U.S. patent application number 09/804780 was filed with the patent office on 2002-01-17 for simultaneous particle separation and chemical reaction.
Invention is credited to Kenny, Margaret A., Weigl, Bernhard, Wu, Caicai, Yager, Paul.
Application Number | 20020006670 09/804780 |
Document ID | / |
Family ID | 25471634 |
Filed Date | 2002-01-17 |
United States Patent
Application |
20020006670 |
Kind Code |
A1 |
Wu, Caicai ; et al. |
January 17, 2002 |
Simultaneous particle separation and chemical reaction
Abstract
This invention provides a method and apparatus for detecting the
presence of analyte particles in a sample fluid also comprising
larger particles, particularly blood. It exploits diffusion to
provide simultaneous filtering of the larger particles and reaction
of the analyte particles. A sample stream and a reagent stream join
on the upstream end of a laminar flow reaction channel and flow in
adjacent laminar streams. The reagents can be in solution or
immobilized on a bead. The analyte particles diffuse from the
sample stream into the reagent stream, leaving behind the larger
particles in the residual sample stream. In the reagent stream the
analyte particles react with reagent particles and form product
particles, thereby creating a product stream. At the downstream end
of the reaction channel, the residual sample stream and the product
stream are divided. The product particles are then detected,
preferably optically, in the product stream. Prior to detection,
the product stream can undergo further filtering or separation, or
can join a second reagent stream to form secondary product
particles. This apparatus and method can be used to implement
competitive immunoassays or sandwich immunoassays using
enzymatically or fluorescently labeled immunoreagents. The
apparatus and method can also be used to synthesize products, in
which case two reagent streams join in the laminar flow reaction
channel.
Inventors: |
Wu, Caicai; (Seattle,
WA) ; Weigl, Bernhard; (Seattle, WA) ; Kenny,
Margaret A.; (Edmonds, WA) ; Yager, Paul;
(Seattle, WA) |
Correspondence
Address: |
GREENLEE WINNER AND SULLIVAN P C
5370 MANHATTAN CIRCLE
SUITE 201
BOULDER
CO
80303
US
|
Family ID: |
25471634 |
Appl. No.: |
09/804780 |
Filed: |
March 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09804780 |
Mar 13, 2001 |
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09501732 |
Feb 10, 2000 |
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6297061 |
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09501732 |
Feb 10, 2000 |
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09335930 |
Jun 18, 1999 |
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6221677 |
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09335930 |
Jun 18, 1999 |
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08938585 |
Sep 26, 1997 |
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Current U.S.
Class: |
436/514 |
Current CPC
Class: |
G01N 33/54313 20130101;
Y10T 436/25375 20150115; Y10T 436/117497 20150115; Y10T 436/2575
20150115; G01N 33/5005 20130101; Y10T 436/118339 20150115; Y10T
436/255 20150115 |
Class at
Publication: |
436/514 |
International
Class: |
G01N 033/558 |
Claims
We claim:
1. A method for reacting first antigens in a stream of blood, said
method comprising the steps of: conducting said blood stream into a
laminar flow reaction channel; separately conducting a reagent
stream comprising first antibodies bound with labeled second
antigens identical to said first antigens into said reaction
channel, such that said blood stream and said reagent stream flow
in adjacent laminar streams; allowing said first antigens to
diffuse from said blood stream into said reagent stream, and to
compete for binding sites on said first antibodies with said
labeled second antigens to form detectable product particles,
thereby converting said reagent stream into a product stream and
said primary stream into a residual primary stream; conducting said
residual primary stream out of said reaction channel; separately
conducting said product stream out of said reaction channel; and
detecting said product particles.
2. The method of claim 1 wherein said first reagent stream further
includes second antibodies, and wherein said first and second
antibodies form a sandwich complex with said first antigens.
3. A method for reacting primary particles from a primary stream,
comprising the steps of: conducting said primary stream into a
first laminar flow reaction channel; separately conducting a first
reagent stream comprising first reagent particles into said first
laminar flow reaction channel, such that said primary stream and
said first reagent stream flow in adjacent laminar streams in said
channel; allowing said primary particles to diffuse from said
primary stream into said first reagent stream, and to react with
said first reagent particles and form first product particles, thus
converting said first reagent stream into a first product stream;
and thereafter conducting a first companion stream into said first
laminar flow reaction channel such that said first product stream
and said first companion stream flow in adjacent laminar streams,
thereby converting said first product stream into a diffused first
product stream and said first companion stream into a diffused
first companion stream.
4. The method of claim 3 further comprising allowing particles in
said diffused first product stream and said diffused first
companion streams to react with each other to form second product
particles.
5. The method of claim 4 further comprising detecting said second
product particles.
6. The method of claim 3 wherein said primary stream contains
larger, nondiffusing particles in addition to said primary
particles.
7. The method of claim 6 wherein said primary stream is blood, said
primary particles are first antigens, and said first reagent
particles are first antibodies.
8. The method of claim 3 wherein said step of detecting said first
product particles comprises a method selected from the group
consisting of optical, electrical, calorimetric and chemical
detection.
9. The method of claim 3 wherein said step of detecting comprises
absorbance, luminescence, fluorescence, or radiation detection.
10. The method of claim 3 wherein said first reagent particles are
immobilized on beads.
11. The method of claim 10 wherein said beads are magnetic.
12. The method of claim 10 further comprising the step of detecting
said first product particles using single particle detection.
13. A method for detecting first antigens in a stream of blood,
comprising the steps of: conducting said blood stream into a first
laminar flow reaction channel; separately conducting a first
reagent stream comprising first reagent particles which are beads
having primary antibodies to said first antigens attached thereto,
into said first laminar flow reaction channel, such that said blood
stream and said first reagent stream flow in adjacent laminar
streams; allowing said first antigens to diffuse from said blood
stream into said first reagent stream, and to react with said
primary antibodies on said beads to form a first complex in said
reaction channel, thereby converting said first reagent stream into
a first product stream comprising said first complex, and said
blood stream into a residual blood stream; thereafter conducting a
first companion stream comprising fluorescently-labeled or
radioactively-labeled secondary antibodies to said first antigens
into said first laminar flow reaction channel such that said first
product stream and said first companion stream flow in adjacent
laminar streams; allowing said fluorescently-labeled or
radioactively-labeled secondary antibodies to diffuse from said
first companion stream into said first product stream and react
with said first complex to form a fluorescently-labeled or
radioactively-labeled second complex attached to said beads; and
detecting said fluorescently-labeled or radioactively-labeled
second complex on said beads.
14. The method of claim 13 wherein said beads having said
fluorescently-labeled second complex attached thereto are detected
in a flow cytometer.
15. The method of claim 13 wherein said beads having said
fluorescently-labeled second complex attached thereto are optically
detected.
16. A method for detecting first antigens in a stream of blood,
comprising the steps of: conducting said blood stream into a first
laminar flow reaction channel; separately conducting a first
reagent stream comprising first reagent particles which are first
complexes comprising second antigens identical to said first
antigens labeled with an enzyme and antibodies to said antigens,
into said first laminar flow reaction channel, such that said blood
stream and said first reagent stream flow in adjacent laminar
streams; allowing said first antigens to diffuse from said blood
stream into said reagent stream to form a mixed stream in which
said first antigens react with said first complexes to bind to said
antibodies and form second complexes comprising said first antigens
complexed with said antibodies, and third complexes comprising said
second antigens labeled with said enzyme, in said reaction channel;
thereafter conducting a first companion stream comprising substrate
for said enzyme into said first laminar flow reaction channel such
that said mixed stream and said first companion stream flow in
adjacent laminar streams; allowing said substrate to react with
said third complex to form product particles in a second laminar
flow channel; and detecting said product particles.
17. The method of claim 16 wherein said second laminar flow channel
is a convoluted channel.
18. A method for detecting first antigens in a stream of blood,
comprising the steps of: conducting said blood stream into a first
laminar flow reaction channel; separately conducting a first
reagent stream comprising first reagent particles which are
magnetic beads having primary antibodies to said first antigens
attached thereto, into said first laminar flow reaction channel,
such that said blood stream and said first reagent stream flow in
adjacent laminar streams; applying a magnetic field to push said
primary antibodies on said magnetic beads into said blood stream
and allowing said antigens to react with said primary antibodies on
said magnetic beads to form a first complex attached to said
magnetic beads in said blood stream; thereafter applying a magnetic
field to push said magnetic beads having said first complex
attached thereto into said reagent stream, thereby converting said
first reagent stream into a first product stream comprising said
first complex, and said blood stream into a residual blood stream;
conducting said first product stream into a second laminar flow
channel; thereafter conducting a first companion stream comprising
labeled secondary antibodies to said first antigens into said
second laminar flow reaction channel such that said first product
stream and said first companion stream flow in adjacent laminar
streams; allowing said labeled secondary antibodies to diffuse from
said first companion stream into said first product stream and
react with said first complex to form a second complex attached to
said magnetic beads; and detecting said labeled second complex on
said beads.
19. The method of claim 18 wherein said magnetic beads having said
labeled second complex attached thereto are detected in a flow
cytometer.
20. A method for reacting primary particles from a primary stream,
comprising the steps of: conducting said primary stream into a
first laminar flow reaction channel; separately conducting a first
reagent stream comprising first reagent particles into said first
laminar flow reaction channel, such that said primary stream and
said first reagent stream flow in adjacent laminar streams;
allowing said primary particles to diffuse from said primary stream
into said first reagent stream, and to react with said first
reagent particles and form first product particles, thereby
converting said first reagent stream into a first product stream
and said primary stream into a residual primary stream; conducting
said residual primary stream out of said first laminar flow
reaction channel; conducting said first product stream into a
second laminar flow channel; and separately conducting a first
companion stream into said second laminar flow channel such that
said first product stream and said first companion stream flow in
adjacent laminar streams thereby converting said first product
stream into a diffused first product stream and said first
companion stream into a diffused first companion stream.
21. The method of claim 20 further comprising the steps of
conducting said diffused first product stream out of said second
laminar flow channel and separately conducting said diffused first
companion stream out of said second laminar flow channel.
22. The method of claim 20 further comprising the steps of:
conducting said diffused first product stream into a third laminar
flow channel; and separately conducting a second companion stream
into said third laminar flow channel.
23. The method of claim 22 further comprising allowing particles in
said diffused first product stream or said second companion stream
to diffuse therebetween and react with each other to form third
product particles.
24. The method of claim 23 further comprising the step of detecting
said third product particles.
25. The method of claim 23 further comprising the step of
separately conducting a stream containing said third product
particles from said third laminar flow channel.
26. The method of claim 20 further comprising the steps of:
conducting said diffused first companion stream into a third
laminar flow channel; and separately conducting a second companion
stream into said third laminar flow channel.
27. The method of claim 26 further comprising allowing particles in
said diffused first companion stream or said second companion
stream to diffuse therebetween and react with each other to form
third product particles.
28. The method of claim 27 further comprising the step of detecting
said third product particles.
29. The method of claim 27 further comprising the step of
separately conducting a stream containing said third product
particles from said third laminar flow channel.
30. The method of claim 27 wherein: said primary stream comprises
whole blood; said primary particles are first antigens; said
reagent particles are first complexes comprising second antigens,
identical to said first antigens, labeled with an enzyme, and
antibodies to said antigens, attached to beads; said first
companion stream is an extraction stream; said second companion
stream comprises a substrate for said enzyme; and said method
comprises the steps of: allowing said first antigens to diffuse
from said primary stream into said reagent stream and react with
said first complex to bind to said antibodies and form second
complexes comprising said first antigens complexed with said
antibodies attached to said beads, and third complexes comprising
said second antigens labeled with said enzyme not attached to said
beads, in said reaction channel; wherein said diffused first
product stream conducted out of said second laminar flow channel
comprises said first antigens complexed with said antibodies
attached to said beads; and said diffused companion stream
comprises said third complex; allowing said substrate to react with
said third complex to form product particles in said third laminar
flow channel; and detecting said product particles.
31. The method of claim 30 wherein said third laminar flow channel
is convoluted.
32. The method of claim 21 wherein: said primary stream comprises
whole blood; said primary particles are first antigens; said
reagent particles are first complexes comprising primary antibodies
to said antigens attached to magnetic beads; said first companion
stream comprises secondary antibodies to said antigens labeled with
an enzyme in buffer; said method further comprises the steps of:
allowing said first antigens to diffuse from said primary stream
into said reagent stream and react with said first complexes to
bind to said antibodies and form second complexes comprising said
first antigens complexed with said antibodies attached to said
beads in said reaction channel; allowing said second complexes to
react with said secondary antibodies labeled with said enzyme to
said antigens in said second laminar flow channel to form third
complexes comprising said first antigens, said primary antibodies,
and said secondary antibodies linked to said enzyme attached to
said magnetic beads; applying a magnetic field across said second
laminar flow channel to maintain said magnetic beads in said
diffused product stream; conducting said diffused product stream
out of said second laminar flow channel into a third laminar flow
channel; separately conducting a second companion stream comprising
a substrate for said enzyme into said third laminar flow channel;
allowing said enzyme to react with said substrate in said third
laminar flow channel to form product particles; and detecting said
product particles.
33. The method of claim 32 wherein said third laminar flow channel
is convoluted.
34. The method of claim 22 wherein said primary stream comprises
whole blood; said primary particles are antigens; said reagent
particles are primary antibodies to said antigens and secondary
antibodies to said antigens linked to an enzyme; said first
companion stream is an extraction stream; said second companion
stream comprises substrate for said enzyme; said method further
comprises the steps of: allowing said antigens to diffuse from said
primary stream into said reagent stream and react with primary
antibodies and said secondary antibodies linked to an enzyme, to
form complexes comprising said antigens, said primary antibodies
and said secondary antibodies linked to an enzyme, in said reaction
channel; allowing unreacted secondary antibodies linked to said
enzyme to diffuse from said first product stream into said
companion stream; allowing said complexes to react with said
substrate in said third laminar flow channel to form product
particles; and detecting said product particles.
35. The method of claim 20 wherein said primary stream comprises
whole blood; said primary particles are antigens; said reagent
particles are primary antibodies to said antigens and
fluorescently-labeled or radioactively-labeled secondary antibodies
to said antigens; said first companion stream is an extraction
stream; said method further comprises the steps of: allowing said
antigens to diffuse from said primary stream into said reagent
stream and react with primary antibodies and fluorescently-labeled
or radioactively-labeled secondary antibodies to form complexes
comprising said antigens, said primary antibodies, and said
secondary antibodies in said reaction channel; allowing unreacted
fluorescently-labeled or radioactively-labeled secondary antibodies
to diffuse from said first product stream into said companion
stream; and detecting said unreacted fluorescently-labeled or
radioactively-labeled antibodies in said diffused first companion
stream.
36. The method of claim 22 wherein: said primary stream comprises
whole blood; said primary particles are antigens; said reagent
particles are primary antibodies to said antigens; said first
companion stream comprises fluorescently-labeled or
radioactively-labeled secondary antibodies to said antigens; said
second companion stream is an extraction stream; and said method
further comprises: allowing said antigens and said primary
antibodies to react in said first reaction channel to form first
complexes in said product stream; allowing said
fluorescently-labeled or radioactively-labeled secondary antibodies
to react with said first complexes in said second laminar flow
channel to form second complexes comprising said antigens, said
primary antibodies and said fluorescently-labeled or
radioactively-labeled secondary antibodies; allowing excess
fluorescently-labeled or radioactively-labeled secondary antibodies
to diffuse from said first diffused product stream into said second
companion stream; and detecting said excess fluorescently-labeled
or radioactively-labeled secondary antibodies in said second
companion stream in said second laminar flow channel.
37. A method for detecting antigens in whole blood comprising the
steps of: conducting a primary stream comprising whole blood
containing first antigens into a laminar flow reaction channel;
separately conducting into said reaction channel a reagent stream
comprising fluorescently-labeled or radioactively-labeled second
antigens identical to said first antigens, complexed with
antibodies to said antigens, to form a complex, such that said
primary stream and said reagent stream flow in adjacent laminar
streams in said channel; allowing first antigens from said blood to
diffuse from said primary stream into said reagent stream, and to
react with said complex to displace fluorescently-labeled or
radioactively-labeled second antigens therefrom, thereby converting
said reagent stream into a product stream and said primary stream
into a residual stream; conducting said residual stream out of said
reaction channel; conducting said product stream into a second
laminar flow channel; conducting a companion stream into said
second laminar flow channel, such that said product stream and said
companion stream flow in adjacent laminar streams; allowing
displaced fluorescently-labeled or radioactively-labeled second
antigens to diffuse into said companion stream; and detecting said
displaced fluorescently-labeled or radioactively-labeled second
antigens in said companion stream.
38. A method for detecting antigens in whole blood comprising the
steps of: conducting a primary stream comprising whole blood
containing first antigens into a laminar flow reaction channel;
separately conducting into said reaction channel a reagent stream
comprising beads having attached thereto antibodies complexed with
fluorescently-labeled or radioactively-labeled second antigens,
identical to said first antigens, to form a complex, such that said
primary stream and said reagent stream flow in adjacent laminar
streams; allowing first antigens from said blood to diffuse from
said primary stream into said reagent stream and to react with said
complex to displace fluorescently-labeled or radioactively-labeled
second antigens on said beads, forming a residual stream comprising
whole blood from which small particles have diffused into said
reagent stream, and a product stream comprising said beads with
antigen-antibody complexes attached thereto; conducting said
residual stream out of said reaction channel; and conducting said
product stream into a second laminar flow channel; conducting a
companion stream into said second laminar flow channel, such that
said product stream and said companion stream flow in adjacent
laminar streams; allowing displaced fluorescently-labeled or
radioactively-labeled second antigens to diffuse into said
companion stream; and detecting said displaced
fluorescently-labeled or radioactively-labeled second antigens in
said companion stream, or detecting fluorescently-labeled or
radioactively-labeled beads and/or fluorescently-labeled or
radioactively-labeled second antigens.
39. A method for detecting antigens from a whole blood stream,
comprising the steps of: conducting a whole blood stream into a
first laminar flow channel; separately conducting an extraction
stream into said first laminar flow channel, such that said whole
blood stream and said extraction stream flow in adjacent laminar
streams in said channel; allowing antigens to diffuse from said
whole blood stream into said extraction stream, thereby converting
said extraction stream into a product stream and said whole blood
stream into a residual primary stream; conducting said residual
primary stream out of said reaction channel; separately conducting
said product stream out of said reaction channel and into a second
laminar flow channel; conducting a first companion stream
comprising enzyme particles, for which said antigens are
substrates, into a second laminar flow channel such that said first
companion stream and said product stream flow in adjacent laminar
streams in said second laminar flow channel; allowing said enzyme
particles and said antigens to diffuse from their respective
streams and react with each other in said second laminar flow
channel to form first product particles, thereby converting said
streams into a diffused stream; and detecting said first product
particles.
40. The method of claim 39 wherein said second laminar flow channel
is convoluted.
41. The method of claim 39 further comprising the steps of:
conducting said diffused stream into a third laminar flow channel;
conducting a second companion stream comprising indicator particles
into said third laminar flow channel such that said diffused stream
and said second companion stream flow in adjacent laminar streams;
allowing particles from said diffused stream to diffuse into said
second companion stream and react with indicator particles to form
second product particles; and detecting said second product
particles.
42. A method for detecting antigens from a whole blood stream,
comprising the steps of: conducting a whole blood stream into a
first laminar flow channel; separately conducting an extraction
stream into said first laminar flow channel, such that said whole
blood stream and said extraction stream flow in adjacent laminar
streams in said channel; allowing antigens to diffuse from said
whole blood stream into said extraction stream, thereby converting
said extraction stream into a product stream and said whole blood
stream into a residual primary stream; conducting said residual
primary stream out of said reaction channel; separately conducting
said product stream out of said reaction channel and into a second
laminar flow channel; conducting a first companion stream
comprising enzyme particles, for which said antigens are
substrates, into a second laminar flow channel such that said first
companion stream and said product stream flow in adjacent laminar
streams in said second laminar flow channel; allowing said enzyme
particles and said antigens to diffuse from their respective
streams and react with each other in said second laminar flow
channel to form first product particles, thereby converting said
streams into a diffused stream; further comprising the steps of:
conducting said diffused stream into a third laminar flow channel;
conducting a second companion stream comprising indicator particles
into said third laminar flow channel such that said diffused stream
and said second companion stream flow in adjacent laminar streams;
allowing particles from said diffused stream to diffuse into said
second companion stream and react with indicator particles to form
second product particles; and detecting said second product
particles.
43. The method of claim 42 wherein said second laminar flow channel
is convoluted.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/501,732 filed Feb. 10, 2000, which is a continuation of U.S.
Ser. No. 09/335,930 filed Jun. 18, 1999, which is a divisional
application of U.S. Ser. No. 08/938,585 filed Sep. 26, 1997, now
abandoned.
FIELD OF THE INVENTION
[0002] This invention relates to simultaneous diffusion based
filtering and chemical reaction of analytes in streams containing
both these analytes and larger particles. The invention is useful,
for example, for analyzing blood to detect the presence of small
particles such as antigens in a stream containing cells, or for
preparing small volumes of fluid products.
BACKGROUND OF THE INVENTION
[0003] It is possible to fabricate intricate fluid systems with
channel sizes as small as a micron. These devices can be
mass-produced inexpensively and are expected to soon be in
widespread use for simple analytical tests. However, in chemical
analysis of turbid fluids, notably blood, filtering of the larger
particles such as cells is generally required prior to analysis,
especially optical analysis. In clinical laboratories this is
generally accomplished by centrifugation. The centrifugal force
generated is a function of distance from the center, and thus
centrifugation is not effective in a small scale apparatus. In
chemical laboratories membrane filters are used to separate the
larger particles. This can be used in microscale apparatus, but
clogging of the filters with use makes them impractical.
[0004] The greater diffusion of small particles relative to larger
particles can be used to partially separate the species. Diffusion
is a process which can easily be neglected at large scales, but
rapidly becomes important at the microscale. Due to extremely small
inertial forces in such structures, practically all flow in
microstructures is laminar. This allows the movement of different
layers of fluid and particles next to each other in a channel
without any mixing other than diffusion. Moreover, due to the small
lateral distances in such channels, diffusion is a powerful tool to
separate molecules and small particles according to their diffusion
coefficients, which is usually a function of their size.
[0005] The present invention exploits diffusion to provide
simultaneous filtering and chemical reaction, which facilitates the
elimination of preprocessing of specimens containing particulate
constituents, thus reducing the sample size and analytical time
required.
SUMMARY OF THE INVENTION
[0006] This invention provides a method and apparatus for reacting
small particles in a fluid also comprising larger particles. It
provides simultaneous filtering of the larger particles and
reaction of the small particles. The reactor can be followed by
collection of or detection of the reaction products. The reactor
exploits diffusion to separate the small primary particles from the
larger particles. It utilizes microscale channels wherein diffusion
becomes a significant factor and wherein the fluid flow is laminar.
The reactor can be simply and inexpensively manufactured and can be
disposed of after use. The reactor is capable of processing a fluid
volume between about 0.01 microliters and about 20 microliters
within a few seconds. Operation with sub-microliter volumes of
sample fluid is a particular advantage for expensive reagents or
for blood analysis. Larger volumes with correspondingly longer
times can be used when preferred, for example viral detection in a
sample with a low viral load.
[0007] The reactor can be used for analysis, in which case the
inlet fluid, termed generically the primary fluid, is a sample
fluid and the small particles, termed generically the small primary
particles, are analyte particles. In this case the reactor is
generally coupled with a detector. Alternatively, the reactor can
be used to rapidly synthesize small volumes of product fluids. In
this case the primary fluid is a reagent fluid and the small
primary particles are reagent particles. This has particular
application to making products starting from natural substances. In
the following, the reactor is described for the analysis
embodiment, but the description also applies to the synthesis
embodiment.
[0008] The invention uses an "H" shaped reactor. In the H-reactor
the crossbar of the H is a laminar flow reaction channel. On the
upstream end of the crossbar a sample (primary) stream and a
reagent stream enter through separate arms of the H, and the sample
stream and the reagent stream flow in adjacent laminar streams in
the crossbar. Because the flow is laminar, there is no turbulent
mixing of the two streams, but the analyte particles diffuse from
the sample stream into the reagent stream, leaving behind the
larger particles in the residual sample stream. In the reagent
stream the analyte particles react with reagent particles and form
product particles, thereby creating a product stream. At the
downstream end of the crossbar, the residual sample stream and the
product stream divide into the two downstream arms of the H. The
product particles can then be detected in the product stream.
[0009] Detection of the product particles can be performed using
optical, electrical, chemical, electrochemical or calorimetric
analysis, or any other technique in the analytical art. More than
one detection technique can be used in the same system. The
preferred embodiments use optical analysis or a combination of
electrochemical and optical analysis. In optical detection, the
product stream can be analyzed by luminescence, fluorescence or
absorbance. To increase the signal in the detection zone the
product stream channel can be broadened or convoluted. The product
stream can connect to a flow cytometer for analysis, particularly a
flow cytometer having a microfabricated flow channel.
[0010] An example of an application of this method is in
competitive immunoassays in solution. The sample stream is whole
blood containing native antigens. The reagent particles are
antibodies bound to a fluorescently labeled antigen. In the
reaction channel, the native antigens diffuse into the reagent
stream and displace the fluorescently labeled antigens. The product
stream contains both native and fluorescently labeled antigens,
some of which are free and some of which remain bound to
antibodies. The relative amounts of free and bound fluorescently
labeled antigen, which is a function of the amount of native
antigen in the blood, can be measured.
[0011] Prior to detection, the product stream can undergo further
filtering or separation. In particular the product stream can join
with an extraction stream in a separation channel such that the
product and extraction streams flow in adjacent laminar flow
streams. Smaller particles in the product stream flow into the
extraction stream for detection, preferably optical detection.
[0012] An example of utilizing the separating channel, is a
competitive immunoassay as above wherein the antibody-fluorescently
labeled antigen complex is immobilized on a microbead. In the
separation channel, the free and bound fluorescently labeled
antigen can be separated by diffusion. The free antigen that enters
the extraction stream can be detected by fluorescence without
interference from the antigen on the beads. In lieu of differential
separation, the product stream can be coupled with a flow cytometer
to measure the fluorescence intensity remaining on the beads. The
bead can be magnetic, and a magnetic field can be used to pin the
bead in the sample stream to allow reaction with the analyte
particles. Following reaction, a reverse field returns the beads to
the reagent stream.
[0013] The detection process can use a second reagent-stream that
joins with the product stream in a "T" configuration. The two
streams flow in adjacent laminar streams, and small product
particles from the product stream diffuse into the second reagent
stream, or small reagent particles from the second reagent stream
diffuse into the product stream. Depending on the diffusion
process, in either or both streams the product particles react with
the second reagent particles to form secondary product particles.
The secondary product particles are detected as described above for
primary product particles.
[0014] First and second reagent streams are useful, for example,
for sandwich immunoassays. The first reagent is a primary antibody
which binds to an antigen from the sample to form a first product.
The first reagent is large enough to change the diffusion
coefficient of the complex. The second reagent is a fluorescently
labeled secondary antibody, which reacts with the first product to
form a sandwich complex. The complex is detected as described above
for primary products. The slower diffusion of the complexed
relative to the uncomplexed labeled antibody is used to distinguish
between the two, either by diffusional separation of the species or
by the extent of depolarization of the fluorescence.
[0015] Second reagent streams are also utilized when the first
reaction involves an enzymatically labeled rather than
fluorescently labeled reagent. The difference in enzymatic activity
of bound and unbound enzymatically labeled reagent particles allows
measurement of the extent of reaction. Through the second reagent
stream, a substrate which is sensitive to the enzyme joins the
first reaction products. Reaction of the substrate and enzyme is
then detected as described above for primary products.
[0016] The first or subsequent product streams can flow through a
delay line to allow the reaction to be completed before detection
or before joining a subsequent reagent stream. The first or
subsequent product streams can also undergo filtering or
diffusional separation before detection or before joining a
subsequent reagent stream. The reagents can be immobilized on
magnetic beads, and a magnetic field can be used to pin the beads
for reaction or flushing steps in any of the reaction channels.
[0017] The sample stream may be any stream containing an analyte
and also containing less diffusive particles, for example blood or
other body fluids, contaminated drinking water, contaminated
organic solvents, biotechnological process samples, e.g.,
fermentation broths, and the like. The analyte can be any smaller
particle in the sample stream which is capable of diffusing into
the reagent stream faster than the larger particles, so as to
substantially leave the larger particles in the residual sample
stream. Examples of analyte particles are hydrogen, calcium, sodium
and other ions, dissolved oxygen, proteins such as albumin, organic
molecules such as alcohols and sugars, drugs such as salicylic
acid, halothane and narcotics, pesticides, heavy metals, organic
and inorganic polymers, viruses, small cells and other particles.
In the preferred embodiment wherein the sample stream is whole
blood, small particles such as antigens diffuse rapidly across the
channel, whereas larger particles such as blood cells diffuse
slowly.
[0018] The larger particles in the sample stream may also be
sensitive to the reagent. Because these do not diffuse into the
reagent stream, they do not interfere with detection of the
analyte. By diffusion of the analyte but not the larger particles,
cross-sensitivities of reagents to larger sample components, a
common problem, can be avoided. Furthermore, the reagent can be
kept in a solution in which it displays its optimal
characteristics. For example, cross-sensitivities to pH or ionic
strength can be suppressed by using strongly buffered reagent
solutions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1, comprising FIGS. 1a-b, is an H reactor illustrated
with a competitive immunoassay. The reactor is shown in (a) plan
view and (b) cross section.
[0020] FIG. 2 is an H reactor illustrated with a sandwich
immunoassay.
[0021] FIG. 3 is an H reactor illustrated with a competitive
immunoassay wherein the antibody is immobilized on a bead.
[0022] FIG. 4 is an H reactor illustrated with a competitive
immunoassay wherein the antibody is immobilized on a magnetic bead
and wherein a magnetic field is applied to pin the beads to one
side of the reaction channel.
[0023] FIG. 5 is a convoluted detection channel for optical
detection.
[0024] FIG. 6 is a broadened detection channel for optical
detection.
[0025] FIG. 7 is a T separator for use with optical detection.
[0026] FIG. 8 is a single particle detection channel for use with
optical detection.
[0027] FIG. 9 is an H reactor with a first reagent inlet followed
by a T reactor with a second reagent inlet, illustrated with a
sandwich immunoassay.
[0028] FIG. 10 is a T reactor for use in combination with an H
reactor, illustrated with the reaction of an enzymatically labeled
antigen.
[0029] FIG. 11 is an H filter and a T reactor for use in
combination with an H reactor, illustrated with a sandwich
immunoassay with subsequent selection of the sandwich complex,
followed by reaction of an enzymatically labeled antibody with a
substrate.
[0030] FIG. 12 is an H filter and a T reactor for use in
combination with an H reactor, illustrated with a sandwich
immunoassay with subsequent selection of the uncomplexed antibody,
followed by reaction of an enzymatically labeled antibody with a
substrate.
[0031] FIG. 13 is an H reactor and subsequent T reactor with
diffusion in the plane perpendicular to the channel cell surface,
illustrated with a competitive immunoassay and subsequent reaction
of an enzymatically labeled antigen with a substrate.
[0032] FIG. 14 shows an embodiment in which an H-filter (not shown)
is connected to two T-reactors in series.
DETAILED DESCRIPTION OF THE INVENTION
[0033] This invention relates to the following co-pending Patent
Applications, all of which are incorporated by reference in their
entirety: U.S. Ser. No. 08/625,808, "Microfabricated
Diffusion-Based Chemical Sensor," filed Mar. 29, 1996, now allowed;
U.S. Ser. No. 08/829,679, "Microfabricated Diffusion-Based Chemical
Sensor," filed Mar. 31, 1997; U.S. patent application Ser. No.
08/900,926, "Simultaneous Analyte Determination and Reference
Balancing in Reference T-Sensor Devices," filed Jul. 25, 1997; U.S.
Ser. No. 08/621,170 "Fluorescent Reporter Beads for Fluid
Analysis," filed Mar. 20, 1996; U.S. Ser. No. 08/663,916,
"Microfabricated Differential Extraction Device and Method," filed
Jun. 14, 1996; U.S. Ser. No. 08/534,515, "Silicon Microchannel
Optical Flow Cytometer," filed Sep. 27, 1995; PCT No. 96/15566
"Silicon Microchannel Optical Flow Cytometer," filed Sep. 27, 1996;
U.S. Ser. No. 08/823,747, "Device and Method For 3-Dimensional
Alignment of Particles in Microfabricated Flow Channels," filed
Mar. 26, 1997; U.S. Ser. No. 08/876,038, "Adsorption-Enhanced
Differential Extraction Device," filed Jun. 13, 1997; U.S. Ser. No.
60/049,533, "Method For Determining Concentration of a Laminar
Sample Stream," filed Jun. 13, 1997; U.S. Ser. No. 08/938,584,
"Device for Rapidly Joining and Splitting Fluid Layers," filed
concurrently herewith; Ser. No. 08/938,093, "Multiple Analyte
Diffusion Based Chemical Sensor," filed concurrently herewith.
[0034] The channel cells and method of this invention are designed
to be carried out such that all flow is laminar. In general, this
is achieved in a device comprising microchannels of a size such
that the Reynolds number for flow within the channel is below about
1, preferably below about 0.1. Reynolds number is the ratio of
inertia to viscosity. Low Reynolds number means that inertia is
essentially negligible, turbulence is essentially negligible, and
the flow of the two adjacent streams is laminar, i.e. the streams
do not mix except for the diffusion of particles as described
above. Flow can be laminar with Reynolds number greater than 1.
However, such systems are prone to developing turbulence when the
flow pattern is disturbed, e.g., when the flow speed of a stream is
changed, or when the viscosity of a stream is changed. The
preferred embodiments of this invention utilize liquid streams,
although the methods and devices are also suitable for use with
gaseous streams.
[0035] The H reactor of this invention is illustrated in plan view
in FIG. 1a and in cross section in FIG. 1b. The channel cell
containing the H reactor comprises substrate plate 1 and coverplate
2. Sample (primary) stream 11 enters sample stream inlet channel 10
through inlet 3. The sample contains analyte (small primary)
particles 12 and larger particles 13. The term "particles" refers
to any species, including dissolved and particulate species such as
molecules, cells, suspended and dissolved particles, ions and
atoms. In this example the sample is whole blood, the analyte is an
antigen, and the larger particles are blood cells. Reagent stream
21, also labeled R1, separately enters reagent stream inlet channel
20 through inlet 4. The term "separately" is used for streams
having individual rather than shared flow channels. In this example
the reagent particles are antibody 23 bound with labeled antigen
22.
[0036] The sample and reagent streams join in reaction channel 30
where they flow in adjacent laminar streams, also called companion
streams. The term "adjacent" is used herein for both side by side
streams, as in FIG. 1, and layered streams, as in FIG. 13. The
sample stream enters one side of the reaction channel and the
reagent stream enters the other side. The term "side" as used
herein refers to both left and right, as in channel 30 of FIG. 1,
and top and bottom, as in channel 30 of FIG. 13.
[0037] Because the flow in the reaction channel is laminar, there
is no turbulent mixing of the streams. However, by diffusional
mixing the small analyte particles diffuse into the reagent stream
and react with the reagent particles to form product particles. The
diffusion direction is termed the depth, labeled d, and the
orthogonal dimension is termed the width, labeled w. In this
example, the native and labeled antigen compete for binding sites
on the antibody. By the end of the reaction channel, the reagent
stream has become a first product stream, P1, and the sample stream
is a residual sample stream. The residual sample stream 41 exits
through residual sample stream outlet channel 40 and outlet 5, and
product stream 51 flows into product stream channel 50.
[0038] In the illustrated embodiment the reaction in channel 30 is
a complex formation between antigen and antibody. The term
"reaction" as used herein includes any interaction between the
analyte particle and reagent particle which leads to a detectable
change. It includes chemical reaction, physical binding,
adsorption, absorption (for example when the analyte particle is
sucked inside a porous reagent particle such as a zeolite),
antibody reaction, nucleic acid binding, ion pairing, ion exchange,
chromatographic type reaction and receptor hormone reaction.
[0039] The product stream flows into a product particle detection
channel. The term product particles refers to all particles in the
product stream. They can be, for example, new species formed from
reaction, or reagent particles the concentration of which depends
on the extent of reaction with analytes. In the example of FIG. 1,
the displaced labeled antigen and the antibody with native antigen
are both product particles. The detection channel of this invention
may be coupled to external detecting means for detecting changes in
the reagent particles carried within the product stream as a result
of contact with analyte particles. Detection and analysis is done
by any means known to the art, including optical means, such as
absorption spectroscopy, luminescence or fluorescence, by chemical
indicators which change color or other properties when exposed to
the analyte, by immunological means, electrical means, e.g.
electrodes inserted into the device, electrochemical means,
radioactive means, or virtually any microanalytical technique known
to the art including magnetic resonance techniques, or other means
known to the art to detect the presence of an analyte such as an
ion, molecule, polymer, virus, DNA sequence, antigen, microorganism
or other factor. Field effects which are ion or chemical sensitive
can be measured in the detection channel. Preferably optical means
are used, and antibodies, DNA sequences and the like are attached
to optical markers. Examples of detection channels are discussed
below, following description of further embodiments of the H
reactor.
[0040] A different reaction scheme is illustrated in FIG. 2.
Analyte 14 is an antigen. Reagent stream 21 contains two types of
reagent particles, primary antibody 26 and labeled secondary
antibody 27. Both antibodies react with the antigen to form product
particles, which exit through product channel 50. The secondary
antibody can be, for example, fluorescently, luminescently or
enzymatically labeled. The first antibody can be sufficiently large
that it reduces the diffusion coefficient of the complex enough to
diffusionally distinguish between the complexed and uncomplexed
labeled antibody.
[0041] The reagent particles can be reporter particles immobilized
on beads to form reporter beads 24, as shown in FIG. 3. Each
reporter bead comprises a bead having a plurality of at least one
type of reporter molecules immobilized thereon. A property of the
reporter bead, such as fluorescence, luminescence, absorbance or
chemical activity, is sensitive to a corresponding analyte. The use
of reporter beads allows for a plurality of analytes to be measured
simultaneously through a single reagent inlet because the beads can
be tagged with different reporter molecules. The reporter bead is
illustrated herein with the competitive immunoassay. It could also
be used with a sandwich immunoassay or with other reporter
molecules.
[0042] The reagent particles can be magnetic reporter beads 25, as
shown in FIG. 4. Within reaction channel 30, transient magnetic
field 35 pulls the beads into the sample stream for reaction with
the analyte. The field is then reversed to pull the beads back to
the product stream for analysis.
[0043] Following the H reactor, the product stream flows into a
detection channel. Although many detection means can be used,
optical detection is preferred. The detection channel can be probed
with absorbance, luminescence or fluorescence measurement. The
absorbance of the reagent particle can change upon reaction and the
detection channel can be monitored in transmission. For this
embodiment, the channel cell is made of an optically transparent
material such as glass or plastic. The external optical apparatus
can be very simple. The sensor can be illuminated on one side with
a light source such as a light bulb and diffuser, and the
absorbance can be detected on the other side with a camera.
Alternatively the fluorescence of reagent particles can change in
response to the analyte, in which case the fluorescence can be
monitored. Alternatively, the reaction product can be luminescent.
For reflection measurements the back side of the channel cell need
not be transparent and is preferably made of a reflective material
such as silicon.
[0044] For optical detection, the signal can be increased by using
convoluted detection channel, as shown in FIG. 5. A product stream,
P, enters the detection channel. There is a difference in optical
properties between bound antigen 22a and unbound antigen 22b. It
can be a difference in, for example, color, fluorescence intensity,
or degree of polarization of the fluorescence. In this embodiment,
detection channel 100 includes a series of turns, making a square
wave geometry. The flow channel can be convoluted in any of a
number of ways. In another embodiment, the flow channel is in the
shape of a coil. In lieu of a convoluted channel, the channel can
include a broadened region, as shown in detection channel 110 of
FIG. 6. An external light source and photodetector are positioned
about the detection channel for absorbance or fluorescence
measurements. Only a photodetector is required for
luminescence.
[0045] In the embodiment of FIG. 7, the optical measurement is
facilitated by diffusional separation of the unbound antigen. The
product stream enters detection channel system 120 through product
stream channel 121 which, if there are no intervening elements, is
product stream channel 50 of the H reactor. Extraction fluid stream
124 enters through extraction stream inlet 122. The extraction
fluid can be any fluid capable of accepting particles diffusing
from the product stream. Preferred extraction streams comprise
water and isotonic solutions such as salt water or organic solvents
like acetone, isopropyl alcohol, ethanol, or any other convenient
liquid which does not interfere with the product particles or the
detection means. The streams join in adjacent laminar flow in
joining channel 123. Both separation and detection take place in
the joining channel. Free antigen 22b diffuses more rapidly than
bound antigen 22a. To select between the bound and unbound antigen,
photo illumination or detection is focused on one side of channel
123.
[0046] Another embodiment of optical detection uses single particle
detection, for example as in flow cytometer detection channel 130,
shown in FIG. 8. The product stream contains particles which are
formed into a single file for the flow cytometer. This is
particularly suitable for product streams containing reporter beads
24. As illustrated, the smaller particles are not necessarily
single file. One embodiment of the flow cytometer uses a v-groove
flow channel. The v-groove channel is described in detail in U.S.
patent application Ser. No. 08/534,515, filed Sep. 27, 1995, which
is incorporated by reference herein in its entirety. The
cross-section of such a channel is like a letter V, and thus is
referred to as a v-groove channel. The v-groove preferably has a
width small enough to force the particles into single file, but
large enough to pass the largest particles without clogging. An
optical head comprising a laser and small and large angle
photodetectors adapted for use with a v-groove flow channel can be
employed.
[0047] An alternative means of achieving single file particle flow
through a flow channel is the sheath flow module disclosed in U.S.
patent application Ser. No. 08/823,747, filed Mar. 26, 1997 and
incorporated in its entirety by reference herein. The sheath flow
module includes sheath fluid inlets before and after, and wider
than, a sample inlet. The product stream is surrounded by sheath
fluid, and the sheathed stream is focused to produce single file
particles.
[0048] In dual detection embodiments of the invention, residual
sample stream 41 is coupled with a flow cytometer. Alternatively,
the fluid stream can flow first through a flow cytometer and then
through the H reactor. This allows independent detection of both
the smaller and larger analyte particles, for example both
undissolved and dissolved analytes or both antigens and cells.
[0049] The channel cell of this invention can be used to introduce
two reagent streams, a first reagent stream in the H reactor and a
second reagent stream in either a T reactor or a second H reactor.
For example, in a sandwich immunoassay primary and secondary
antibodies can be separately introduced, as shown in FIG. 9. The H
reactor comprises sample stream inlet channel 10, first reagent
stream inlet channel 20, reaction channel 30, residual sample
stream outlet channel 40 and product stream channel 50. The first
reagent stream contains primary antibody 26, which reacts with
antigen in the sample to form a first product stream, P1.
[0050] A second reagent is introduced to the first product in
reagent stream 61 (R2) through second reagent stream inlet channel
60. The second reagent stream contains labeled secondary antibodies
27. The first product stream and second reagent stream flow in
adjacent laminar streams in joining channel 70, which functions as
a reaction channel. In this illustration, the reaction channel is
sufficiently long to allow both the first product particles and the
second reagent particles to diffuse to the adjacent stream. In one
embodiment, the first or second reagent particles are immobilized
on magnetic beads and a magnetic field is used to pull the beads to
one side of channel 70 for reaction. The beads can remain on that
side or be pulled to the other side with a reversed field.
[0051] A second product stream, P2, exits in stream 71 through
channel 70. Having first and second reagent inlets can be useful,
for example, to allow undesirable side reactions to go to
completion before the addition of the second reagent. Particles
diffuse between the first product and second reagent streams to
form a second product. The second product stream 71 the enters a
detection channel, for example an optical detection channel as
illustrated in FIGS. 5-8.
[0052] In generic terms, stream 61 is a companion stream to the
first product stream. After diffusion of small particles between
the companion stream and the first product stream, which takes
place in second laminar flow channel 70, the streams are termed
diffused first product and diffused companion streams.
[0053] Another application of a second reagent channel is for
chemical detection of product particles. In the above examples, the
reagent particles are fluorescently labeled. They can alternatively
be chemically labeled, for example enzymatically labeled. In the
embodiment of FIG. 10, the antigen in the first reagent is
enzymatically labeled. The first product stream flows out of the H
reactor (not shown) in product stream channel 50. It contains some
bound antigen 22a and some unbound antigen 22b which has been
displaced from the antibody by the native antigen from the sample.
The enzymatic activity is different in the bound and unbound
antigen, typically the unbound antigen is more active. Enzyme
substrate particles 62 in second reagent stream 61 enter through
second reagent stream inlet channel 60. In joining (reaction)
channel 70 they react with the labeled antigen to produce enzyme
product particles 72. Second reaction stream 71 flows into a
detection channel to detect the enzyme product optically or
otherwise. From the amount of enzyme product detected, the amount
of antigen in the sample stream can be calculated.
[0054] In yet another embodiment, reagent particles 62 react with a
first product particle to form a chemiluminescent or bioluminescent
product. The luminescence is optically detected. Chemiluminescent
reagents are readily available (see, for example, "Tropix
Luminescence Products", 1997, Perkin Elmer Applied Biosystems,
Bedford, Mass.). Luminescent reagents can also be bound to
antibodies and antigens to make luminescently labeled reagent
particles.
[0055] In a sandwich immunoassay the secondary antibody can
likewise be enzymatically labeled as shown in FIGS. 11-12. These
embodiments further illustrate an H separator between the H reactor
and the T reactor. First product stream P1 leaves the H reactor
(not shown) through first product stream channel 50. In the
illustrated embodiment one type of product particles is a sandwich
of native antigen between primary antibody 26 and enzymatically
labeled secondary antibody 27a. The sandwich product particles can
be formed in a single step, as in FIG. 2, or in two steps, as in
FIG. 9. The product stream contains both bound labeled antibodies
27a and unbound labeled antibodies 27b. Rather than distinguish
them based on relative chemical activity, they can be separated by
diffusion prior to the second reaction.
[0056] These embodiments include an H separator. The product stream
enters through channel 50 and a companion stream, extraction stream
81, enters through extraction stream inlet 80. The two streams flow
in adjacent laminar streams in separation channel 85. The smaller
product particles, in this case the unbound antibodies, diffuse
into the extraction stream faster than the sandwich complex. The
two product streams, the residual first product stream P1'
containing the larger particles and the diffused first product
stream P1" containing the smaller particles, are separated into
channels 92 and 90, respectively.
[0057] In the embodiment of FIG. 11 the larger particles enter a T
reactor. In reaction channel 70, the product stream flows adjacent
to a companion stream, the second reagent stream, which enters
through inlet channel 60. Enzyme substrate 62 is converted into
enzyme product 72, which flows out in product stream P2 for
subsequent detection. In the embodiment of FIG. 12, the lighter
product stream in channel 90 meets the second reagent stream, which
enters through channel 60, in reaction channel 70. Again the enzyme
substrate is converted to enzyme product, which is subsequently
detected.
[0058] In addition to the product stream outlets illustrated above,
additional outlets can be provided for conducting specimen streams
from the product stream channel, or at successive intervals along
the length of the reaction channel. The specimen channels can be,
for example, smaller channels branching from the reaction or
product channels. Analyte concentration can be measured in the
specimen streams by means such as viewports, fluorescence detectors
or flow cytometers.
[0059] The length of the reaction channels and the distance
traveled by the product stream prior to detection can be selected
to allow reactions to go to completion, to limit the sampling of
constituents based on their diffusion constants, and to alter the
efficiency of separation of particles. The reaction channel is long
enough to permit small analyte particles to diffuse from the sample
stream and have a detectable effect on reagent particles,
preferably at least about 2 mm long. The diffusion time required
depends on the diffusion coefficient of the analyte particles. The
reaction time required depends on the reaction rate. Some
reactions, such as ion reactions, are completed within
microseconds. Some reactions, such as competitive immunoassays that
involve unloading a bound antigen, require minutes. To allow
greater time for reaction between the analyte particles and the
reagent particles, the length of the product stream channel can be
increased.
[0060] The length of the flow channel depends on its geometry. The
flow channel can be straight or convoluted. Convoluted channels
provide longer distances for diffusion or reaction to occur without
increasing the size of the substrate plate in which the channel is
formed, thereby allowing for measurement of analytes with smaller
diffusion coefficients or reaction rates. The diffusion coefficient
of the analyte, which is usually inversely proportional to the size
of the analyte, affects the desired reaction channel length. For a
given flow speed, particles with smaller diffusion coefficients
require a longer flow channel to have time to diffuse into the
reagent stream. In preferred embodiments of this invention the
channel length of a straight reaction channel is between about 5 mm
and about 50 mm. In embodiments of this invention wherein the
reaction channel is convoluted, the length of the channel is
defined or limited only by the size of the microchip or other
material into which the channel is etched or otherwise formed.
[0061] As an alternative to increasing channel length to allow more
diffusion or reaction of analyte particles, the flow rate can be
decreased or the flow may be stopped to allow reactions to proceed
and then restarted. However, several factors limit the minimum flow
rate. First, the flow rate is typically achieved by a pumping means
and some types of pumps cannot produce as low a pressure and flow
rate as may be desired to allow enough time for diffusion of the
particles. Second, if the flow rate is too slow, particles more
dense than the surrounding fluid may sink to the bottom of the flow
channel and particles less dense than the surrounding fluid may
float to the top of the flow channel. It is preferable that the
flow rate be fast enough that hydrodynamic forces substantially
prevent particles from sinking to the bottom, floating to the top,
or sticking to the walls of the flow channel. In some applications,
notably use in space, sedimentation is not a factor. Sedimentation
can be avoided by orienting the channel cell with the laminar flow
reaction channel vertical.
[0062] The flow rate of the input streams is preferably between
about 5 micrometers/second and about 5000 micrometers/second, more
preferably about 25 micrometers/second. Preferably the flow rate
for both the sample and reagent streams is the same.
[0063] By adjusting the configuration of the channels in accordance
with the principles discussed above to provide an appropriate
channel length, flow velocity and contact time between the sample
stream and the reagent stream, the size of the particles remaining
in the sample stream and the particles diffusing into the reagent
stream can be controlled. The contact time required can be
calculated as a function of the diffusion coefficient of the
particle and the distance over which the particle must diffuse. If
the diffusion coefficient of the larger particles is about ten
times smaller than the coefficient for the analytes, the product
stream should be substantially free of the large particles.
[0064] The channel cell of this invention has been demonstrated
with diffusional separation occurring in a plane parallel to the
channel cell surface, termed the parallel embodiment. The channels
can alternatively be formed so that the diffusional separation
takes place in a plane orthogonal to the channel cell surface. FIG.
13 is a cross section of an H reactor and a T reactor formed in the
orthogonal plane, termed the orthogonal embodiment. The channels
are formed between substrate plate 1 and coverplate 2. The H
reactor is formed by sample (primary) stream inlet channel 10,
first reagent stream inlet channel 20, reaction channel 30,
residual sample stream outlet channel 40 and first product stream
channel 50. The first reagent stream inlet can, like the sample
inlet, feed through the substrate plate.
[0065] As in the parallel configuration, the diffusion direction is
termed the depth, labeled d, but note that the diffusion direction,
and hence the depth, in FIG. 13 is orthogonal to the diffusion
direction in FIG. 1. The depth of channel 30 is optionally greater
than the depth of channels 20 and 50 to accommodate two streams.
Although this H reactor does not have the visual appearance of the
letter "H", it has the functional criteria of two laminar flow
channels joining in the upstream end of a reaction channel to form
adjacent flow streams, layered in this case rather than side by
side, and two laminar flow channels branching from the downstream
end of the reaction channel.
[0066] The product stream of the H reactor of FIG. 13 enters a T
reactor comprising product stream channel 50, second reagent stream
inlet channel 60, and reaction channel 70. The depth of channel 70
is optionally greater than the depth of channel 50 to accommodate
two streams. In the previous embodiments (see FIG. 9, for example),
channels 50 and 60 were collinear; in this embodiment they join at
a right angle. As in the case of the H reactor, it is not the
visual appearance of the letter "T" that defines the T reactor, but
rather the functional criteria of the product stream channel
joining a companion stream inlet channel to form adjacent laminar
streams in the reaction channel.
[0067] The perpendicular embodiment can have a larger contact area
between the sample and reagent streams than the parallel version.
The width of the flow channel in the perpendicular embodiment can
be increased to increase the contact area while maintaining laminar
flow. This allows a greater reaction volume, which is particularly
advantageous for the synthesis application of the device. The
parallel embodiment is cheaper and easier to fabricate, which is
particularly advantageous in the analysis application of the
device.
[0068] In either the parallel or perpendicular embodiment, the
channel cell is generally formed by two plates with abutting
surfaces. The channels may be formed in both plates, or one plate
can contain the channels and the other can be a flat cover plate.
The channel cells of this invention may be formed by any techniques
known to the art. Silicon channel plates are preferably formed by
etching the flow channels onto the horizontal surface of a silicon
microchip and placing a cover plate, preferably of an optically
clear material such as glass or a silicone rubber sheet, on the
etched substrate plate. To promote flow, the comers can be etched.
For non-silicon channel plates, other means for manufacturing the
channel cells of this invention include molding the device in
plastic, micromachining, and other techniques known to the art.
Precision injection molded plastics can also be used to form the
devices. In a preferred embodiment of this invention, channel cells
have hydrophilic surfaces to facilitate flow of liquid therein and
allow operation of the device without the necessity for
pressurization. The substrate may be treated by means known to the
art following fabrication of the channels to render it hydrophilic.
The cover plate is also preferably treated to render it
hydrophilic.
[0069] For optical detection in transmission, such as absorbance
detection, the analyte detection area, and optionally other parts
of the channel cell system, are optically accessible. Typically the
detection area lies between optically transparent plates. Analyte
detection area as used herein refers to that portion of a flow
channel where changes in the analyte particles or the reagent
particles are measured. For detection with reflection, such as
fluorescence or luminescence detection, only one plate need be
transparent, typically the cover plate. For product synthesis, the
channel system need not be transparent in any portion.
[0070] The preferred channel dimensions depend on the application,
with the criterion that laminar flow must be maintained. The
channel depth (diffusion direction) is preferably between about 10
and 1000 .mu.m, and most preferably around 400 .mu.m, in both the
parallel and perpendicular embodiments. The channel width is about
10-200 .mu.m in the parallel embodiment. In the perpendicular
embodiment, it can be more than several millimeters wide and still
maintain laminar flow.
[0071] Means for applying pressure to the flow of the feed fluids
through the device can also be provided. Such means can be provided
at the inlets and/or the outlets (e.g. as vacuum exerted by
chemical or mechanical means). Means for applying such pressure are
known to the art, and include the use of a column of water or other
means of applying water pressure, electroendoosmotic forces,
optical forces, gravitational forces, and surface tension forces.
The outlets can be connected to fluid receptacles. Such receptacles
may be coupled to an analytical or detection device.
[0072] FIG. 14 shows an embodiment in which the product stream
channel 50 of an H-filter (not shown) is connected to the upstream
end of a second laminar flow channel 200 at the opposite side of
which is provided a second companion reagent stream inlet channel
210. The downstream end of second laminar flow channel 200 is
connected to the upstream end of third laminar flow channel 230.
Connected to the upstream end of third laminar flow channel 230 is
third companion reagent stream inlet 220.
[0073] Various aspects of this invention have been illustrated with
specific examples. Combinations and variations of these embodiments
will be readily apparent to those skilled in the art and fall
within the spirit and scope of this invention. For example, any of
the exemplified configurations and reaction schemes can be
implemented with reagent particles immobilized on beads. The beads
can be magnetic and magnetic fields can be used to manipulate the
beads. Filters, diffusion based or otherwise, can be placed before
or after the H reactor, and can be positioned between the H reactor
and subsequent reactors, separators and detectors. Each reagent
stream can contain more than one type of reagent particle for
detection of a single type of analyte particle or for simultaneous
detection of multiple analytes. More than one reagent stream
channel can join the upstream end of the reaction channel, or more
than one reagent stream channel can merge prior to joining the
reaction channel. More than one product stream channel can leave
the downstream end of the reaction channel. In addition to
detecting species in the product stream, the residual sample stream
and any of the companion streams can be analyzed. The angles of the
"H" and "T" are not limited to right angles. Parallel and
perpendicular geometries can be combined in one channel system.
This reactor can be used in combination with other sample
preparation and analysis apparatus.
EXAMPLE 1
[0074] Tests performed by EMIT (Enzyme Multiplied Immunoassay
Technique) can be carried out in the H reactor combined with the T
reactor of this invention. EMIT is a homogeneous immunoassay for
low-molecular-weight ligands. The assay is based on binding of
antibody to an enzyme labeled ligand in order to change the enzyme
activity. The competitive binding of antibody bound and unbound
ligands is used to measure the concentration of unbound ligand.
Here, digoxin, a drug used to control cardiac arrhythmia and
requiring frequent concentration analysis in case of intoxication,
is selected as an example test. The EMIT assay for digoxin is based
on the competitive binding between drug in the sample and drug
labeled with glucose-6-phosphate dehydrogenase made using
recombinant DNA technology (rG6P-DH) for antibody binding sites.
The drug concentration is measured through enzyme activity which
decreases upon binding to the antibody. Active enzyme reduces NAD
to NADH.
[0075] The reaction is illustrated in FIG. I combined with FIG. 10.
In this assay, reagent stream R1 contains digoxin labeled with
glucose-6-phosphate dehydrogenase 22 and antibody 23. Reagent
stream R1 is imported through channel 20 and contacts the sample
stream from channel 10. Digoxin in the sample 12 diffuses into the
reagent stream in channel 30, binds with antibody and is
transported to channel 50, while cellular components are
transported to channel 40. The more digoxin molecules in the sample
stream, the more antibody binds with free digoxin instead of enzyme
labeled digoxin. As a result, the more enzyme is freed from
antibody binding.
[0076] In channel 70, the product stream encounters reagent stream
R2, containing two types of reagent particles, the substrate
glucose-6-phosphate (not shown) and NAD 62. Freed enzyme oxidizes
glucose-6-phosphate and reduces NAD to NADH 72. In the second
product stream, the residual enzyme activity is measured by
spectroscopy through the change in absorbance by NADH at 340
nm.
EXAMPLE 2
[0077] In another embodiment using multiple reagents in series, the
sample stream is blood to be analyzed for glucose, the first
reagent stream R1 contains glucose oxidase, and the second reagent
stream R2 contains a pH sensitive dye. In channel 30 glucose
particles from the blood diffuse into the reagent stream and are
changed to gluconic acid. In channel 70 the gluconic acid reacts
with the pH-sensitive dye. In the second product stream, the
reaction is detected by changes in the dye absorbance.
* * * * *