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Claims  |
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What is claimed is:
1. A method of separating a target subpopulation of cells in a
cell-containing liquid sample comprising the steps of:
(A) providing a mesoscale sample flow passage comprising a solid wall
having immobilized thereon a binding protein specific for a cell
membrane-bound protein characteristic of said target population;
(B) passing a cell-containing liquid sample through said passage under
conditions to permit capture of members of the cell target subpopulation
by reversible cell surface protein-immobilized protein binding, while
permitting other cells to pass therethrough; and
(C) changing the conditions in said flow passage to release said target
subpopulation of cells.
2. The method of claim 1 wherein said conditions to permit capture comprise
a flow rate of fluid in said flow passage that permits said capture and
wherein said flow rate is increased in step C to shear cells off said
solid wall.
3. The method of claim 1 wherein step C is conducted by introducing a
solvent in said flow channel which desorbs said cells from said solid
wall.
4. A method for detecting an analyte in a fluid, cell-containing sample,
the method comprising the steps of:
(i) providing a 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;
(ii) delivering a cell-containing fluid sample to the inlet port and
through the flow system to the cell lysing structure in the cell handling
region thereby to lyse cells in the sample producing a lysed cell sample;
and
(iii) detecting an analyte in the fluid sample in the flow system with the
detection means.
5. The method of claim 4 wherein the device, provided in step (i), further
comprises means for inducing flow of cells in a sample through the
mesoscale flow channel and the cell handling region to force cells in the
sample into contact with the cell lysing structure, thereby to lyse cells
in the sample; and
wherein step (ii) comprises delivering a cell-containing fluid sample to
the inlet port and through the flow system to the cell lysing structure in
the cell handling region with the flow inducing means.
6. The method of claim 4 wherein the cell lysing structure, in the device
provided in step (i), comprises a portion of a flow channel having cell
membrane piercing protrusions extending from a wall thereof; and
wherein step (ii) comprises delivering the cell-containing fluid sample to
the cell membrane piercing protrusions in the cell lysing structure,
thereby to lyse cells in the sample.
7. The method of claim 4 wherein the cell lysing structure, in the device
provided in step (i), comprises sharp edged particles trapped within the
cell handling region; and
wherein step (ii) comprises delivering the cell-containing fluid sample to
the sharp edged particles in the cell handling region, thereby to lyse
cells in the sample.
8. The method of claim 4 wherein the cell lysing structure, in the device
provided in step (i), comprises a region of restricted cross-sectional
dimension sufficient to permit passage of intracellular molecules while
prohibiting passage of cells; and
wherein step (ii) comprises delivering the cell-containing fluid sample to
the region of restricted cross-sectional dimension, thereby to lyse cells
in the sample.
9. The method of claim 4 wherein step (iii) comprises detecting the
presence of an intracellular molecular component of a cell in the sample.
10. The method of claim 4, further comprising, prior to step (iii), the
step of collecting insoluble cellular debris in the lysed cell sample
obtained in step (ii).
11. The method of claim 4, further comprising, prior to step (iii), the
step of filtering the lysed cell sample obtained in step (ii).
12. A method for analyzing a fluid, cell-containing sample, the method
comprising:
(i) providing a 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; and
means for detecting an analyte in a fluid sample in said flow system;
(ii) delivering a fluid, cell containing sample to the inlet port;
(iii) inducing flow of the cell-containing sample through the flow system
within the flow inducing means:
at a first flow rate sufficiently slow to permit capture of cells in a cell
population in the sample by the binding sites, thereby to separate the
cell population from the sample; and
at a second flow rate, higher than the first flow rate, and sufficient to
release the separated cells from the capture region; and
(iv) detecting an analyte in the fluid sample in the flow system with the
detection means.
13. The method of claim 12 wherein step (iv) comprises detecting the
presence of an analyte comprising an extracellular component in the sample
with the detection means.
14. The method of claim 12 wherein the method further comprises, prior to
step (iv), the step of lysing the cells; and
wherein step (iv) comprises detecting an analyte comprising an
intracellular component of the lysed cells with the detection means.
15. The method of claim 14 further comprising filtering the lysed cell
sample prior to step (iv).
16. A method for analyzing a fluid, cell-containing sample, the method
comprising:
(i) providing a 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 structure 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;
(ii) delivering a fluid, cell-containing sample to the inlet port through
the flow system with the flow inducing means to the cell handling
structure, to permit cell sorting of the sample in the cell sieve, and
cell lysis of the cell sample in the cell lysing structure; and
(iii) detecting an analyte in the sample in the flow system with the
detection means.
17. The method of claim 5, 12 or 16 wherein the method comprises utilizing
a pump to deliver the cell-containing sample to the flow system in the
device.
18. The method of claim 4, 12 or 16, wherein said detecting step comprises
the step of optically viewing the contents of the mesoscale flow system in
the substrate.
19. The method of claim 16 wherein the method comprises detecting an
analyte comprising an intracellular component of the lysed cells with the
detection means.
20. A method for analyzing a cell-containing fluid sample, the method
comprising:
(i) providing a 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;
(ii) delivering a cell-containing fluid sample to said inlet port, and
through the cell handling structure and the mesoscale flow systems with
the flow inducing means; and
(iii) detecting and comparing data from the detection regions of said flow
systems.
21. The method of claim 4, 12, or 16 wherein said detecting step comprises
optically or electrically gathering data within the mesoscale flow system,
said data being indicative of the presence or concentration of an analyte
in a sample contained within the flow system.
22. The method of claim 12 or 16 wherein said detecting step comprises
detecting a particular cell type in a heterogeneous cell population in the
sample.
23. The method of claim 4, 12, 16 or 20 wherein, in the device provided in
step (i), 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.
24. The method of claim 23 wherein the channel width in said portion is
between 2.0 and 500 .mu.m.
25. The method of claim 23 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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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, now abandoned, 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, now abandoned, 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 analyses. 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 10 according to the invention
that includes a solid substrate 14, on which are machined entry ports 16A
and 16B, mesoscale flow channel 20A-20C, cell lysis chamber 22 with
protrusion 24 disposed therein, filter 28, and fractal region 40, with a
transparent cover 12 adhered to the surface of the substrate. A portion of
cover 12 is shown broken away for purposes of illustration.
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 28
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 mesoscale flow systems 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 activation of silaceous surfaces, and the
subsequent attachment of a binding moiety to the surfaces. (See, e.g.,
Haller in: Solid Phase Biochemistry, W. H. Scouten, Ed., John Wiley, New
York, pp 535-597 (1983); and Mandenius et al., Anal. Biochem.,
137:106-114(1984), and Anal. Biochem., 170:68-72 (1988)).
The binding moiety in the detection region may comprise, e.g., an antigen
binding protein, a DNA probe, or one of a ligand/receptor pair, to enable
the detection of a preselected cellular, intracellular, or other analyte,
such as an antigen, a polynucleotide or a cell surface molecule. The
binding assays available in the art which may be utilized in the detection
region include immunoassays, enzymatic assays, ligand/binder assays and
DNA hybridization assays. The detection of a particular intracellular
analyte may be implemented by the selection of an appropriate binding
moiety in detection region. The detection region may be fabricated
according to methods disclosed in U.S. Ser. No. 07/877,701, filed May 1,
1992, now abandoned, the disclosure of which is incorporated herein by
reference.
The mesoscale detection region may also comprise a region sensitive to
changes in flow properties induced by the presence of a preselected
cellular, intracellular or other analyte in the fluid sample. The flow
sensitive region may comprise, e.g., a fractal region, comprising
bifurcations leading to plural secondary flow channels. The flow sensitive
region, e.g., the fractal region, may be constructed in accordance with
the copending related application U.S. Ser. No. 07/877,701 filed May 1,
1992, now abandoned, the disclosure of which is incorporated herein by
reference.
The devices may comprise a plurali | | |
