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Claims  |
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What is claimed is:
1. A device for analyzing a fluid, cell-containing sample, the device
comprising:
a solid substrate microfabricated to define:
a sample inlet port; and
a mesoscale flow system comprising:
a sample flow channel extending from said inlet port; and
a cell handling region for treating cells disposed in fluid communication
with said flow channel, said cell handling region comprising a cell lysing
structure; and
means for detecting an analyte in a fluid sample in said flow system.
2. The device of claim 1 further comprising means for inducing flow of
cells in a sample through said mesoscale flow channel and said cell
handling region to force cells in said sample into contact with said cell
lysing structure, thereby to lyse cells in said sample.
3. The device of claim 1 wherein said cell lysing structure comprises a
portion of a flow channel having cell membrane piercing protrusions
extending from a wall thereof.
4. The device of claim 1 wherein said cell lysing structure comprises sharp
edged particles trapped within said cell handling region; and
wherein said device further comprises means for inducing flow to force
cells in said sample into contact with said sharp edged particles, thereby
to lyse said cells.
5. The device of claim 1 wherein said cell lysing structure comprises a
region of restricted cross-sectional dimension sufficient to permit
passage of intracellular molecules while prohibiting passage of cells.
6. The device of claim 1 wherein said means for detecting comprises means
downstream of said cell lysing structure for detecting the presence of an
intracellular molecular component of a cell in said sample.
7. The device of claim 1 further comprising means disposed downstream of
said cell lysing structure for collecting insoluble cellular debris.
8. The device of claim 1 further comprising a filter means disposed
downstream of said cell lysing structure.
9. The device of claim 1, wherein said substrate comprises microfabricated
silicon.
10. A device for analyzing a fluid, cell-containing sample, the device
comprising:
a solid substrate microfabricated to define:
a sample inlet port; and
a mesoscale flow system comprising:
a sample flow channel extending from said inlet port; and
a cell handling region for treating cells disposed in fluid communication
with said flow channel, said cell handling region comprising a cell
capture region comprising immobilized binding sites which reversibly bind
a preselected cell surface molecule of a cell population in a
cell-containing fluid sample; and
means for inducing flow of cells in said sample through said mesoscale flow
channel and said cell handling region;
wherein said means for inducing flow of said cell-containing sample is used
to induce flow:
at a first flow rate sufficiently slow to permit capture of cells in said
cell population by said binding sites, thereby to separate said cell
population from said sample; and
at a second flow rate, higher than said first flow rate, and sufficient to
release said separated cells from said capture region; and
wherein said device further comprises means for detecting an analyte in a
fluid sample in said flow system.
11. The device of claim 10 wherein said detecting means comprises means
downstream of said cell capture region for determining the presence of an
extracellular component of said sample.
12. The device of claim 10 wherein said flow system further comprises:
cell lysing means downstream from said cell capture region and wherein said
flow inducing means includes means for forcing cells into said cell lysing
means, thereby to lyse cells in said sample; and
wherein said detecting means comprises means for detecting the presence of
an intracellular component in said captured cells.
13. The device of claim 12 further comprising filter means, disposed
between said cell-lysing means and said detection means, for filtering
cellular debris from said sample.
14. The device of claim 13 further comprising a sump for collecting
insoluble debris disposed adjacent said filter.
15. A device for analyzing a fluid, cell-containing sample, the device
comprising:
a solid substrate microfabricated to define:
a sample inlet port; and
a mesoscale flow system comprising:
a sample flow channel extending from said inlet port; and
a cell handling region for treating cells disposed in fluid communication
with said flow channel, said cell handling structure defining:
a cell sieve comprising means defining a plurality of flow passages of
restricted size allowing only cells of a sufficiently small diameter to
pass therethrough; and
a cell lysing structure;
means for inducing flow of cells in a sample through said mesoscale flow
channel and said cell handling structure; and
means for detecting an analyte in a fluid sample in said flow system.
16. The device of claim 2, 10 or 15 further comprising an appliance for use
in combination with said substrate, said appliance comprising:
means for holding said substrate; and
fluid input means interfitting with an inlet port on said substrate; and
wherein said means for inducing flow comprises pump means, disposed in said
appliance, for passing fluid through the flow system of said substrate
when it is held in said housing means.
17. The device of claim 1, 10 or 15, wherein said means for detecting
comprises an appliance for use in combination with said substrate, said
appliance comprising:
means for holding said substrate; and
optical means for viewing the contents of said mesoscale flow system in
said substrate.
18. The device of claim 17, wherein said optical means comprises magnifying
optics and a video camera, and wherein said appliance further comprises:
a tilt mechanism for manually adjusting the angle and location of the
device; and
a video screen for viewing the contents of said flow system.
19. The device of claim 2, 10 or 15, wherein said:
a solid substrate microfabricated to define:
a sample inlet port;
a mesoscale flow system further comprises:
a branching channel in fluid communication with said flow channel; and
at least two additional ports communicating between said flow channel and
said branching channel, respectively, and the exterior of said flow
system; and
wherein said device further comprises valve means for directing flow
through said flow system to a selected one of said additional ports.
20. The device of claim 19 wherein said detecting means comprises a
detection region within said mesoscale flow system for optically or
electrically gathering data indicative of the presence or concentration of
an analyte in a sample contained within said flow system.
21. The device of claim 19 further comprising an appliance for use in
combination with said substrate, said appliance comprising:
means for holding said substrate;
fluid flow channels interfitting with at least two of said ports when said
substrate is held in said holding means; and
wherein said means for inducing flow comprises pump means disposed within
said appliance in fluid communication with said inlet ports for inducing
flow within said flow system.
22. The device of claim 21 wherein said valve means is disposed within said
appliance.
23. A device for analyzing a cell-containing fluid sample, the device
comprising:
a solid substrate microfabricated to define:
a mesoscale cell handling structure; and
at least two mesoscale flow systems, each of which comprise a flow channel
and an analyte detection region, one of said flow systems being adapted to
analyze a sample, the other being adapted as a control, and said flow
systems being in fluid communication with said cell handling structure;
and
means for inducing flow of a sample through said cell handling structure
and then through both said flow systems, thereby to permit comparison of
data from the detection regions of said systems.
24. The device of claim 1, 10, 15 or 23 wherein, within at least a portion
of a channel in a said flow system, the channel width and channel depth
each are between 0.1 .mu.m and 500 .mu.m.
25. The device of claim 24 wherein the channel width in said portion is
between 2.0 and 500 .mu.m.
26. The device of claim 24 wherein the channel depth in said portion is
between 0.1 and 100 .mu.m. |
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Claims |
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Description |
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REFERENCE TO RELATED APPLICATIONS
This application is being filed contemporaneously with the following
related copending applications: U.S. Ser. No. 07/877,702 filed May 1,
1992, U.S. Ser. No. 07/877,701 filed May 1, 1992; U.S. Ser. No. 07/877,662
filed May 1, 1992 and U.S. Ser. No. 07/877,661, filed May 1, 1992, the
disclosures of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
This invention relates generally to methods and apparatus for conducting
analyses. More particularly, the invention relates to the design and
construction of small, typically single-use, modules capable of analyzing
a fluid sample.
In recent decades the art has developed a very large number of protocols,
test kits, and cartridges for conducting analyses on biological samples
for various diagnostic and monitoring purposes. Immunoassays,
agglutination assays, and analyses based on polymerase chain reaction,
various ligand-receptor interactions, and differential migration of
species in a complex sample all have been used to determine the presence
or concentration of various biological compounds or contaminants, or the
presence of particular cell types.
Recently, small, disposable devices have been developed for handling
biological samples and for conducting certain clinical tests. Shoji et al.
reported the use of a miniature blood gas analyzer fabricated on a silicon
wafer. Shoji et al., Sensors and Actuators, 15:101-107 (1988). Sato et al.
reported a cell fusion technique using micromechanical silicon devices.
Sato et al., Sensors and Actuators, A21-A23:948-953 (1990). Ciba Corning
Diagnostics Corp. (USA) has manufactured a microprocessor-controlled laser
photometer for detecting blood clotting.
Micromachining technology originated in the microelectronics industry.
Angell et al., Scientific American, 248:44-55 (1983). Micromachining
technology has enabled the manufacture of microengineered devices having
structural elements with minimal dimensions ranging from tens of microns
(the dimensions of biological cells) to nanometers (the dimensions of some
biological macromolecules). This scale is referred to herein as
"mesoscale". Most experiments involving mesoscale structures have involved
studies of micromechanics, i.e., mechanical motion and flow properties.
The potential capability of mesoscale structures has not been exploited
fully in the life sciences.
Brunette (Exper. Cell Res., 167:203-217 (1986) and 164:11-26 (1986))
studied the behavior of fibroblasts and epithelial cells in grooves in
silicon, titanium-coated polymers and the like. McCartney et al. (Cancer
Res., 41:3046-3051 (1981)) examined the behavior of tumor cells in grooved
plastic substrates. LaCelle (Blood Cells, 12:179-189 (1986)) studied
leukocyte and erythrocyte flow in microcapillaries to gain insight into
microcirculation. Hung and Weissman reported a study of fluid dynamics in
micromachined channels, but did not produce data associated with an
analytic device. Hung et al., Med. and Biol. Engineering, 9:237-245
(1971); and Weissman et al., Am. Inst. Chem. Eng. J., 17:25-30 (1971).
Columbus et al. utilized a sandwich composed of two orthogonally
orientated v-grooved embossed sheets in the control of capillary flow of
biological fluids to discrete ion-selective electrodes in an experimental
multi-channel test device. Columbus et al., Clin. Chem., 33:1531-1537
(1987). Masuda et al. and Washizu et al. have reported the use of a fluid
flow chamber for the manipulation of cells (e.g. cell fusion). Masuda et
al., Proceedings IEEE/IAS Meeting, pp. 1549-1553 (1987); and Washizu et
al., Proceedings IEEE/IAS Meeting pp. 1735-1740 (1988). The art has not
fully explored the potential of using mesoscale devices for the analyses
of biological fluids and detection of microorganisms.
The current analytical techniques utilized for the detection of
microorganisms are rarely automated, usually require incubation in a
suitable medium to increase the number of organisms, and invariably employ
visual and/or chemical methods to identify the strain or sub-species. The
inherent delay in such methods frequently necessitates medical
intervention prior to definitive identification of the nature of an
infection. In industrial, public health or clinical environments, such
delays may have serious consequences. There is a need for convenient
systems for the rapid detection of microorganisms.
An object of the invention is to provide analytical systems with optimal
reaction environments that can analyze microvolumes of sample, detect
substances present in very low concentrations, and produce analytical
results rapidly. Another object is to provide easily mass produced,
disposable, small (e.g., less than 1 cc in volume) devices having
mesoscale functional elements capable of rapid, automated analyses in a
range of biological and other applications. It is a further object of the
invention to provide a family of such devices that individually can be
used to implement a range of rapid clinical tests, e.g., tests for
bacterial contamination, virus infection, sperm motility, blood
parameters, contaminants in food, water, or body fluids, and the like.
SUMMARY OF THE INVENTION
The invention provides methods and devices for the analysis of a fluid
sample. The device comprises a solid substrate, typically on the order of
a few millimeters thick and approximately 0.2 to 2.0 centimeters square,
micro-fabricated to define a sample inlet port and a mesoscale flow
system. The mesoscale flow system includes a sample flow channel,
extending from the inlet port, and a fluid handling region, in fluid
communication with the flow channel. The term "mesoscale" is used herein
to define chambers and flow passages having cross-sectional dimensions on
the order of 0.1 .mu.m to 500 .mu.m. The mesoscale flow channels and fluid
handling regions have preferred depths on the order of 0.1 .mu.m to 100
.mu.m, typically 2-50 .mu.m. The channels have preferred widths on the
order of 2.0 to 500 .mu.m, more preferably 3-100 .mu.m. For many
applications, channels of 5-50 .mu.m widths will be useful. Chambers in
the substrates often will have larger dimensions, e.g., a few millimeters.
In one embodiment, the device may be utilized to analyze a cell containing
fluid sample, and the fluid handling region may comprise a cell handling
region. The device may further include means for inducing flow of cells in
the sample through the mesoscale flow system. The cell handling region may
comprise a cell lysis means. The flow inducing means may be utilized to
force a cell sample through the cell lysis means to rupture the cells.
Means may also be provided in the device for detecting the presence of an
intracellular molecular component of a cell in the cell sample. The cell
lysis means may comprise, e.g., sharp-edged pieces of silicon trapped
within the cell handling region, or cell membrane piercing protrusions
extending from a wall of the cell handling region of the mesoscale flow
system. Alternatively, a region of reduced cross-sectional area may
comprise the cell lysis means. The flow system may further comprise a
microfabricated filter for, e.g., filtering cellular debris from the
sample, prior to analysis for the presence of an intracellular analyte.
The cell handling region may also comprise a cell capture region comprising
binding sites capable of reversibly binding a cell surface molecule to
enable the selective isolation of a cell population from a cell sample.
Means may also be provided downstream of the cell capture region for
determining the presence of a cell or cell surface molecule in the sample.
In another embodiment, the cell handling region may comprise an inert
barrier, such as posts extending from a wall of the region, to enable the
sorting of cells by size. The posts also may comprise, e.g., a barrier to
the flow of a sperm sample, to enable the assessment of sperm motility.
Generally, as disclosed herein, the solid substrate comprises a chip
containing the mesoscale flow system. The mesoscale flow system may be
designed and fabricated from silicon and other solid substrates using
established micromachining methods. The mesoscale flow systems in the
devices may be constructed by microfabricating flow channels and one or
more fluid handling regions into the surface of the substrate, and then
adhering a cover, e.g., a transparent glass cover, over the surface. The
devices typically are designed on a scale suitable to analyze microvolumes
(<10 .mu.L) of sample, introduced into the flow system through an inlet
port defined, e.g., by a hole communicating with the flow system through
the substrate or the cover. The volume of the mesoscale flow system
typically will be <5 .mu.m, and the volume of individual channels,
chambers, or other functional elements are often less than 1 .mu.m, e.g.,
in the nL or pL range. Cells or other components present in very low
concentrations (e.g., nanogram quantities) in microvolumes of a sample
fluid can be rapidly analyzed (e.g., <10 minutes).
The chips typically will be used with an appliance which contains a nesting
site for holding the chip, and which mates one or more input ports on the
chip with one or more flow lines in the appliance. After a fluid sample,
e.g., a cell-containing fluid sample, suspected to contain a particular
cell type, or molecular component, is applied to the inlet port of the
substrate, the chip is placed in the appliance, and a pump, e.g., in the
appliance, is actuated to force the sample through the flow system.
Alternatively, a sample may be injected into the chip by the appliance.
The sample also may enter the flow system by capillary action.
In one embodiment, the fluid handling chamber of the device may include a
mesoscale detection region, downstream from the fluid handling region, for
detecting the presence of an analyte in the fluid sample such as a
cellular, intracellular, or other fluid sample component. The detection
region may be constructed in accordance with U.S. Ser. No. 07/877,702
filed May 1, 1992, the disclosure of which is incorporated herein by
reference. The appliance may be designed to receive electronic or
spectrophotometric signals in the detection region, to indicate the
presence of the preselected component in the cell sample. The presence of
a cellular, intracellular or other analyte in the detection region may
also be detected optically, e.g., through a transparent or translucent
window, such as a transparent cover, over the detection region, or through
a translucent section of the substrate itself. The appliance may include
sensing equipment such as a spectrophotometer, capable of detecting the
presence of a preselected analyte in the detection region. In one
embodiment, the detection region may comprise binding moieties, capable of
binding to the analyte to be detected, thereby to enhance and facilitate
detection. The detection region also may comprise a fractal region, i.e.,
a region of serially bifurcating flow channels, sensitive to changes in
flow properties of a fluid sample, as is disclosed in U.S. Ser. No.
07/877,701, filed May 1, 1992, the disclosure of which is incorporated
herein by reference. The device also may be fabricated with at least three
inlet ports, in fluid communication with the flow system, provided with
valves, e.g., in an appliance used in combination with the device, for
closing and opening the ports to enable the control of fluid flow through
the mesoscale flow system.
The mesoscale devices can be adapted to perform a wide range of biological
tests. Some of the features and benefits of the devices are summarized in
Table 1. A device may include two or more separated flow systems, e.g.,
fed by a common inlet port, with different cell handling chambers in each
of the systems to enable two or more analyses to be conducted
simultaneously. The devices can be utilized to implement a range of rapid
tests, e.g., to detect the presence of a cellular or intracellular
component of a fluid sample. The devices may be utilized to detect, e.g.,
a pathogenic bacteria or virus, or for cell sorting. The invention
provides methods and devices for a wide range of possible analysis. Assays
may be completed rapidly, and at the conclusion of the assay the chip can
be discarded, which advantageously prevents contamination between samples,
entombs potentially hazardous materials, and provides inexpensive,
microsample analyses.
TABLE 1
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Feature Benefit
______________________________________
Flexibility No limits to the number of chip
designs or applications available.
Reproducible
Allows reliable, standardized, mass
production of chips.
Low Cost Allows competitive pricing with
Production existing systems. Disposable nature
for single-use processes.
Small Size No bulky instrumentation required.
Lends itself to portable units and
systems designed for use in non-
conventional lab environments.
Minimal storage and shipping costs.
Microscale Minimal sample and reagent volumes
required. Reduces reagent costs,
especially for more expensive,
specialized test procedures. Allows
simplified instrumentation schemes.
Sterility Chips can be sterilized for use in
microbiological assays and other
procedures requiring clean environments.
Sealed System
Minimizes biohazards. Ensures
process integrity.
Multiple Circuit
Can perform multiple processes or
Capabilities
analyses on a single chip. Allows
panel assays.
Multiple Expands capabilities for assay and
Detector process monitoring to virtually any
Capabilities
system. Allows broad range of
applications.
Reuseable Chips
Reduces per process cost to the user
for certain applications.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a magnified plan view of a device according to the invention that
includes a solid substrate 14, on which are machined entry ports 16,
mesoscale flow channel 20, cell lysis chamber 22, and fractal region 40,
with a transparent cover 12 adhered to the surface of the substrate.
FIG. 2 is a longitudinal cross sectional view of the device shown in FIG.
1.
FIG. 3 is a perspective view of the device of FIG. 1.
FIG. 4 is a schematic illustration of analytical device 10 nested within
appliance 50, which is used to support the device 10 and to regulate and
detect the pressure of sample fluids in device 10.
FIG. 5 is a cross sectional perspective view of a fluid handling region 22
on the inert substrate 14 with cell or debris filtering protrusions 26
extending from the wall of the flow channel.
FIG. 6 is a cross sectional view of a fluid handling region 22 on the inert
substrate 14 with cell piercing protrusions 24 extending from the wall of
the channel.
FIG. 7 is a schematic top view of an analytical device 10 fabricated with a
series of mesoscale chambers suitable for implementing a variety of
functions including cell sorting, cell lysing and PCR analysis.
FIGS. 8 through 10 illustrate different embodiments of a filter
microfabricated in a mesoscale flow channel 20.
FIGS. 11 is schematic perspective view of an apparatus 60 used in
combination with device 10 for viewing the contents of device 10.
FIG. 12 is a schematic cross sectional view of the apparatus 60 of FIG. 11.
Like reference characters in the respective drawn figures indicate
corresponding parts.
DETAILED DESCRIPTION
The invention provides methods and apparatus for the analysis of a fluid
sample. The device comprises a solid substrate, microfabricated to define
a sample inlet port and a mesoscale flow system. The mesoscale flow system
comprises a sample flow channel extending from the inlet port, and a fluid
handling region in fluid communication with the flow channel. In one
embodiment, the devices may be utilized to analyse a cell-containing fluid
sample. The devices may be used, e.g., to detect the presence of a
cellular or intracellular component in a cell sample.
Analytical devices having mesoscale flow channels and cell handling
chambers can be designed and fabricated in large quantities from a solid
substrate material. They can be sterilized easily. Silicon is a preferred
substrate material because of the well-developed technology permitting its
precise and efficient fabrication, but other materials may be used,
including polymers such as polytetrafluoroethylenes. The sample inlet port
and other ports, the mesoscale flow system, including the sample flow
channel(s) and the fluid handling region(s), and other functional
elements, may be fabricated inexpensively in large quantities from a
silicon substrate by any of a variety of micromachining methods known to
those skilled in the art. The micromachining methods available include
film deposition processes such as spin coating and chemical vapor
deposition, laser fabrication or photolithographic techniques such as UV
or X-ray processes, or etching methods which may be performed by either
wet chemical processes or plasma processes. (See, e.g., Manz et al.,
Trends in Analytical Chemistry 10: 144-149 (1991)).
Flow channels of varying widths and depths can be fabricated with mesoscale
dimensions. The silicon substrate containing a fabricated mesoscale flow
channel may be covered and sealed with a thin anodically bonded glass
cover. Other clear or opaque cover materials may be used. Alternatively,
two silicon substrates can be sandwiched, or a silicon substrate can be
sandwiched between two glass covers. The use of a transparent cover
results in a window which facilitates dynamic viewing of the channel
contents, and allows optical probing of the mesoscale flow system either
visually or by machine. Other fabrication approaches may be used.
The capacity of the devices is very small, and therefore the amount of
sample fluid required for an analysis is low. For example, in a 1
cm.times.1 cm silicon substrate, having on its surface an array of 500
grooves which are 10 microns wide.times.10 microns deep.times.1 cm
(10.sup.4 microns) long, the volume of each groove is 10.sup.-3 .mu.L and
the total volume of the 500 grooves is 0.5 .mu.L. The low volume of the
mesoscale flow systems allows assays to be performed on very small amounts
of a liquid sample (<5 .mu.l). The mesoscal flow system of the devices may
be microfabricated with microliter volumes or alternatively nanoliter
volumes or less, which advantageously limits the amount of sample and/or
reagent fluids required for the assay. In one embodiment, electron
micrographs of biological structures such as circulatory networks may be
used as masks for fabricating mesoscale flow systems on the substrate.
Mesoscale flow systems may be fabricated in a range of sizes and
conformations.
In one embodiment, the devices may be utilized to analyze a cell-containing
fluid sample. The fluid handling region may comprise, in one embodiment, a
cell lysing means, to allow cells in a fluid sample to be lysed prior to
analysis for an intracellular molecule such as an mRNA or DNA molecule. As
illustrated in FIG. 6, the cell lysing means may comprise cell membrane
piercing protrusions 24, extending from a surface of cell handling region
22. The device may include means, such as a pump for inducing flow through
the flow system. As fluid flow is forced through the piercing protrusions
24, cells are ruptured. Cell debris may be filtered off using a filter
microfabricated in the flow system downstream from the cell lysis means.
The cell lysis region may also comprise sharp edged particles, e.g.,
fabricated from silicon, trapped within the cell handling region. In
addition, the cell lysis means may comprise a region of restricted
cross-sectional dimension, which implements cell lysis upon application of
sufficient flow pressure. In another embodiment, the cell lysis means may
comprise a cell lysing agent.
The devices may include a mesoscale detection region microfabricated in the
mesoscale flow system, in fluid communication with a cell lysis region,
comprising binding moieties capable of binding to a selected intracellular
molecular component in the cell sample. Binding moieties may be introduced
into the detection region via an inlet port in fluid communication with
the detection region. Alternatively, binding moieties may be immobilized
in the detection region either by physical absorption onto the channel
surfaces, or by covalent attachment to the channel surfaces, or to solid
phase reactant such as a polymeric bead. Techniques available in the art
may be utilized for the chemical activ | | |
