate. The solubilizing fluid dissolves at least
a portion of the first compound, and at least a portion of the
dissolved first compound is drawn into the capillary element.
[0010] In another aspect, the present invention provides methods of
sampling compounds into a microfluidic channel, which, in addition
to providing the compounds reversibly immobilized on a substrate,
and a capillary, as above, also comprises a drop of solubilizing
fluid suspended from the open end of the capillary. The drop of
solubilizing fluid suspended from the open end of the capillary
element is moved relative to the substrate to place the drop into
contact with a first compound immobilized on the first surface of
the substrate. At least a portion of the compound solubilized by
the drop of solubilizing fluid is then drawn into the microfluidic
channel within the capillary element. These steps may be repeated
multiple times with respect to one or multiple compounds on the
substrate surface.
[0011] The present invention also generally provides devices and
systems for carrying out the methods described herein or methods
similar thereto. For example, in one aspect, the present invention
provides systems for analyzing a plurality of different sample
materials. The systems typically comprise a microfluidic element
comprising a capillary element having at least a first microfluidic
channel disposed therethrough, the capillary element having at
least one open end The system also typically includes a sample
substrate comprising a plurality of different sample materials
reversibly immobilized thereon, each different sample being
immobilized in a different discrete region of the first surface. A
translation system is provided attached to at least one of the
substrate or the microfluidic element, for moving either the
microfluidic element relative to the substrate surface or vice
versa. The system provides for a sensing system for sensing when a
volume of fluid at the open end of the capillary element contacts
the first surface of the substrate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 schematically illustrates an overall microfluidic
system including a microfluidic device, controller, computer and
sample material substrate.
[0013] FIG. 2 schematically illustrates a microfluidic device for
use in high throughput analytical operations.
[0014] FIG. 3 illustrates one aspect of the operation of the
sampling systems of the present invention using a hanging drop of
fluid on the end of a capillary element to resolubilize sample
material on a substrate surface.
[0015] FIG. 4 illustrates an alternate aspect of the operation of
the sampling systems of the present invention using an expelled
fluid volume to resolubilize sample material on the sample
substrate.
[0016] FIG. 5 illustrates a dual capillary embodiment of the
sampling systems of the present invention.
[0017] FIG. 6 schematically illustrates an electrical sensing
system for sensing contact between the fluid at the end of the
capillary and the sample substrate.
[0018] FIGS. 7A, 7B and 7C schematically illustrate alternate
optical sensing systems for sensing contact between the fluid in
the capillary and the surface of the sample substrate.
[0019] FIGS. 8A, 8B and 8C are plots of capacitance phase vs.
distance from the substrate surface for a model capillary
system.
[0020] FIGS. 9A and 9B illustrate two model systems used to
demonstrate the efficacy of certain aspects of the systems of the
present invention. FIG. 9C illustrates the channel layout of an
NS71 sipper chip.
[0021] FIG. 10 shows fluorescent intensity changes as rhodamine
labeled DNA is sipped from a polypropylene card. Sixty spots were
dissolved in total, while 20 peaks are shown in FIG. 10.
[0022] FIG. 11A and B illustrate resolubilization and sampling of
rhodamine labeled DNA from Teflon cards using a single capillary
system and a sipper chip system, respectively.
[0023] FIG. 12 illustrates discrimination of single base mismatches
between the molecular beacon sequence and four liquid
oligonucleotide targets in a microfluidic device.
[0024] FIG. 13 illustrates discrimination of single base mismatches
between the molecular beacon sequence and four dried
oligonucleotide targets that were dried onto a solid substrate,
where the dried reagents were reconstituted and aspirated into a
microfluidic device for the analysis.
[0025] FIG. 14 illustrates discrimination of single base mismatches
between an oligonucleotide target sequence and two molecular beacon
sequences that were dried onto a surface of a solid support, where
the dried reagents were reconstituted and aspirated into a
microfluidic device for analysis.
[0026] FIG. 15 shows measurements from an electrical sensing system
coupled to a microfluidic capillary element and planar solid
substrate during a multiple accession run where the capillary
element was repeatedly contacted with the surface of the planar
solid substrate.
[0027] FIG. 16 illustrates sensing data from an optical sensing
system of the present invention. Specifically shown is a plot of
fluorescence intensity vs. distance between a substrate and
capillary, while a library substrate is moved into and out of
contact with a capillary element of a microfluidic device.
[0028] FIG. 17 schematically illustrates a sleeve offset structure
for providing a fixed offset of sampling elements from reagent
array substrates.
[0029] FIG. 18 is a plot of fluorescence vs. time from a channel in
a microfluidic device which was used to repeatedly sip dried
fluorescent compounds from the surface of a substrate card.
[0030] FIG. 19 is a plot of fluorescence vs. time for repeatedly
sipping multiple different dried fluorescent compounds from the
surface of a substrate card.
[0031] FIG. 20 is a plot of fluorescence vs. time for a continuous
flow HSA binding assay, where fluorescence level is indicative of
the amount of a fluorescent dye bound to HSA, and reductions in
fluorescence are indicative of displacement of that dye.
[0032] FIG. 21 illustrates a TCPTP enzymatic reaction using the
sample substrate array as a source of inhibitors.
DETAILED DESCRIPTION OF THE INVENTION
[0033] I. General System Description
[0034] The present invention generally provides novel devices for
containing large numbers of potential pharmaceutical compounds in a
stabilized addressable format for use in high throughput screening
applications, as well as providing systems that integrate these
devices with microfluidic elements, control elements and data
acquisition and analysis elements, in high throughput screening
applications.
[0035] Generally, the present invention provides a microfluidic
device in which the fundamental aspects of the screening assay are
carried out. Microfluidic devices for use in high throughput
screening applications have been described in detail in U.S. Pat.
No. 5,942,443, which is incorporated herein by reference for all
purposes. Typically, such devices include a main analysis channel
disposed within a substrate or body. The assay reactants are flowed
along the analysis channels along with, at least periodically, a
quantity or plug of a compound that is to be screened ("the test
compound"). The effect of the test compound on the assay reactants
is then ascertained. Although described as screening test compounds
for effects against pharmaceutically relevant targets, it should be
readily appreciated that the devices, methods and systems of the
present invention are broadly applicable to a wide range of
different high throughput analyses, e.g. diagnostic evaluations,
nucleic acid analyses.
[0036] In order to bring large numbers of diverse test compounds
into the channels of the microfluidic device, these devices are
typically outfitted with a pipettor or sampling capillary element.
Specifically, a pipettor element, i.e., a capillary tube, is
typically provided extending from the body of the microfluidic
device, and wherein the lumen or channel of the capillary element
is in fluid communication with the analysis channel of the device,
or another channel of the device that is in fluid communication
with the analysis channel. The sampling element then samples the
test compounds from the library storage component.
[0037] Library storage components have typically comprised
multiwell plates filled with fluid reagents. The sampling element
would simply be dipped into the wells of the multiwell plates and a
sample of each test compound would be drawn into the sampling
element. In accordance with aspects of the present invention
however, the storage component comprises a collection of test
compounds that are removably immobilized within separate discrete
regions of a planar or substantially planar substrate, e.g., a
sheet or card. The sampling element subjects these test compounds
to appropriate conditions to remove the compounds from the surface
of the substrate, e.g., by solubilizing dried compounds. The
solubilized compounds are then individually drawn up into the
sampling element and the microfluidic device, for analysis.
[0038] The overall systems of the present invention also typically
comprise ancillary elements useful in carrying out the screening
analyses, such as material transport systems and controls for
directing material movement through the various fluid conduits of
the system, robotic elements for controlling the relative
positioning of the library storage element and the analysis system,
e.g., the microfluidic device, detection systems, e.g., optical,
electrochemical, thermal, etc., for detecting the results of the
analysis that is being carried out within the microfluidic device,
and a computer or processor for both controlling the operation of
the system as a whole, and for recording and/or analyzing the data
generated by the system.
[0039] FIG. 1 schematically illustrates an overall system in
accordance with the present invention. As shown, the system 100
includes a microfluidic device 102 having a main analysis channel
104 disposed within its interior. A sampling pipettor or capillary
106 is attached to the device 102 such that the channel within the
capillary (not shown) is in fluid communication with the analysis
channel 104. A library storage substrate 108 is provided so as to
be accessible by the capillary element 106. Typically, one or both
of the device 102 and the library substrate 108 are provided
mounted on an x-y-z translation stage 110 that moves one or both of
these components relative to the other. Typically, the x-y-z
translation stage 110 is automatically controlled, e.g., by a
robotic positioning system (not shown). Such robotic x-y-z
translation systems are generally commercially available from,
e.g., Parker-Hannefin, Corp. In the case of preferred aspects of
the invention, the x-y-z translation stage is optionally coupled to
a sensor, illustrated as a box 150, that senses when the capillary
element 106 is sufficiently proximal to the library storage
substrate 108. The sensor 150 may be a stand-alone instrument or
system, or may be incorporated into or made up of other components
of the system, e.g., controller 120, computer 140 or detector
130.
[0040] The system also typically includes a controller instrument
120 operably coupled to the device 102 that controls and directs
the movement of material through the channel or channels of the
device 102. As described in greater detail below, the controller
120 may be an electrical controller, a pressure controller or the
like. In the case of an electrical controller, operable coupling of
the controller to the microfluidic device is typically accomplished
via electrical leads and electrodes placed into contact with fluid
reservoirs 112 in the device. In the case of a pressure based flow
controller, the operable connection is typically provided by one or
more vacuum or pressure lines coupled to the termini of one or more
channels of the device 102.
[0041] A detection system 130 is also typically provided within
sensory communication of the one or more analysis channels 104 of
the device 102. The detection system detects signals from the
analysis channel and the data is collected, stored and/or analyzed
by a computer or processor 140 that is operably coupled to the
detector. As used herein, the phrase "within sensory communication"
refers to a detector that is positioned within or sufficiently
proximal to the analysis channel so as to receive a detectable
signal from the contents of the channel. The computer 140 (or
optionally, an additional computer, not shown) is also coupled to
the controller 120 to control the movement of material within the
channel(s) of the device 102 in accordance with a preprogrammed set
of instructions.
[0042] While typically described herein as an overall system, it
will be appreciated however that the present invention encompasses
all of the components of the overall system, whether in the
aggregate, or as separate and discrete elements. The various
elements of the overall system are each described in greater
detail, below.
[0043] II. Microfluidic Assay Devices
[0044] As noted above, the screening assay methods of the present
invention are generally carried out within one or more microfluidic
channels. As used herein, the term "microfluidic" refers to a
fluidic component, e.g. a channel, chamber, reservoir, or the like,
that includes at least one cross-sectional dimension, e.g., depth,
width, length, diameter, of from about 0.1 .mu.m to about 500
.mu.m. Microfluidic devices having dimensions in these ranges are
described in U.S. Pat. Nos. 5,942,443 and 5,880,071, each of which
is incorporated herein by reference. Typically, such devices are
planar in structure and are fabricated from an aggregation of
planar substrate layers where the fluidic elements are defined in
the interface of the various layers. Specifically, channels and
chambers are typically etched, embossed, molded, ablated or
otherwise fabricated into a surface of a first substrate layer as
grooves and/or depressions. A second substrate layer is then
overlaid and bonded to the first to cover the grooves creating
sealed channels within the interior portion of the device.
[0045] In the case of planar microfluidic devices, substrate
materials can be selected from a wide variety of different
materials, provided such materials are compatible with the desired
analysis to be carried out within the devices, and such substrates
are compatible with microfabrication techniques. In preferred
aspects, the substrate layers are individually selected from
silica-based substrates (e.g., glass, quartz, silicon, fused
silica, etc.), polymeric substrates (e.g., polymethylmethacrylate,
polycarbonate, polytetrafluoroethylene, polyvinylchloride,
polydimethylsiloxane, polysulfone, polystyrene, polymethylpentene,
polypropylene, polyethylene, polyvinylidine fluoride, and
acrylonitrile-butadiene-styrene copolymer, parylene), ceramic
substrates, or the like.
[0046] Although described in terms of a layered planar body
structure, it will be appreciated that microfluidic devices in
accordance with the present invention may take a variety of forms,
including aggregations of fluidic components, e.g., capillary
tubes, individual chambers, etc., that are pieced together to
provide the integrated fluidic elements of the complete device.
[0047] The microfluidic devices of the invention typically include
at least one main analysis channel, but may include two or more
main analysis channels in order to multiplex the number of analyses
being carried out in the microfluidic device at any given time.
Typically, a single device will include from about 1 to about 100
separate analysis channels. Preferably, each device will include
more than 1, more preferably, 4 or more, still more preferably, 8
or more and often, 12 or more analysis channels.
[0048] In most cases, the analysis channel or channel is
intersected by at least one other microscale channel disposed
within the body of the device. Typically, the one or more
additional channels are used to bring the test compounds and assay
reagents into the main analysis channel, in order to carry out the
desired assay.
[0049] One example of a microfluidic device for carrying out
high-throughput assays is shown from a top and end view in FIG. 2.
As shown, the overall device 102 is planar in structure and is
fabricated as an aggregation of substrate layers, e.g., layers 102a
and 102b. The fluidic elements of the device, e.g., channels 104,
206 and 210, are defined in the space at the interface of the two
substrate layers. Typically, this is carried out by etching,
ablating, molding, or embossing one or more grooves into the
surface of one or both of the two substrate layers which are
typically either polymeric, e.g., plastic (see, e.g., U.S. Pat. No.
5,885,470), or silica based, e.g., glass, fused silica, quartz,
silicon, or the like. When the second substrate layer is mated with
and bonded or fused to the first substrate layer, these grooves are
sealed to form conduits or channels within the interior of the body
of the device.
[0050] Sampling capillary 106 is also provided attached to the
device for accessing externally located sources of sample material,
e.g., samples, test compounds, etc., that are being subjected to
the assay in question. As shown, the sampling capillary 106 is open
at one end 106a for accessing external materials, and is fluidly
coupled to at least one channel in the body of the device 102 at
the other end 106b.
[0051] In operation, assay reagents, i.e., enzyme and substrate,
are typically concurrently flowed into the main analysis channel
104 from reservoirs 204 and 208 via channels 206 and 210,
respectively. These reagents are optionally combined with other
reagents, buffers or other diluents from reservoirs 212 and 214,
respectively, brought into the main channel 104 via channels 216
and 218, respectively. Periodically, test compounds, sample
materials or the like are introduced into the main analysis channel
104 from an external library via the external sampling capillary
106. For example, in one aspect, assay reagents are continuously
flowed along the main analysis channel 104 producing a steady state
signal at detection window 220 that is indicative of the
functioning of the assayed system. When a test compound is
introduced that has an effect on the assay system, e.g., as an
inhibitor or enhancer of activity, it produces a deviation from the
steady state signal.
[0052] The structure and/or operation of the sampling capillary or
pipettor element 106 may vary depending upon the specific process
that is to be used to sample materials from the library storage
component. As described in greater detail below, the library
storage component typically comprises substrate or substrate matrix
that includes a large number of different compounds, samples or
other materials to be assayed or screened that are reversibly
immobilized upon its surface in discrete locations. As such, the
pipettor element typically has the ability to present a volume of
fluid at its open end, which fluid is used to solubilize or
otherwise release the sample or test compound material from the
library storage component.
[0053] In its first and simplest aspect, a single capillary or
pipettor element is provided attached to the body structure of the
device, e.g., as shown in FIGS. 1 and 2. As shown in FIG. 3, in
operation, a spacer or solubilizing fluid is introduced into the
capillary element by placing the open end 106a of the capillary
element 106 into contact with a source, well or reservoir 302 of
the spacer or solubilizing fluid, and drawing that fluid into the
capillary channel 106c (Panel A). When the capillary is removed
from the solubilizing fluid well 302 (Panel B), the surface tension
on the fluid results in a small amount or drop 304 of fluid that
remains suspended from the open end 106a of the capillary 106. This
residual fluid or "hanging drop" is then moved over to an
appropriate location on the library substrate 306 (Panel C). The
hanging drop is then moved into contact with the surface of the
library substrate 306 (and the compound or sample immobilized
thereon), at which point the compound or sample on the library
substrate dissolves or is otherwise released into the small volume
of fluid that was the hanging drop. Once released from the
substrate 306, the compound or sample material is drawn into the
capillary channel 106c (Panel D) and then into the analysis channel
of the device (e.g., channel 104 in FIG. 2) whereupon it is
screened in the assay of interest.
[0054] FIG. 4 illustrates a variation of the simple method
illustrated in FIG. 3. In particular, instead of relying upon a
residual amount of resolubilizing fluid that adheres to the end of
the capillary, the system is operated so as to expel a small volume
of the resolubilizing fluid from the end of the capillary. This is
useful in cases where larger amounts of fluid are desired for
resolubilization than may be provided in a hanging drop. Typically,
the fluid is expelled from the capillary end 106a when the
capillary 106 is positioned over the appropriate location on the
sample substrate 306 (Panel C, FIG. 4). Expulsion of a small amount
of fluid is easily accomplished by simply reversing the flow
direction in the capillary channel 106c. Typically, this is
accomplished by either applying a slight positive pressure to the
waste well of the device, e.g., well 222 in FIG. 2. Alternatively,
in electrokinetically controlled aspects, the polarity of an
electric field can be modulated to cause electroosmotic fluid flow
in the desired direction, e.g., out of the open end of the
capillary channel. Again, as with FIG. 3, the material released
from the surface of the substrate is drawn into the capillary
channel 106c and into the analysis channel for analysis.
[0055] Alternatively, a dual channel or dual capillary system can
be used to provide the resolubilizing fluid onto the surface of the
sample substrate. A second channel or capillary element is then
used to draw the material from the surface of the substrate and
into the analysis channel of the microfluidic device. An example of
such a dual channel system is illustrated in FIG. 5.
[0056] As shown in panel A, the overall device (not shown) is
similar to that shown in FIG. 2 except that an additional capillary
element is provided fluidly coupled to a resolubilizing fluid
reservoir. In particular, the device includes a main sampling
capillary 502 and a fluid delivery capillary 504. Each of the fluid
delivery capillary 504 and the sampling capillary 502 have an open
end (504a and 502a, respectively) for expelling fluid onto the
substrate surface 506 and drawing resolubilized sample materials
508 into the sampling capillary. Typically, the sampling and fluid
delivery capillaries are disposed adjacent one another, e.g., as
shown, so that fluid is delivered from the delivery capillary 504
and drawn up into the sampling capillary 502 without moving the
overall device or library substrate 506. The fluid delivery
capillary is fluidically coupled to a source of the resolubilizing
fluid whereas the sampling capillary is fluidly connected to the
analysis channel within the device (e.g., as shown for capillary
element 106 in FIG. 2). The source of resolubilizing fluid may be
integrated within the overall microfluidic device, e.g., as a well
or reservoir in the overall body structure. Alternatively, the
source of resolubilization may be partially or entirely separate
from the microfluidic device. Although described as two discrete
capillary elements, it will be appreciated that two capillary sized
channels could be provided within one element that is attached to
the body of the device where the sampling capillary channel is
connected to the analysis channel, while the fluid delivery
capillary channel is connected to a channel that leads to a
resolubilizing fluid reservoir or well, e.g., as shown for reagent
well 204 and channel 206 in FIG. 2. In particular, the sampling
channel and the fluid delivery channel would be disposed within a
single capillary or pipettor element that is attached to the body
structure of the overall device, itself.
[0057] The operation of this system is also schematically
illustrated in FIG. 5. In particular, the capillaries are
positioned adjacent to an immobilized compound or sample 508 on the
library substrate 506 (Panel A). Fluid 510 is expelled from the
fluid delivery capillary 504 onto the substrate surface, whereupon
the compound or sample material 508 is at least partially
resolubilized into the expelled fluid (Panel B and C). A portion of
the fluid on the substrate is then drawn into the sampling
capillary 502 and into the analysis channel, as described above
(Panel C). The capillaries 502 and 504 are then moved away from the
substrate 506 (Panel D) to either draw in a spacer fluid plug to
separate the resolubilized material from a subsequent sample
material, or positioned adjacent another immobilized test compound
for sampling.
[0058] As noted above, the particular spot or area of sample
material, e.g., sample material 508, may be provided such that is
substantially entirely solubilized and drawn into the sampling
capillary 502. However, preferably, each sample material region
includes sufficient material such that it can be sampled multiple
times, e.g., 2, 3, 5, 10 or more times. This is discussed in
greater detail herein, with respect to library substrates.
[0059] Although primarily described in terms of the preferred
embodiments where the sampling pipettor or capillary is fluidly
coupled to an analysis channel within a microfluidic device to
which the capillary is attached, it will be appreciated that the
methods and systems described above work equally well in
non-integrated assay systems. For example, in one aspect, the
solubilized sample material drawn into the sampling capillary is
transported to a discrete and separate reaction vessel for
analysis, e.g., by moving the pipettor or capillary over or within
the reaction vessel or well, e.g., a well in a multiwell plate, or
the like. The positioning of the vessel and/or the sampling
capillary is typically accomplished by placing one or both of the
vessel and/or the sampling capillary on a translation stage, e.g.,
a x-y-z translation stage, to move the vessel or capillary into the
appropriate position relative to the other. Once positioned, the
sampling capillary expels the solubilized sample material into the
reaction vessel or well in which the desired analysis is carried
out.
[0060] III. Sample Accession
[0061] As noted previously, the above-described microfluidic device
carries out high-throughput experimentation by accessing a large
number of diverse reagents from outside the device itself, e.g.,
from a reagent library. In the case of the present invention, this
library typically takes the form of a card or substrate that has a
large number of discrete quantities of different test compounds
removably immobilized thereon. By "removably immobilized," is meant
that the sample materials are present upon the sample substrate in
an immobilized format, e.g., confined in a discrete region, but are
removable from that substrate through appropriate action.
[0062] By way of example, samples that are deposited and dried upon
the sample substrate are removably immobilized in that the dried
reagents remain within their confined space, but are removable by
dissolving them in fluid and pulling the fluid off of the sample
substrate. A variety of other types of removable immobilization are
optionally used in conjunction with the present invention. For
example, structural barriers are used to confine liquid sample
materials within a confined region of the sample substrate while
permitting removal of those liquid sample materials. Similarly,
porous sample matrices are optionally used to retain fluid reagents
within a confined space on the sample substrate. Such sample
materials are then removable by withdrawing the fluids from the
pores of the substrate. Alternatively, sample materials may be
coupled to substrate matrices, e.g., through ionic, hydrophobic or
hydrophilic interactions, covalent but severable interactions,
which couplings are severable by exposing the substrate to an
appropriate environment, e.g., high or low salt buffer solution,
organic buffer, thermal dissociation or release (e.g., where the
matrix incorporates a thermally responsive hydrogel, which expands
or contracts upon heating, to expel entrained compounds), light or
other electromagnetic radiation (e.g., in the case of photolabile
linker groups) etc. Such selectively releasable compound materials
are also particularly useful in applications where particular
compound locations are to be revisited multiple times, as discussed
further herein. For example, a limited quantity of material can be
released by the controlled exposure of the material to the cleaving
agent or environmental conditions, e.g., heat, light, etc.
Additional material is then released upon a subsequent visit using
additional cleaving agents or environmental conditions. By way of
example, if a particular compound deposit is tethered to the
substrate using a photocleavable linker, a relatively precise
amount of material could be released upon each visit by adjusting
the intensity or duration of photoexposure of the compound
deposit.
[0063] In particularly preferred aspects, the sample materials are
provided dried upon or within the sample substrate. Typically, such
sample substrates are readily prepared by any of a variety of
different methods. In particular, simple pipetting methods are
optionally used to spot the sample materials in discrete regions of
the sample substrate. Alternatively, for higher density collections
of sample materials, ink-jet printing methods are readily employed
to print or direct fluid sample materials onto discrete regions of
the sample substrate, whereupon they are lyophilized in place.
These various methods optionally benefit from the presence of
constraining regions on the surface of the sample substrate, e.g.,
raise barriers/depressions on the surface, hydrophobic barriers
surrounding hydrophilic regions or vice versa.
[0064] In certain preferred aspects, the discrete quantities of
sample material on the sample substrate are present in sufficient
quantities or over a sufficiently large area so as to permit
multiple samplings of each different sample material. In
particularly preferred aspects, each discrete quantity of material
on the sample substrate contains a sufficient amount of material to
permit sampling from the material spot more than one time,
preferably, more than two times, more than three times, often more
than five times, and in some cases, more than ten times. Typically,
the amount of material required for multiple samplings is dependent
upon the nature of the sampling system used. Typically, each
sampling will deposit an amount of solubilizing fluid that is 100
nl or less, preferably, 10 nl or less, and often, 1 nl or less. In
accordance with the multiple sampling aspects of the system, the
quantity of sample material should be only partially dissolved in
the solubilizing fluid. By "partially dissolved," is meant that
only a portion of the material in a discrete sample quantity is
solubilized at a given time. The partial dissolution includes
instances where the solubilizing fluid is only deposited upon a
portion of the sample material region dissolving all of the
material in that portion of the sample material region, or
alternatively, deposited upon the entire sample material region but
wherein the material is not completely dissolved.
[0065] Typically, screening assays are performed on compounds that
are present at concentrations in the micromolar range, e.g., from
about 1 to about 20 .mu.M. In the present invention, compounds are
typically sipped into the assay system in nanoliter range volumes.
As such, if one assumes the dissolution volume, e.g., from a
hanging drop is approximately 10 nl, then it is generally desirable
to have the compounds present upon the surface in amounts at or in
excess of about 5 femtomoles. Of course, depending upon the
activity or efficacy of a given compound in a system, this amount
can vary greatly. Similarly, these amounts can change significantly
depending upon the number of times a particular sample spot is
accessed. In general, each discrete quantity of sample material
will contain between 0.5 pg and 100 ng of sample material,
preferably, between about 5 pg and about 10 ng or between about 10
femtomole and about 20 picomoles of material and preferably between
about 10 and about 1000 femtomoles of material. Typically,
materials that are present in these amounts are more than adequate
for 1.times., 2.times., and 3.times. and even upwards of 5.times.
to 10.times. sampling from each spot. The spots are typically
deposited in solutions that range from about 10 nM to about 10
.mu.M. The concentration and amounts of compounds deposited upon
the substrate surface typically depends upon the amount of material
that is to be sampled, e.g., per sampling or in a number of
samplings. Typically deposited compounds are present at quantities
that are greater than or equal to about 1 pmole/mm.sup.2.
[0066] In order to facilitate rapid solubilization of the sample
material, in certain aspects, it is preferred to provide the sample
material in a thin layer on the surface of the substrate (or its
pores). For example, materials are typically deposited upon the
substrate at concentrations and quantities calculated substantially
to provide a molecular monolayer or near monolayer of the compound
species. In some cases, materials are deposited at greater than
monolayer quantities, often falling between about one and twenty
times monolayer quantities.
[0067] For a porous substrate, e.g., a honeycomb matrix, because
the sample material is entrained in the porous substrate matrix,
the amount of surface area covered by a particular sample material
is much greater per unit of external surface area than in the case
of non-porous substrates. As such, much greater amounts of sample
material can be provided in the same or smaller external surface
area than in non-porous substrates.
[0068] The cards described herein are typically fabricated from any
number of different materials, depending upon the nature of the
material to be deposited thereon, the desired quantity of material
to be deposited thereon, etc. For example, for some applications,
the card comprises a solid non-porous substrate where the sample
materials are spotted or deposited upon one planar surface of the
card. Such substrates are typically suitable where it is less
important to maximize the amount of material on the sample
substrate. Examples of these non-porous substrates include, e.g.,
metal substrates, glass, quartz or silicon substrates, polymer
substrates or polymer coated substrates including, e.g.,
polystyrene, polypropylene, polyethylene, polytetrafluoroethylene,
polycarbonate, acrylics, i.e., polymethylmethacrylate, and the
like.
[0069] Substrate surfaces may be of the base substrate material or
may comprise a coating on a rigid substrate base. For example, in
the case of glass substrates, the surface of the base glass
substrate may be treated to provide surface properties that are
compatible and/or beneficial to the reagents deposited thereon.
Such treatments include derivatization of the glass surface, e.g.,
through silanization or the like, or through coating of the surface
using, e.g., a thin layer of other material such as a polymeric or
metallic material. Derivatization using silane chemistry is well
known to those of skill in the art and can be readily employed to
add amine, aldehyde, diol or other functional groups to the surface
of the glass substrate, depending upon the desired surface
properties. Alternatively, a glass substrate layer may be provided
over the surface of another base substrate, e.g., silicon, metal,
ceramic, or the like.
[0070] In the case of polymer substrates, as with the glass or
other silica based substrates described above, the sample substrate
may be entirely comprised of these polymer materials, or such
materials may be provided as a coating over a support element,
e.g., metal, silicon, ceramic, glass or other polymer or plastic
card, e.g., to provide sufficient rigidity to the library
substrate. In some cases, metal substrates are used either coated
or uncoated, in order to take advantage of their conductivity, as
described in greater detail below.
[0071] Further, in the case of metal substrates, metals that are
not easily corroded under potentially high salt conditions, applied
electric fields, and the like are preferred. For example, titanium
substrates, platinum substrates and gold substrates are generally
suitable for this reason, although other metals, e.g., aluminum,
stainless steel, and the like, are also useful. Of course, for cost
reasons, titanium metal substrates are generally preferred where no
external coating is being applied.
[0072] Alternatively, where greater amounts of material are desired
to be immobilized upon the card, porous materials are used. In
particular, porous materials provide an increased surface area upon
which sample materials may be immobilized, dried or otherwise
disposed. Porous substrates include membranes, scintered materials,
e.g., metal, glass, polymers, etc., spun polymer materials, or the
like.
[0073] Examples of particularly useful porous substrate materials
include substrate matrices such as aluminum oxide, etched
polycarbonate substrates, etched silicon (optionally including a
polymer or other compound compatible coating, and like substrates
that comprise arrayed honeycomb pores, e.g., hexagonal pores. Such
substrates are particularly preferred for their ability to maintain
liquid samples within a confined area. Specifically, because of
their porous nature, fluids deposited upon a surface of the matrix
do not laterally diffuse across the substrate surface to any great
extent. Instead, the fluids wick into the pores in the substrate.
This property allows sample materials to be deposited upon the
substrate matrix in relatively high densities without concern for
samples diffusing together across the substrate surface or through
the interstices of the matrix. In addition, the pores in the sample
substrate provide a greatly increased surface area as compared to
non-porous substrates, upon which greater quantities of sample
material may be deposited in a monolayer or otherwise thin coating,
as described in greater detail herein.
[0074] Other useful substrate materials include conventional porous
membrane materials, i.e., nitrocellulose, polyvinylidine difluoride
(PVDF), polysulfone, polyvinyl chloride, spun polypropylene,
polytetrafluoroethylene (PTFE), and the like. However, honeycombed
matrices are generally more preferred as far as porous matrices are
concerned, due to their ability to contain the spotted materials
within discrete sets of pores, rather than permitting their
diffusion across or through the substrate.
[0075] As noted above, the type of sample substrate often depends
upon the nature of the sample material that is to be deposited upon
it. In turn, the sample materials to be deposited upon the sample
substrate or card depend, of course, upon the type of screening
application one is performing. For example, in pharmaceutical
screening operations, the test compounds will range from complex
organic substances to peptides, proteins, carbohydrates, nucleic
acids and the like, which may have been produced in combinatorial
synthetic processes or isolated from natural sources. It is then
desirable to individually assay each material on the substrate in
order to determine whether that compound possesses any useful
pharmacological activity.
[0076] For other screening applications, sample materials in
accordance with the present invention include biological
macromolecules, e.g., proteins, peptides, nucleic acids or
fragments thereof, including, DNA, RNA, double or single stranded,
peptide nucleic acids (PNA), lipids, etc. In the case of these
latter compounds, the sample substrate also can serve as an array
of sources of material for analysis of a particular sample
material. Specifically, sample substrates may be provided with
arrays or collections of different oligonucleotide probes, primers,
or the like. Such collections or arrays are then selectively
accessible by a microfluidic device or system whereby the probes or
primers can be used to examine a sample material, e.g., a target
nucleic acid, for identification, sequencing or the like (see,
e.g., Published International Patent Application No. WO 98/45481,
which is incorporated herein by reference in its entirety for all
purposes. Such collections of materials are useful in a variety of
research and diagnostic fields, including nucleic acid sequencing,
characterization, diagnostics and the like.
[0077] The sample substrates of the present invention typically
include relatively large numbers of different sample materials
within relatively small substrate areas. In particular, the sample
substrates described herein typically include at least 10 different
and discrete quantities of sample material immobilized, dried or
otherwise contained within a square cm of substrate surface area.
In preferred aspects, the sample materials are present at a density
greater than 100 samples/cm.sup.2, preferably, greater than 500
samples/cm.sup.2, often greater than 1000 samples/cm.sup.2, and in
some cases, more than 10,000 samples/cm.sup.2. As noted above,
preparation of high-density arrays of sample materials is
facilitated by the use of ink-jet or related fluid direction
systems (see, e.g., U.S. Pat. No. 5,474,796, as well as by the use
of appropriate low diffusion sample substrate materials.
Alternatively, pin or quill based contact printing or spotting
methods may be readily employed, where a pin or quill is first
dipped into the reagent of interest. The pin, with a quantity of
material on its end, is then contacted with the surface of the
reagent array substrate, whereupon the material is transferred to
that surface. Arrays of pins/quills are used simultaneously to
sample from multiple reagent sources, e.g., wells in a 96, 384 or
1536 well plate and transfer material to the surface of the reagent
array substrate.
[0078] In certain preferred aspects, the samples that are
reversibly immobilized on the surface of the sample substrate are
provided in a form that permits easier deposition, drying, release
and/or solubilization of those compounds from that surface,
depending upon the particular application that is contemplated. For
example, in one optional embodiment, compounds that are spotted and
dried onto the substrate surface comprise, in addition to the
particular compound or compound mixture, include at least one
excipient material that enhances one or more of the deposition
and/or the solubilization of the compound in the appropriate
solubilization liquid. Such excipients also function as binding
agents for the dried compound to enhance the deposition of the
compound material on the substrate. Similarly, excipient materials
can aid in the controlled dispersion of liquid on the surface of
the substrates during the spotting operation. Examples of
excipients include starches, dextrans, glycols, e.g., PEG, other
polymers, e.g., polyethylene oxide, polyvinylpyrrolidone,
detergents as well as simple sugars, e.g., sucrose, fructose,
maltose, trehelose, and modified versions of these, and the like.
The excipient material is typically provided as a mixture with the
various test compounds or compound mixtures, which are then spotted
onto the substrate surface and dried.
[0079] Alternatively, or additionally, the test compounds are dried
on the substrate surface by a freeze drying that yields test
compounds that are generally in a more soluble format, e.g., by
virtue of their greater surface area. In particular, freeze drying
techniques typically result in materials that are "fluffier" in
terms of their physical state, and are therefore more easily
dissolved. A variety of other drying methods may be employed
depending upon the nature of the reagents being provided on the
array substrate, including heat drying, vacuum drying, drying under
controlled atmosphere, e.g., alkane or alcohol vapor, or the
like.
[0080] In addition to providing a source of different test
compounds, reagents or the like, in some aspects, the sample
substrate optionally functions as an intermediate staging area for
the operations that are performed within the channels of a
microfluidic device or as a holding area for slower reactions, and
the like. Specifically, a portion of, or an entire sample substrate
can be used to, e.g., premix several reagents, stage randomly
accessed reagents, and/or provide multiple dilutions of particular
reagents, prior to introduction into the microfluidic device.
[0081] In some cases, reagents that are to be combined in a
particular reaction are less compatible with the mixing kinetics of
a microfluidic device, e.g., they diffuse or react so slowly that
there is insufficient time to mix and react reagents during the
rapid processing operations of a microfluidic system. For example,
molecules having slow diffusion kinetics include large molecules or
molecules in viscous medium. Reagents that have slower reaction
kinetics, e.g., certain enzymes and substrates, are also optionally
mixed outside of the channels of a microfluidic device, and allowed
to react for a set time before being sampled into the channels of
the microfluidic device. Alternatively, one may wish to mix several
different reagents for a particular analysis, which in a capillary
channel can require substantial diffusion times, e.g., for serial
plugs of material to completely diffuse into each other. As such,
in accordance with certain aspects of the invention, one can
deposit various reagents that are to be mixed upon a portion of a
sample substrate where the reagents are permitted to mix for
sufficient time. The resulting mixture is then sampled into a
microfluidic device as described herein. Typically, such mixtures
can include a first reagent combined with a second reagent, and
alternatively a third reagent, fourth reagent and fifth reagent can
be added, either to the first and second reagent as a complete
mixture, or as separate combinations in different portions of the
intermediate sample substrate.
[0082] Relatedly, one can mix different combinations of reagents
onto a portion of a sample substrate prior to introducing the
mixtures into a microfluidic device. For example, one could deposit
a first reagent in multiple regions of a sample substrate, and add
to that reagent multiple different reagents, which would then be
introduced into a microfluidic device. This allows one to perform a
pseudo-combinatorial process of reagent combination and addition.
Additionally, such staging allows for mixing different reagents for
introduction into a multiple sipper capillary system, e.g., where
fixed capillary elements cannot randomly access all reagents in a
sample array. In particular where a microfluidic device employs
multiple accession capillaries having regular rigid spacing, one
cannot randomly access different samples with each of the different
capillaries, e.g., each sampling accesses multiple samples that are
on the same spacing as the capillaries, which spacing does not
change. Accordingly, in order to access a different combination of
samples in a multiple capillary system, it is useful to reposition
one or more samples on an intermediate staging substrate in an
orientation that is different from the orientation that such
samples had in the original sample substrate. The repositioned
samples, test compounds, reagents, etc., then may be simultaneously
sampled by a multiple capillary device where such simultaneous
sampling could not have occurred in the original orientation of the
samples in the original sample substrate array.
[0083] In a further related aspect, one can separately hydrate
compounds before sipping them into the capillary element as
adjacent plugs, e.g., to allow mixing and reaction within the
capillary element or any associated channel. Extrapolating this,
one can also hydrate and sip a first reagent into a capillary
element, and then use that hydrated reagent to hydrate a subsequent
reagent, e.g., through aspiration of the hydrated first reagent
from the capillary or as a hanging droplet of the first
reagent.
[0084] Finally, in addition to mixing reagents with other reagents,
one can use the intermediate staging process to mix reagents with
diluent in order to provide one or multiple different dilutions of
the particular reagent or reagents prior to introducing it into the
microfluidic device.
[0085] The portion of the sample substrate that is to be used as
the staging area can be on a discrete sample substrate, or it can
be a previously unused region of the sample substrate from which
test compounds are originally obtained. In order to utilize the
staging aspect of the sample substrate, one can deposit the
reagents into particular regions of the sample substrate using a
variety of methods. For example, well-known pipetting methods may
be utilized to add reagents to the surface of the sample substrate.
Alternatively, printing techniques, e.g., ink-jet printing
techniques can be used for this reagent staging in much the same
way such methods are utilized in spotting test compounds onto
sample substrates, as described herein. In certain preferred
aspects, a pipetting system similar to the accession systems
employed in conjunction with the microfluidic devices described
herein is employed. Specifically, a microscale capillary element is
used in conjunction with a pumping system, e.g., a vacuum pump to
draw different reagents into the capillary and then dispense those
fluids onto the intermediate staging substrate. The different
reagents are optionally sampled iteratively into the capillary and
then dispensed en masse, onto the substrate, or they are
individually sampled and dispensed successively onto the
substrate.
[0086] The present invention also provides the above-described
sample substrates in conjunction with a high-speed, highly accurate
sampling system for sampling the sample materials from the sample
substrate, and transporting those materials to an analytical
element where the sample materials are scrutinized, e.g., for
content, make-up, or effect on other systems, e.g., biological
systems. In particular, the sampling system typically comprises a
pipetting or capillary element, e.g., as described in substantial
detail, above.
[0087] As alluded to above, because the sampling systems of the
present invention contain large numbers of discrete sample
materials for assaying in very small areas, the system for
accessing these materials must be highly accurate and very fast.
For example, in contacting a droplet on the end of a capillary
element with a substrate, it is often necessary to position the
capillary to within a matter of microns, e.g., 10 to 500 .mu.m from
the substrate surface, to allow the droplet to contact that
surface. However, surface variations in substrates typically makes
it difficult to program such positioning ahead of time, so as to
yield consistent positioning of the capillary element relative to
the substrate.
[0088] Accordingly, the systems of the present invention include a
sensor component for detecting when the droplet on the capillary
end has contacted or is positioned sufficiently proximal to the
surface of the substrate. Generally, a capillary element or the
drop disposed thereon is sufficiently proximal to the substrate if
is within 1 mm or less, typically, 0.5 mm, 0.2 mm, 0.1 mm or even
less, and typically is actually contacted with the substrate. In
preferred aspects, optical or electrical sensor systems are
utilized in performing this function, e.g., to sense whether the
droplet has been contacted with or moved sufficiently proximal to
the substrate surface.
[0089] For example, in at least one aspect, an electrical signal is
used to sense when the drop contacts the surface of the card. FIG.
6 is a schematic illustration of an example of a sensing system
according to the present invention. As shown, the system 600
includes a microfluidic device 602 that comprises a capillary
element 604. A lock-in amplifier 606 is also provided which is
connected to the fluid within the microfluidic device and capillary
element, e.g., via an electrical lead 614 to a reservoir or well
608. Optionally, the lock-in amplifier is substituted with a
capacitance or conductivity meter. The lock-in amplifier is also
connected to the sample substrate 610, or the support element 612
underlying the sample substrate, e.g., via lead 616.
[0090] In general operation, a current is applied to the circuit
shown in FIG. 6, e.g., through leads 614 and 616. In this system,
the "droplet-air gap-sample substrate" functions as a capacitor.
The phase of the current relative to the current in a reference
channel is a function of the relative impedances of the capacitor
and any resistive impedances (such as the resistance represented by
the fluid in a channel or capillary) in the circuit. As used
herein, a reference channel typically includes a simple electrical
circuit that is independent of the circuit through which
capacitance is being measured, and lacks the varying capacitance of
the fluid channel-droplet-air gap-substrate capacitor. In the case
of the particular circuit shown, the lock-in amplifier 606 applies
an alternating voltage to lead 616, the capacitance and/or
resistance between the substrate and the droplet on the capillary
604 and fluid results in a current in lead 614, which is measured
by the lock-in amplifier via a low impedance input. The magnitude
and phase of the current in lead 614, compared to the voltage in
lead 616 indicates the capacitive and/or resistive coupling from
the substrate through the capillary. Optionally, the voltage can be
applied on lead 614 and current can be measured on lead 616 or the
current can be measured on the voltage applying lead.
[0091] Besides simply looking at the phase of the current
travelling through the circuit, one can also monitor the component
of the current that is 90 degrees ahead in phase of the reference
current or applied voltage, since this corresponds to the
capacitive portion of the circuit. The presence of capacitance in
the circuit results in a shift in the phase of an alternating
current coupled through the circuit, e.g., relative to a reference
signal/applied voltage. Thus, where the droplet is separated from
the substrate surface, e.g., by moving the substrate relative to
the droplet on x-y-z translation stage 110, the capacitance of the
"droplet-air gap-sample substrate" portion of the circuit is
changing. The change in capacitance becomes increasingly fast as
the drop approaches the surface of the sample substrate, thus
yielding an increasingly fast change in the phase of the current.
Contact between the drop and the substrate, which is also
accompanied by a change in the geometry of the drop, yields further
changes in the phase of the current. The sum of these changes is a
rapid and detectable change in the phase (or equivalently, the
component of the current 90 degrees ahead in phase of the reference
signal/applied voltage) that occurs when the drop contacts the
surface of the substrate. By monitoring the phase shift of the
current through the circuit as noted above, one can monitor the
relative proximity and even contact of the droplet with the
surface. In FIG. 6, the system is illustrated as having the
electrical connection to a reservoir of the overall microfluidic
device such that the sensing current is applied through the
channels of the device and the capillary element. However, it will
be readily appreciated that this is primarily for convenience.
Specifically, in certain aspects, it may be desirable to provide
the electrical connection for applying this current into the
droplet of fluid at the end of the capillary, e.g., through an
electrode arrangement. Typically, this is accomplished by providing
an electrically conductive layer along the outer surface of the
capillary element such that a fluid droplet at the end of the
capillary element will be in contact with the conductive layer.
This layer may be a coating over the outer surface of the capillary
element or may be patterned or otherwise deposited on that outer
surface or a portion thereof.
[0092] Although described in terms of measuring the phase of the
current applied through the capacitive portion of the circuit, it
will be appreciated that the presently described sensing methods
rely either directly or indirectly on a measurement of the changes
in the capacitance of the overall circuit as a measure of the
proximity of the drop to the surface. Capacitance measurements may
take the form of phase measurements as described herein, or may be
direct measurements of the capacitance of the circuit, e.g., using
a capacitance meter. As described in greater detail herein, other
electrical parameters also provide a basis for measurement.
[0093] Because the system relies upon the capacitance of the
circuit, it enables the surface of the substrate to be a thin,
non-conductive layer overlaid upon a conductive supporting member,
e.g., from about 10 .mu.m to about 1000 .mu.m, and preferably from
about 10 to about 500 .mu.m thick. In particular, it is often
desirable to retain the sample materials on inert substrates so as
to avoid any adverse interactions between the sample material and
the substrate surface. Typically preferred inert layers include,
e.g., polytetrafluoroethylene (Teflon.TM.), acrylic, e.g., PMMA,
polypropylene, polystyrene, polycarbonate, metal oxides, SiO.sub.2,
Si.sub.3N.sub.4, silicon oxynitride and the like. In alternate
aspects, however, conductivity is optionally used as the electrical
signal. In particular, completion or closing of the overall circuit
by contacting the drop of fluid with the substrate surface is
detected and used as the indicative signal. In such cases, it is
generally preferred to utilize a substrate surface that is
conductive, e.g., a metallic substrate such as aluminum, titanium,
platinum, gold, stainless steel, or the like, or semiconductive,
e.g., silicon. In these cases, a current applied to the circuit can
be alternating or direct. In addition, the system, while clearly
capable of sensing actual contact between the droplet and the
substrate surface also is capable if sensing when the droplet
approaches the surface, allowing one to sense an electrical signal
that is indicative of the distance between the fluid and the
substrate, rather than sensing actual contact.
[0094] In addition to electrical sensing systems, optical sensing
systems may also be used in this aspect of the invention, e.g.,
optically sensing when the drop has contacted the surface of the
substrate. As used herein, the term "optical sensing" specifically
excludes the direct observation of the contact between the drop and
the substrate by the human eye. Instead, an automatic and/or remote
sensing operation is envisioned. In at least one aspect, light from
a light source is directed down the capillary element. As the light
exits the capillary, it diverges. The light exiting the capillary
reflects from, or in some cases excites fluorescence on the surface
of the substrate.
[0095] In preferred aspects, the substrate surface is itself
fluorescent or has a fluorescent material associated with it, e.g.,
coated thereon. The fluorescent material may be coated directly on
the surface or it may be incorporated within another surface
coating, e.g., a polymeric material. In some aspects, e.g., where a
very thin translucent polymeric layer is applied as the surface of
the substrate, the fluorescent coating may be applied underneath
the polymeric layer, so as to not interfere or intermingle with any
of the chemical compounds or other materials on the surface of the
substrate.
[0096] While the capillary is distant from the substrate surface,
the reflected or fluoresced light collected back through the
capillary is significantly reduced by divergence of the light upon
leaving the capillary, which results in a lower power density of
incident light on the surface of the substrate. Divergence of the
reflected or fluoresced light from the surface of the substrate
also results in lower levels of recollected reflected or emitted
light from the surface. However, as the capillary element is moved
closer to the substrate, the light exiting the capillary is not
permitted to diverge as far before being incident on the substrate
surface. Further, the capillary collects a larger percentage of the
reflected or fluoresced light, which is then detected. By setting a
minimum threshold of collected light, or sensing a dramatic change
in the collected light, one can then determine that the capillary
is sufficiently close to the substrate surface. Changes in the
geometry or shape of a drop at the end of the capillary also can
have an effect on the level of collected light.
[0097] A schematic illustration of an exemplary optical detection
system is illustrated in FIG. 7A. As shown, the system 700 includes
a microfluidic device 102, e.g., as shown in FIG. 1, which includes
a capillary element 106. Light from a light source 702 is directed
down through the capillary element 106 which functions as a light
pipe. Upon exiting the open end of the capillary 106a, the light
diverges and is incident upon the surface of substrate 704, upon
which reagent and/or sample materials are spotted or otherwise
deposited. Reflected light and/or emitted fluorescence from the
surface of the substrate also diverges, with a fraction of the
reflected or emitted light being collected back through the
capillary element 106. This fraction of collected light is then
passed up through the capillary element 106, through the top layer
of the microfluidic device 102, and directed to a detector 706,
through an optical train, as represented by beamsplitter 708
(however, more complex optical trains are also envisioned,
including objective lenses, spatial and spectral filters,
additional dichroic beamsplitters and the like). When the substrate
is moved closer to the open end of the capillary element 106, the
light is not permitted to diverge as far before being incident upon
the surface of substrate 704. This results in a higher power
density for the light incident upon the substrate surface, which in
turn yields greater reflected or fluoresced light. Further, because
the capillary 106 is closer to the surface of substrate 704, the
reflected or emitted light is not permitted to diverge as far
before being collected by the capillary 106, resulting in a higher
percentage of collected light. This higher percentage is then
detected and compared to a threshold level to determine whether the
capillary is sufficiently close to the substrate surface.
[0098] Relatedly, once the substrate is contacted, the amount of
fluorescence emitted from the surface and collected through the
capillary will not change, despite movement of the substrate
relative to the surface. Thus, monitoring a stabilization of
fluorescence levels also provides an indication of contact between
the capillary end and the substrate surface. Of course, this method
is less preferred as it requires contact between the capillary end
and the substrate surface, which can potentially have adverse
effects on the capillary, the substrate surface and/or materials
deposited on the surface.
[0099] Although generally described in terms of methods of
measuring light transmission of the capillary/drop/substrate system
as a method of monitoring the relative positions of the capillary
and the substrate, optical methods also include imaging systems.
Specifically, in certain aspects, an optical sensing system
comprises an imaging detector, e.g., a camera, CCD, or the like,
disposed adjacent to and focused upon the capillary element and
substrate surface. The imaging detector is operably coupled to a
computer. The computer, programmed with image analysis software,
recognizes from the image detector when the capillary element is
sufficiently proximal to or contacting the substrate surface. One
example of an imaging based system for monitoring sampling from a
library card is illustrated in FIGS. 7B and 7C, from a perspective
and side view, respectively.
[0100] As illustrated in FIGS. 7B and 7C, the overall system
includes a reagent library card or array substrate 704 as described
above upon which is deposited a number of discrete quantities of
material or materials (not shown). As described above, such
materials may be dried or otherwise reversibly immobilized on the
surface of the reagent array. In the particular embodiment
illustrated in FIGS. 7B and 7C, the reagent array substrate 704 is
fabricated from a transparent material, e.g., glass, quartz, or a
transparent or translucent polymeric material, e.g., polycarbonate,
acrylic (e.g., PMMA), polystyrene, or the like. A microfluidic
device 102 that includes one or more external sampling capillary
elements 106 is positioned above the array's surface such that the
sampling element(s) are capable of being moved down to sample the
materials from the surface of the array substrate 704. As shown,
the microfluidic device 102 is placed onto a platform 720 that
supports the device 102 over the array substrate 704, while the
array substrate is placed on platform 722 that supports the array
substrate 704 beneath the microfluidic device 102. One or both of
platforms 720 and 722 are mounted on an x-y-z translation robot
(partially shown as robot arm 724) to move the array and the
microfluidic device relative to each other. As shown, platform 722
also includes an optional reagent trough 730 for sampling reagents,
buffers or the like that are used consistently throughout a number
of separate analyses.
[0101] A video imaging system, e.g., a camera 726, CCD, or the
like, is positioned below the array substrate and images the array
substrate 704 from the underside. As shown, the camera images the
underside of the array substrate 704 via an angled mirror 728 that
reflects the underside of the array toward the camera 726. When the
sampling element is robotically moved down to the surface of the
array, such that a droplet of fluid at the end of the sampling
element contacts the material on the array surface, the imaging
system records the event through the transparent array substrate
704. In alternate aspects, one or more additional imaging systems
or cameras may be provided, e.g., directed at the sampling element
and sample array substrate from the side, to permit more accurate
imaging of the distances, and thus, the contacting event, between
the sampling element and the substrate surface. It will be
understood that either the array or the microfluidic device or both
may be moved to contact the sampling element with the surface of
the array. In preferred aspects, it is the reagent array substrate
that is primarily moved toward the sampling element of the
microfluidic device, so as to avoid moving the detection and
control elements associated with the microfluidic device. Image
analysis software then recognizes that the sampling element has
contacted the surface of the array. This is preferably accomplished
by the system recognizing the contact of fluid at the end of the
capillary element with the surface of the array. However, in those
cases where the camera or imaging system is focused on the end of
the sampling element, e.g., for position alignment as described
below, contact or near contact with the surface of the array may be
indicated when the spotted materials on the surface of the array
come into focus in the imaging system, e.g., the sampling element
has moved sufficiently close to the surface such that both the end
of the sampling element and the surface of the array are in the
focal plane of the imaging system. Again, image analysis software
is readily configured to recognize these events.
[0102] Recognition of contact or near contact with the array
surface then stops the advancement of the robot. The sample
material is then drawn into the sampling element for analysis
within the microfluidic device, and the robot then lifts the sample
away from the card surface and moves the sampling element to sample
additional materials, e.g., spacer buffers and/or additional
immobilized samples.
[0103] The imaging system is also used in conjunction with
alignment of the sampling elements to the reagent regions on the
surface of the array substrate. In accordance with preferred
aspects of the present invention, the reagent materials that are
immobilized on the array are positioned in a regularly spaced
rectangular grid, e.g., spotted in one or more rows of compounds in
a gridded format at regular intervals. The spacing of the materials
typically depends upon the number of different material that are to
be spotted onto the array, the available surface area of the array,
the desired quantity of material in each spot, etc. As such,
specific dimensions for spacing can vary greatly. Typically,
materials will be positioned to be within one to several
millimeters of adjacent spots, and will be oriented in rows and/or
columns of spots. Because of their regular gridded spacing, the
system is aligned by locating outer spots in the grid and
interpolating those that fall between. For example, in multiple row
grids, location of the various compound locations on the substrate
involves location of the four corner most spots on the array, e.g.,
through manual alignment or through the inclusion of markers in the
spots or on the substrate that permit their automatic location, and
calculating or interpolating the position of all of the spots that
are located between those four corners. In general, alignment can
rely upon location of two points on a particular row, or on
location of any three spots on an array of multiple rows to
identify where the remaining spots are located, although using the
four corner spots permits the highest confidence in the alignment
procedure. In some cases, where one has identified the relative
position of the first spot accessed, one can extrapolate the
location of all other spots in the array. For example, if one first
locates one or more given spots, knowing the relative location of
those spots, the orientation of the array, etc., one can then
interpolate and extrapolate the location of the other spots on an
array. In such cases, it is not necessary that the first identified
spots be located at the ends of any given row or rows in the array,
e.g., the four corners.
[0104] In operation, the system moves the sampling element to a
position that is close to the expected position of one of the
corner spots (the "first position"). The sampling element is then
aligned to the first spot by locating that spot with the imaging
system and analysis software, and adjusting the positioning of the
array using the x-y translation of the robotics system. The first
spot may be located by virtue of its own optical properties, e.g.,
fluorescence, opacity, etc., or it may be located by virtue of a
fabricated alignment mark on the array, e.g., a mark in the array
or on its upper or lower surface that corresponds to the location
of the first spot. The location of the first spot is then recorded
and the process is repeated with the second and third corners. As
noted, this provides sufficient information to locate all of the
spots in the array. In order to verify that the positions have been
properly identified, the fourth corner is then located (by virtue
of the calculations made from the first three corner positions.
This allows a positive confirmation of positioning process.
[0105] As noted previously, contact between the end of the sampling
element or capillary and the surface of the array substrate can
result in a number of problems, including inconsistencies in
sampling materials from the array surface. Further, in the case of
multiplexed systems, multiple capillary elements may be required to
simultaneously access multiple reagent spots on an array. Due to
variations in manufacturing of both the array surface and the
length of the sampling elements, it would generally be very
difficult to ensure the appropriate tolerances required to
guarantee access to multiple reagent spots simultaneously, e.g.,
some capillaries may contact the surface of the reagent array while
others remain a small distance from the surface. As noted, this can
result in differing abilities of the capillary to pick up
solubilized reagents from the surface of the reagent array
substrate.
[0106] In order to optimize consistency in sampling, both in a
single capillary from spot to spot, as well as from capillary to
capillary, e.g., in multiple capillary systems, certain embodiments
of the microfluidic devices of the present invention are provided
with a fixed offset sleeve or frame (both are referred to herein as
a sleeve), that prevents the capillary end from directly contacting
the surface of the array, and maintains the end at a fixed distance
from the surface of the array during the sampling operation, either
by virtue of en enclosed cylinder of the extended sleeve, or
through a tripod or dipod-like structure at the open end of the
sleeve. In the case where a sampling element contacts the surface,
the sleeve or frame maintains the open end of the sampling element
a set fixed distance from the surface. In the case of multiple
sampling elements, the sleeve maintains the open end of each
element a set fixed distance from the surface. Even if one element
is longer than another, the system moves the substrate and/or
microfluidic device, relative to the other until all of the sleeves
on the sampling elements have contacted the surface of the reagent
array substrate. Where one element is longer than the other, that
element merely deflects in response to the pressure of contact with
the surface until the other sampling elements are brought into
contact with the reagent array substrate surface. This portion of
the present invention is illustrated in FIGS. 17A, B, C and D.
Elements that are common to FIGS. 1 and 2 and FIG. 17 are
referenced with common reference numerals.
[0107] As shown in FIG. 17A, a microfluidic device 102 includes an
external sampling element or capillary 106 that includes a channel
106c that is in fluid communication with a channel 104 in device
102. A sleeve 1702 is provided fitted onto the open end 106a of
sampling capillary 106, such that the sleeve extends a set distance
(as shown by the arrows) beyond the end 106a of capillary 106.
FIGS. 17b, C and D illustrate a device 102 that includes multiple
capillary elements, e.g., sampling elements 106 and 150, where the
lengths of the various sampling elements have differing lengths,
making simultaneous sampling with each element difficult. Each of
the multiple sampling elements is provided with a sleeve 1702 that
provides the same degree of offset for each sampling element, e.g.,
each sleeve extends a set distance beyond the end of each sampling
element. In FIG. 17C, the longer sampling element 150 is shown
contacting the surface of the reagent array substrate 108.
Continued movement of the substrate 108 toward the microfluidic
device 102 then causes the deflection of the longer capillary 150
until the sleeve 1702 on the shorter sampling elements (e.g. 106)
contacts the surface of the array substrate 108 (FIG. 17D). Once
that occurs, the sleeves ensure that the open end 106a and 150a of
each sampling element (106 and 150) is the same distance from the
array surface, thus ensuring consistent sampling of materials from
the array substrate surface.
[0108] The amount of offset for a sleeve structure can vary
depending upon the needs of the particular application. In some
case that offset can be relatively small, e.g., 10 to 100 .mu.m,
whereas other applications can have a larger offset, e.g., 100
.mu.m to 1 mm or more.
[0109] The present invention is further illustrated with reference
to the following non-limiting examples.
EXAMPLES
Example 1
Reconstituting and Sampling of Nucleic Acids System Set-Up and
Operation
[0110] Two different system set-ups were used to demonstrate the
sampling and analysis systems of the present invention. These are
illustrated in FIGS. 9A and 9B. FIG. 9A shows a system 900 that
uses a single capillary 902 to dissolve and aspirate the dried
reagents 904 from the surface of a substrate 906. The system
included a library substrate holder (not shown) that fixed the
polymeric substrate card 906 between two pieces of aluminum. The
aluminum holder was then attached to a robot arm, which allowed the
card to move in three dimensions (+/-0.5 .mu.m resolution). The
capillary 902 was fixed above the card 906 and fed into a waste
well 910. Vacuum and pressure (indicated by the arrow) were applied
through the waste well using a syringe pump (not shown), to expel a
buffer droplet 912 from the capillary tip and sip up rehydrated
compound 904 from the surface of the card 906. For detection
purposes, a small window was burned in the capillary polyimide
coating. An optical system 914 consisting of an arc lamp, PMT and
30x objective, was then focused through the window so that
rhodamine labeled molecules could be detected. The measured signal
from the PMT was transmitted to a computer 916 for recording and
documentation of the experiment.
[0111] The second system 950 shown in FIG. 9B, utilized the same
substrate configuration. The capillary, however, was replaced by an
NS71 sipper chip 952 (shown in FIG. 9C and substantially similar to
the chip shown in FIGS. 1 and 2), which includes an integrated
capillary element 954 in communication with channels in the
interior portion of the device 952 (the point of communication
between the external capillary and the internal channels is shown
from the top view as a black spot in FIG. 9C). The syringe pump
(not shown) was attached to the waste port on the chip and
controlled both pressure and vacuum (as shown by the arrow). The
detection system 912 was also the same but was re-oriented to
detect the fluorescent signal of molecules flowing through the
central channel of the chip 952.
[0112] All reagent deposition were achieved using the single
capillary setup shown in FIG. 9A. Once the capillary and substrate
were in place, the computer was programmed to move the substrate
relative to the capillary, and expel a certain volume of fluid from
the capillary in order to generate a desired pattern of material
spots on the surface of the substrate, e.g., number and spacing of
spots. Typical numbers were 2000 .mu.m center to center spot
spacing and +/-2.0 psig through an 8 inch, 50 .mu.m I.D. glass
capillary. The capillary was usually about 50 .mu.m from the card
surface during deposition. Once the appropriate data was entered
and the program was initiated, the robot moved the substrate card
holder so that the capillary was in the reagent well. The reagent
was pulled into the capillary for 15 seconds, after which, the
substrates card holder moved so that the capillary was positioned
over the first spot. Pressure was then applied to the capillary for
13 seconds so that a drop of reagent was formed on the card. After
13 seconds, the substrate card holder moved back to the reagent
well to pull up the material for the second spot. This was repeated
as necessary.
[0113] To dissolve and aspirate the dried reagents, the single
capillary and the sipper chip method (FIGS. 9A and 9B) were both
used. Regardless of the method, the setup was essentially the same.
The program was told the location of the buffer well, the first
spot location, the spot spacing and the pressure/vacuum necessary
to rehydrate and aspirate the dried material. Where the capillary
method was used, the spot location was known from the deposition
process. The method of redissolving the reagent was then done one
of two ways. In the first case, a positive pressure method was used
where the syringe pump pulled up buffer for 25 seconds at -2.0 psig
and then dispensed a small amount of that buffer at the reagent
spot (2.0 psig for 9 seconds). Once the drop was dispensed, it was
pulled back into the capillary (-2.0 psig) for 8 seconds. The robot
then moved back to the starting position so that the capillary was
in the buffer well. This was repeated for each spot. As the
dissolved reagents passed by the capillary window, they were
detected by the PMT and displayed by the computer.
[0114] The second method of dissolving the compound used a hanging
drop from the capillary element. The system was setup the same as
the positive pressure method except that no positive pressure was
used. The capillary sipped up buffer when in the buffer well for 12
seconds at -2.0 psig. The pressure then changed to 0 psig and the
capillary moved to the reagent spot. As the capillary left the
buffer well, a hemispherical drop of reagent remained suspended
from the capillary end. The radius of the drop matches the radius
of the capillary and is approximately 13 nl in volume. When the
drop touched the reagent spot, the reagent dissolved. The system
waited for three seconds while the reagent dissolved in the drop
before aspirating the drop into the capillary at -0.5 psig for 5
seconds.
[0115] When using a sipper chip to dissolve the reagents, the
process was very similar to using a capillary. However, in order to
avoid backflow along any of the side channels of the sipper chip,
it was desirable to avoid applying a positive pressure to the waste
well of the chip. As such, the hanging drop method was found to be
most suitable with these chips. The back-flow problem could also be
addressed using control of pressure at the various ports/reservoirs
of the device. It was also noted that the system performed
optimally when the pressure was kept constant for the duration of
the assay, e.g., -0.5 psig. During sipper chip setup, the location
of the buffer well and first spot on the card were reset as
compared to the single capillary system, as the capillary used to
deposit the spots was in a different placement from the capillary
element integrated into a chip. Typical parameters used with the
sipper chip were as follows: constant vacuum of -0.5 psig; buffer
dwell time of 90 seconds; sample dwell time of 6 seconds.
[0116] Sampling Rhodamine and Rhodamine-Labeled DNA
[0117] Initial tests were performed by simply spotting Rhodamine
B/Rhodamine labeled DNA and redissolving it in water. Each of the
three methods described above were tested this way. In FIG. 10,
Rhodamine labeled DNA was spotted onto polypropylene as outlined
above. Sixty spots were retrieved with the data from 20, as shown.
A single capillary was used to retrieve the compounds in 50 mM
HEPES using a positive pressure method. On average the
concentration of material retrieved was at 60 .mu.M. This
concentration varied by +/-8% (1 s.d.) between peaks.
[0118] As shown in FIG. 10, the fluorescent intensity changes as
rhodamine labeled DNA is sipped from a polypropylene card.
[0119] The data in FIGS. 11A and 11B illustrate use of the hanging
drop method to dissolve spots of rhodamine labeled DNA on Teflon
using both a single capillary set-up (FIG. 11A) and a sipper chip
method (FIG. 11B). In FIG. 10, a single fused silica capillary is
used. In FIGS. 11A and 11B, a sipper chip was used. As shown in
FIG. 11A, peak heights are reproducible to within 20% (1 S.D.)
using this particular method. Variability in sampling efficiencies
in these plots appears to stem from surface variations in the
library substrate surfaces, and static electrical interactions, and
is easily remedied by appropriate selection and treatment of the
library substrates. As shown in FIG. 11B, Rhodamine labeled DNA was
spotted onto Teflon, and the spots were redissolved and aspirated
onto an NS71 sipper chip using the method described above.
Example 2
Integrated Sampling and SNP Hybridization Analysis
[0120] Hybridization reactions were used to demonstrate an
integrated sampling and reaction operation on a chip from dried
reagents. Molecular beacons were used as indicators of
hybridization. These molecular beacons are DNA molecules where the
5' end has a dabcyl quenching group and the 3' end has a
fluorescent moiety. The last five bases on both the 3' and 5' ends
are complimentary and thus the DNA strand can wrap around and
hybridize to itself. This conformation causes the dabcyl and
fluorescent moiety to be sufficiently close that the fluorescence
is quenched. The remaining unhybridized region of the DNA molecule
is on average 15 to 25 nucleotides in length. When a target DNA
molecule, which is complimentary to this region, is in solution,
the target will hybridize to the beacon and cause the loop to open.
The dabcyl and fluorescent molecule will then be sufficiently far
apart that the hybrid will fluoresce. Single nucleotide
polymorphisms have been detected using molecular beacons with
greater discrimination than that seen using linear DNA molecules
(Tyagi et al. Nature Biotechnology, 16, 1998).
[0121] In the demonstrating the efficacy of the sampling and
reaction system of the invention, the molecular beacon protocol
outlined by Tyagi et al. was followed. The beacon sequence was 5'
Tamra-gcg aga agt taa gac cta tgc tcg c-dabcyl 3' and the perfect
match target sequence was 5'-cat agg tct taa ctt-3'. Three central
position mismatch sequences were also used to demonstrate SNP
discrimination. Two types of experiments were run. In one case, the
targets were spotted onto the card or substrate and in the other
the beacon was spotted onto the card or substrate. Before spotting
either reagent, the concentrations of reagents needed for the
reaction were calculated considering several factors, including the
concentration ratio of target to beacon. For purposes of this
experiment, a three-fold excess of target to beacon was determined
to give reasonable discrimination. Second, the chip design was
examined to determine what percentage of each reagent would be
present in the reaction channel. The channel geometry or mask
layout of the device used is illustrated in FIG. 9C. In the device
used, the side arms contribute approximately 30% of the flow down
the central channel, whereas the capillary element contributes
approximately 70%. Third, because of the spotting and
reconstitution methods, the concentration of dissolved material on
the card was determined to be approximately 10.times. the spotting
concentration. Once all this was considered, the appropriate
concentrations were determined for the reactions. Typical
concentrations on the chip were 30 .mu.M beacon and 100 .mu.M
target. This translates into 100 .mu.M beacon in the side arm and
reagent wells and library substrate spots made with 15 .mu.M
target.
[0122] FIG. 12 shows discrimination of the molecular beacon
sequence when the beacon is in the side arm wells at 100 .mu.M and
the 4 possible targets (perfect match and three mismatches are all
liquid in this assay, not dried) are in the buffer wells at 150
.mu.M. As indicated by the plot of FIG. 12, SNP discrimination is
very clear using this method. Specifically, as shown, the perfect
hybrid match and the three possible central position mismatches
(150 uM in the buffer well, 100 uM in the reaction channel) are
clearly distinguishable. The first (lack of) peaks which are at
2800 seconds represent the `A` mismatch, the peaks at approximately
2900 seconds represent the `G` mismatch and the peaks just after
3000 seconds represent the `T` mismatch.
[0123] FIG. 13 illustrates a similar example to that shown in FIG.
12 except that the sampled materials are dried, rather than in
fluid form. Specifically shown is the hybridization of a molecular
beacon (concentration in side arm wells at 100 .mu.M) to the
perfect match DNA target and the three possible central position
mismatch targets. The three pairs of SNP peaks above correspond to
the `A` mismatch, `G` mismatch and `T` mismatch accordingly. The
DNA targets were spotted onto the library card at 150 .mu.M and
were therefore approximately 1 mM in the reaction channel.
[0124] This concentration of target is also readily and routinely
adjustable to optimize for maximum discrimination between
mismatches. Specifically, as shown, target concentrations were
somewhat higher than optimum, to ensure sufficient material was
accessed. However, this higher concentration resulted in a reduced
level of discrimination between variant sequences.
[0125] In FIG. 14, the experiment was reversed, such that the two
different beacons were spotted onto the card and the target to one
beacon was placed in the side arm wells of the chip. The background
fluorescence of the unreacted beacon is shown in the plot. The
target was deposited into the chip in the side arms at 150 .mu.M
and the beacons were spotted onto the card at 50 .mu.M. Only one
beacon was a perfect match to the target, which is evident from the
two different peak heights in the plot of FIG. 14. The two peaks
shown at approximately 750 seconds represent control peaks, e.g.,
perfect match hybridization and a background fluorescence level of
the beacon, alone. The more rounded peak shows the background
fluorescence of each sipped unreacted beacon, which is used in
order to provide meaningful discrimination in the hybridization
assays. The subsequent peaks all show reduced levels of
fluorescence, e.g., reduced beacon hybridization, representing
lower hybridization efficiencies resulting from the single base
mismatches.
Example 3
Electrical Sensing System
[0126] As described above, in at least certain aspects of the
instant invention, a sensing system is utilized to facilitate
sampling of materials from the library substrate. An electrical
sensing system was modeled on treatment of the fluid drop-air
gap-substrate as a capacitor. This circuit is illustrated in FIG.
6. When an AC current is applied through the circuit, the phase
angle of a simple model system is given by the equation:
.theta.=arctan(1/.omega.C/R)
[0127] Where C is capacitance and R is resistance. FIGS. 8A and 8B
illustrate theoretical calculations of the phase angle and
impedance as a function of the distance for a system having 1
gigaohm of resistance in the capillary and a 1 .mu.m thick Teflon
coating covering a metal plate as the substrate. In FIG. 8A, the
thin line represents capacitance for a Teflon.RTM. thickness of 1
uM and the thick line represents capacitive impedance (Ohms) at 103
Hz. In FIG. 8B, the thin line represents phase and the thick line
represents resistance. FIG. 8C shows actual impedance measurements
made using a model capillary system. The thick line represents the
signal and the thin line represents phase. The fluid used was 25
.mu.M HEPES buffer, and the capillary was 2 cm long with an inner
diameter of 20 .mu.m and an outer diameter of 360 .mu.m. An
aluminum plate overlaid with a 25 .mu.m thick sheet of Saran.RTM.
wrap was used to simulate a Teflon.RTM. coating.
[0128] FIG. 15 illustrates a plot of impedance representative of
multiple accession events, e.g., where a fluid drop at the end of
the capillary is contacted with a metal substrate card, using this
electrical sensing method. As can be seen, a number of accession
events can be carried out relatively quickly (as shown, intervals
are approximately 20 seconds, but could readily be shortened) and
accurately using this method.
Example 4
Optical Sensing System
[0129] A sampling system was set-up substantially as shown in FIG.
7. A red diode laser (632 nm) was used as the light source 702, and
a photodiode was used as the detector 706. The optical train also
included the following filters: 1) excitation filter=634 nm; 2)
beamsplitter=670 nm; and emission filter=700 nm. The chip was
substantially the same as that described in the above examples
except that the capillary element was Teflon coated rather than
polyimide coated, as polyimide has a relatively high fluorescence
level which would contribute to background fluorescence levels.
[0130] The library substrate was simulated using a glass microscope
slide covered with vinyl tape (Scotch 35 vinyl tape (orange)),
which fluoresces brightly at the wavelength of the diode laser. The
library substrate was placed upon a standard x-y-z translation
stage (Parker) for movement relative to the chip. The objective was
placed approximately 1 mm above the upper surface of the chip,
giving what appeared to be maximum coupling of the laser into the
capillary channel and maximum observed changes in fluorescence
levels.
[0131] The experiment was commenced by placing the open end of the
capillary element into contact with the surface of the library
substrate (as confirmed by magnified visual inspection). The data
from the experiment are plotted in FIG. 16. As shown, the
fluorescent signal is a reasonably sensitive function of the
distance to the surface, with the slope near contact being equal to
about 0.2 fluorescence units/.mu.m. The fluorescence then stayed
constant once contact was made with the surface. The plots shown
correspond to movement of the capillary toward the substrate
(dashed line) and moving away from the substrate (solid line). As
can be seen, this method provides a useful method of ascertaining
distance of the capillary element from the substrate surface,
and/or contact with that surface.
Example 5
Reproducibility of Sampling and Assay Results
[0132] The chip based assay system, e.g., as shown in FIG. 9B was
employed in monitoring the reproducibility of the overall system in
both simple compound sampling and assay performance.
[0133] In a first experiment, a fluorescent compound, Edans, was
spotted at 100 .mu.M onto an aminopropyl silanated glass microscope
slide, where the spotted solution comprised Edans, DMSO, PEG (10 Kd
M.W. at 1%) and dextran (65 Kd at 0.1%). Approximately 5 nl spots
were deposited in a standard grid format, and evaporatively dried.
The sampling capillary of the chip was aligned using the four
corner optical alignment method described herein. Following
alignment, more than 50 spots were visited, redissolved by a buffer
drop from the sampling capillary, and drawn into the channel
network in the interior of the chip. The fluorescent signal from
each sampled spot, as it passed through the main channel of the
chip, is shown in FIG. 18. As can be seen, fluorescent intensity
for each spot is highly regular and always detectable. The average
background-subtracted intensity was approximately 29,000 counts
with a CV of 12%.
[0134] A second experiment employed a different spotting solution
and different set of compounds from the first. Specifically, 19
fluorescent compound library compounds were separately spotted onto
an aminopropyl silanated glass slide as described above, where the
spotting solution, in addition to the individual compound, also
included DMSO, dextran (65 Kd MW, 1%) and dextran sulfate (500 Kd
MW, 0.1%). The compounds were then sipped in 20 separate runs,
e.g., all 19 compounds were sipped in 20 sets. FIG. 19 illustrates
the fluorescent intensity data from a portion of the overall
screen. As can be seen, from set to set, the 19 different compounds
gave similar results. The background-subtracted peak heights were
determined and analyzed to determine a standard deviation for each
compound. For less fluorescent compounds, CVs were relatively high,
due to the lower fluorescent intensity, whereas higher fluorescence
compounds had CVs that were substantially lower. In general, the
CVs ranged from approximately 6 to 41%, with the average being
approximately 20%.
[0135] A third experiment combined sampling with an assay
application within the chip. In particular, the chip was used to
run a human serum albumin (HSA) binding assay in continuous flow
mode, while different compounds were sipped into the device and
their ability to bind HSA (as measured by displacement of a
fluorescent dye from HSA) was determined. Three different compounds
with known binding activity to HSA were spotted repeatedly onto a
Teflon.RTM. substrate as described above, and dried. The different
compounds were then sipped by aspirating 1.times. PBS onto the
compounds from the sampling capillary and drawing the compound into
the chip. In the main channel of the chip, HSA was continually
mixed with a fluorogenic HSA binding dye (dansylsarcosine)
whereupon the compounds bind, and the mixture was flowed along the
main channel past a detector. Displacement of the dye results in a
reduction in the amount of fluorescence emitted by the dye. FIG. 20
shows the fluorescent signal of the HSA/dye mixture with the
periodic introduction of the different HSA binding compounds that
were sipped into the main channel. As can be seen, each different
compound gave a highly reproducible fluorescent dip with each spot
sipped.
[0136] The efficacy of the systems of the invention with respect to
enzyme assays is further illustrated in FIG. 21 illustrates a data
plot of a T-cell protein tyrosine phosphatase (TCPTP) when screened
against three known inhibitors that were provided dried on a solid
substrate along with negative control compounds. Each inhibitor was
spotted at two different concentrations (100 and 25 .mu.M) in DMSO
with 1% Dextran (65 kD MW) as an excipient. Using the assay formats
and microfluidic devices (NS-71) described above, the various
spotted compounds were hydrated from the capillary tip and drawn
into the microfluidic device where they were combined with the
assay components (enzyme and substrate (DiFMUP). Approximately 25%
of each compound spot was dissolved in each sampling with the same
approximate volume of hydrating fluid as deposition fluid, implying
an approximate concentration of 25 .mu.M and 6 .mu.M for the higher
and lower concentration spots, respectively. At these approximate
concentrations, the inhibitory response as shown in FIG. 21 is
comparable to that seen in purely liquid formats, e.g., samples
from a multiwell plate.
[0137] All publications and patent applications are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference. Although the present
invention has been described in some detail by way of illustration
and example for purposes of clarity and understanding, it will be
apparent that certain changes and modifications may be practiced
within the scope of the appended claims.
* * * * *