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Claims  |
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What is claimed is:
1. A device for detecting the presence of an analyte in a fluid sample, the
device comprising:
a solid substrate microfabricated to define:
a sample inlet port; and
a mesoscale flow system comprising:
a primary sample flow channel extending from said inlet port; and
a fractal region, in fluid communication with said primary flow channel,
comprising bifurcations leading to plural secondary flow channels; and
means for detecting a flow property of a fluid sample in said flow system
as an indication of the presence of an analyte in the fluid sample.
2. The device of claim 1 wherein said fractal region further comprises
junctions in fluid communication with said secondary flow channels leading
to a third flow channel in said mesoscale flow system.
3. The device of claim 1 further comprising means for inducing flow of said
sample through said mesoscale flow system.
4. The device of claim 2 wherein said fractal region comprises equal
numbers of bifurcations and junctions disposed serially along the
direction of flow of the sample through said fractal region.
5. The device of claim 4, wherein the fractal region has a reduced
cross-sectional area relative to said primary flow channel and said third
flow channel.
6. The device of claim 3, wherein said means for detecting comprises means
for detecting analyte induced restriction of flow through said flow
system.
7. The device of claim 2 wherein said means for detecting comprises means
for detecting a parameter in said third flow channel.
8. The device of claim 2, wherein said means for detecting comprises means
for detecting and comparing a parameter in said primary sample flow
channel with a parameter in said third flow channel.
9. The device of claim 7 or 8 wherein said means for detecting comprises an
electrical detection means.
10. The device of claim 7 wherein said parameter is fluid pressure.
11. The device of claim 7 wherein said parameter is fluid conductivity.
12. The device of claim, 1 wherein said means for detecting comprises means
for detecting a parameter in said fractal region.
13. The device of claim 12 wherein said detecting means comprises means
defining an optical path to said fractal region.
14. The device claim 1 further comprising a binding moiety disposed within
said fractal region for binding the analyte of said sample.
15. The device of claim 14 wherein said binding moiety comprises particles
which bind with the analyte of said sample to induce particle
agglomeration.
16. The device of claim 1 wherein said means for detecting comprises a
magnetic detection means.
17. The device of claim 1 wherein said substrate defines a plurality of
said flow systems.
18. The device of claim 1 wherein said means for detecting comprises means
for detecting the growth of an organism in said flow system.
19. The device of claim 1 wherein the sample is a sperm sample and wherein
flow of sperm through the fractal region provides an indication of sperm
motility.
20. The device of claim 19, wherein a channel in said substrate is disposed
at an angle with respect to a horizontal plane.
21. The device of claim 1 where said solid substrate comprises
microfabricated silicon.
22. The device of claim 1, further comprising an appliance for use in
combination with said substrate, said appliance comprising:
means for holding said substrate;
fluid input means interfitting with the inlet port on said substrate; and
pump means for passing fluid through the flow system of said substrate when
said substrate is held in said holding means.
23. The device of claim 1, 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.
24. The device of claim 23, 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.
25. The device of claim 1 wherein said flow system includes a control
fractal region permitting comparison of flow of said sample in said
fractal region and said control fractal region.
26. A method for detecting the presence or absence of an analyte in a fluid
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 primary sample flow channel extending from said inlet port; and
a fractal region, in fluid communication with said primary flow channel,
comprising bifurcations leading to plural secondary flow channels;
(ii) passing a fluid sample suspected to contain an analyte through said
mesoscale flow system;
(iii) detecting the restriction or blockage of flow of the fluid sample
through said system; and
(iv) correlating the detected restriction or blockage of flow, or lack of
said detected restriction or blockage of flow, to the presence or absence
of an analyte in said sample.
27. The method of claim 26, wherein said flow system comprises a binding
moiety, capable of binding said analyte in said sample, to promote said
restriction or blockage of flow through said system.
28. The method of claim 27 wherein said binding moiety is disposed on
particles which bind with said analyte in said sample to induce particle
agglomeration, thereby to promote said restriction or blockage of flow.
29. The method of claim 27 wherein said analyte is a cell population in
said sample;
said binding moiety comprises a crosslinker of cells in said population;
and
said restriction of flow is caused by crosslinker-induced cell aggregation.
30. The method of claim 27 wherein said binding moiety is immobilized
within said flow system.
31. The method of claim 30 wherein flow is restricted by the build-up of a
macromolecular surface layer on a surface of said flow system.
32. The method of claim 26 wherein said fractal region further comprises
junctions in fluid communication with said secondary flow channels leading
to a third channel in said flow system, to amplify the effect of occlusion
of said flow system or increase in viscosity within said substrate.
33. The method of claim 26 wherein, in step (iii), restriction or blockage
is detected electrically.
34. The method of claim 26 wherein, in step (iii), restriction or blockage
is detected optically.
35. The method of claim 32 wherein said fractal region further comprises a
binding moiety for binding the analyte of said sample.
36. The method of claim 35 wherein said binding moiety contains particles
which bind with the analyte of said sample to induce particle
agglomeration.
37. The method of claim 26, wherein said substrate, provided in step (i),
further comprises a control region in fluid communication with said sample
inlet port; and
wherein, in step (iii), flow of said sample in said fractal region and said
control region is detected and compared.
38. The method of claim 26 wherein the analyte comprises a replicable
procaryotic organism; and
wherein, in step (iii), the restriction or blockage of flow of said
organism through said flow system serves as an indication of the presence
of said organism.
39. A method for detecting the presence of an analyte in a fluid 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 primary sample flow channel extending from said inlet port; and
a fractal region, in fluid communication with said primary flow channel,
comprising bifurcations leading to plural secondary flow channels;
(ii) passing a fluid sample suspected to contain an analyte through said
mesoscale flow system;
(iii) detecting a flow property of the fluid sample in the flow system; and
(iv) correlating the detected flow property of the fluid sample to the
presence or absence of an analyte in the sample.
40. The method of claim 39 wherein said fractal region further comprises
junctions, in fluid communication with the secondary flow channels,
leading to a third flow channel.
41. The method of claim 32 or 40 wherein said fractal region comprises
equal numbers of bifurcations and junctions disposed serially along the
direction of flow of the sample through said fractal region.
42. The device of claim 1, 4 or 25 wherein, within at least a portion of a
channel in said flow system, each of the channel width and channel depth
is between 0.1 .mu.m and 500 .mu.m.
43. The device of claim 42 wherein the channel width in said portion is
between 2.0 and 300 .mu.m.
44. The device of claim 42 wherein the channel depth in said portion is
between 0.1 and 100 .mu.m.
45. The method of claim 26, 39 or 40 wherein, within at least a portion of
a channel in said flow system, the channel width and channel depth each
are between 0.1 .mu.m and 500 .mu.m.
46. The method of claim 45 wherein the channel width in said portion is
between 2.0 and 300 .mu.m.
47. The method of claim 45 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 applications: U.S. Ser. No. 07/877,702, filed May 1, 1992,
Mesoscale Detection Structures; U.S. Ser. No. 07/877,536, filed May 1,
1992, now U.S. Pat. No. 5,304,487, Fluid Handling in Mesoscale Analytical
Devices; U.S. Ser. No. 07/877,667, filed May 1, 1992, Mesoscale
Polynucleotide Amplification Analysis; and U.S. Ser. No. 07/877,661, filed
May 1, 1992, now U.S. Pat. No. 5,296,375, Mesoscale Sperm Handling
Devices, 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 rapidly
determining the presence of an analyte in 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 that can
analyze microvolumes of sample 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 of preselected molecular or
cellular analytes, in a range of 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 tests, e.g., tests for bacterial or
viral infection, sperm motility, blood parameters, contaminants in food,
water, or body fluids, and the like. Yet another object is to provide a
family of analytical assay protocols for detecting the presence of an
analyte wherein the information indicative of a positive assay is obtained
by measuring directly or indirectly alteration of flow properties of fluid
flowing through a restricted passage.
SUMMARY OF THE INVENTION
The invention provides methods and apparatus for detecting the presence of
an analyte in a fluid sample. In one embodiment, the invention provides a
device comprising a solid substrate, typically on the order of a few
millimeters thick and approximately a 0.2 to 2.0 centimeters square,
microfabricated to define a sample inlet port and a mesoscale flow system.
The invention provides a method wherein a sample fluid is passed through
the mesoscale flow system, and the analyte induced restriction or blockage
of flow through the system is detected as a positive indication of the
presence of the analyte. In one embodiment, the mesoscale flow system
includes a primary sample flow channel, extending from the inlet port, and
a fractal region, in fluid communication with the primary flow channel,
comprising bifurcations leading to plural secondary flow channels. The
term "mesoscale" is used herein to define flow passages having
cross-sectional dimensions on the order of approximately 0.1 .mu.m to 500
.mu.m, with 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 may have larger
dimensions, e.g., widths and lengths of 1-5 mm. Preferred depths are on
the order of 0.1 to 100 .mu.m, typically 2-50 .mu.m.
The fractal region typically further comprises junctions, in fluid
communication with the secondary flow channels, leading to a third flow
channel. The fractal region may comprise equal numbers of bifurcations and
junctions disposed serially along the direction of flow. Preferably, but
not necessarily, the branching channels in the fractal region
progressively decrease in cross-sectional area at each bifurcation and
increase at each junction. The fractal flow region is very sensitive to
the flow properties of a sample. Means may be provided in the device for
inducing flow of the sample through the flow system. Means also may be
provided in the device for detecting changes in flow properties, such as
restriction or blockage of flow, induced by the presence of an analyte.
The devices and methods of the invention may be used to implement a
variety of automated, sensitive and rapid tests including analyses for the
presence of particular types of cells or macromolecules, for monitoring
reactions or cell growth, or for conducting sperm motility testing.
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 fractal 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 (<5
.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. Analytes present in very low concentrations (e.g.,
nanogram quantities) can be rapidly detected (<10 minutes). After an assay
is complete, the devices can be discarded.
In one embodiment, a specific binding moiety may be provided in the
mesoscale flow system, e.g., in the fractal region, to enhance restriction
or blockage of sample flow through the flow system. The binding moieties
may comprise particles which bind with a component of the sample to induce
detectable particle agglomeration. Optionally, the binding moiety may be
immobilized on the internal surfaces of the mesoscale flow system, so that
binding induces stenosis of the passage.
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 biological fluid sample, suspected to contain a particular
analyte, such as a cellular contaminant, or toxin, 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 simply by capillary
action through an inlet port.
The presence of a preselected analyte in a fluid sample may be detected by
sensing analyte-induced changes in sample fluid flow properties, such as
changes in the pressure or electrical conductivity, at different points in
the flow system. In one embodiment, analyte induced restriction or
blockage of flow in the mesoscale flow system, e.g., in the fractal
region, may be detected by pressure detectors, e.g., in the appliance used
in combination with the device. In another embodiment, analyte-induced
changes in conductivity in a region of the flow system caused by
introduction of a sample fluid may be readily detected through electrical
conductivity sensors in contact with the flow system. For example, the
presence of analyte may cause clogging of a restricted flow passage, and
beyond the passage, the absence of liquid can be detected by measuring
conductivity. The appliance also may include electrical contacts in the
nesting region which mate with contacts integrated into the structure of
the chip to, e.g., receive electrical signals indicative of a pressure
reading, conductivity, or the like, sensed in some region of the flow
system to indicate flow restriction, as a positive indication of the
presence of the analyte.
Analyte induced changes in flow properties of a sample fluid also may be
detected optically, e.g., through a transparent or translucent window,
such as a transparent cover over the flow system, or through a translucent
section of the substrate itself. The appliance may include sensing
equipment, such as a spectrophotometer, capable of detecting analyte
induced changes in flow properties of a sample through an optical window
in a chip.
The devices of the invention 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, each with different binding
moieties in, e.g., different fractal detection regions, to enable the
detection of two or more analytes simultaneously. The device may also
comprise a control flow system so that data from the sample region and the
control region may be detected and compared. The devices can provide rapid
clinical tests for the detection of, e.g., pathogenic bacteria, or
viruses, or to test, e.g., the motility of a sperm sample. The invention
provides methods and devices for use in a wide range of possible assays.
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 biologically hazardous material, and provides
an inexpensive, microsample analysis.
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
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 Reduces per process cost to the user
Chips for certain applications.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a magnified plan view of device 10 according to the invention
that comprises substrate 14 microfabricated with ports 16, mesoscale flow
channel 20, and a fractally bifurcating system of flow channels 40.
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 cross sectional view of an analytical device 10
nested within an 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 schematic plan view of a substrate 14 microfabricated with a
fractally bifurcating system of flow channels 40 symmetrically disposed on
the substrate, and tapering to a narrower diameter towards the center of
the fractal system.
FIG. 6 is a schematic plan view of device 10 that includes substrate 14
microfabricated with entry ports 16, mesoscale flow channel 20, and a
fractally bifurcating system of flow channels 40, provided with beads 42
to enhance flow restriction and agglomeration in the fractal.
FIG. 7 is a schematic longitudinal cross-sectional view of a device
according to the invention which includes electrical conductors 17 and 18
for measuring conductivity of fluids in the device.
FIG. 8 is a perspective view of the device shown in FIG. 7.
FIG. 9 is a schematic plan view of a multitest apparatus constructed in
accordance with the invention.
FIG. 10 is a schematic plan view of an analytical device fabricated with a
series of mesoscale chambers suitable for implementing a variety of
functions including cell sorting, cell lysing, PCR analysis, and detection
of PCR products in the fractal region 40.
FIG. 11 is a schematic plan view of device 10 according to the invention
that includes substrate 14 microfabricated with ports 16, mesoscale flow
channels 20, and a pair of fractal flow channels 40.
FIG. 12 is a schematic perspective view of an apparatus 60 used in
combination with device 10 for viewing the contents of device 10.
FIG. 13 is a schematic cross sectional view of the apparatus 60 of FIG. 12.
Like reference characters in the respective drawn figures indicate
corresponding parts.
DETAILED DESCRIPTION
The invention provides methods and apparatus for detecting the presence of
an analyte in a fluid sample. In one embodiment, the invention provides a
device comprising a solid substrate, typically on the order of a few
millimeters thick and 0.2 to 2.0 centimeters square, microfabricated to
define a sample inlet port and a mesoscale flow system. A sample fluid is
passed through the mesoscale flow system, and the analyte induced
restriction or blockage of flow through the system is detected as a
positive indication of the presence of the analyte.
Analytical devices having mesoscale flow channels and fractal regions 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 and other
ports, the mesoscale flow system, including the sample flow channel(s),
the fractal 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. 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. The flow system may comprise a
nonbranching channel, or alternatively, in a preferred embodiment, the
flow system may comprise a fractal region including bifurications leading
to plural secondary channels. In the devices, flow restriction in the
mesoscale flow system serves as a positive indicator of the presence of an
analyte.
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 (<10 .mu.L). The volume of the flow system typically
will be <5 .mu.L, and the volume of individual channels, chambers, or
other functional elements are often less than 1 .mu.l, e.g., in the
nanoliter or picoliter range. 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.
An important consequence and advantage of employing flow channels having
mesoscale dimensions is that alterations in the flow properties of
macromolecules, particles, and cells entrained or dissolved in aqueous
liquids within the channels is easily influenced by stenosis, i. e.,
narrowing of the flow channels, and easily detected. The provision of the
fractal region serves to simplify alteration in flow. Thus, for example, a
sample suspected to be contaminated with bacteria can be cultured in the
device and the presence of a multiplicity of the organism can be detected
by determining whether fluid can be forced through the system at a given
pressure. Where no bacteria is present, fluid would flow easily; a large
number of cells would serve to partially or totally occlude the fractal
region. As another example, accretion of macromolecules onto specific
binding proteins immobilized on the walls of the flow channel is
sufficient to inhibit liquid flow through the channel provided its
dimensions are small enough. In still another example, the presence of a
target polynucleotide in a polynucleotide sample may be indicated by
flowing the contents of a chamber after a suitable number of PCR cycles
through a fractal region, as the viscosity of a solution laden with a
large amount of polynucleotides will be larger than a solution of
nucleotides.
In one embodiment, illustrated schematically in FIGS. 1, 2 and 3, the
device 10 may include a silicon substrate 14 microfabricated with ports
16, primary sample flow channel 20A, and a fractal system of flow channels
40. The ports may be microfabricated with mesoscale or larger dimensions.
The fractal region 40 in this case comprises equal numbers of bifurcations
and junctions, disposed serially along the direction of flow through the
fractal region, leading to a third flow channel 20B. The substrate 14 is
covered with a clear glass or plastic window 12 to close the channels. In
operation, a fluid sample enters the device through inlet port 16A and
flow channel 20A, and then flows through the fractal region 40 to flow
channel 20B and port 16B. The fractal region 40 is very sensitive to the
flow properties of a sample. Restriction or blockage of flow of a sample
through the fractal region 40 can serve as an indicator of the presence of
an analyte in the sample and may be detected, e.g., optically through the
window 12.
In another embodiment, the fractal system 40 may be fabricated on a silicon
substrate with reduced dimensions at each bifurcation, providing
sequentially narrower flow channels, as illustrated schematically in FIG.
5. FIG. 5 shows device 10, which comprises substrate 14 microfabricated
with fractal flow channels 40, which have a reduced cross-sectional area
relative to the primary flow channel 20A and the third flow channel 20B.
In operation, a sample fluid enters the device 10 through inlet port 16A
and channel 20A, and then flows through the fractal region 40 to flow
channel 20B and port 16B. Fluid flow through this fractal region 40 is
very sensitive to changes in fluid viscosity and to | | |
