U.S. patent application number 09/863835 was filed with the patent office on 2002-04-11 for devices for analyzing the presence and concentration of multiple analytes using a diffusion-based chemical sensor.
This patent application is currently assigned to The University of Washington. Invention is credited to Weigl , Bernhard H, Yager , Paul.
Application Number | 20020041827 09/863835 |
Document ID | / |
Family ID | 25470879 |
Filed Date | 2002-04-11 |
United States Patent
Application |
20020041827 |
Kind Code |
A1 |
Yager , Paul ; et
al. |
April 11, 2002 |
Devices for Analyzing the Presence and Concentration of Multiple
Analytes Using a Diffusion-based Chemical Sensor
Abstract
The present invention provides a microfabricated sensor and a
method capable of rapid simultaneous measurement of multiple
analytes in a fluid sample. The sensor is inexpensive, disposable
and portable, and requires only microliters of sample, a particular
advantage with precious fluids such as blood. The sensor utilizes
diffusion between layered laminar streams rather than side by side
streams. This allows multiple side by side channels for
simultaneous detection of multiple analytes. In the sensor, a
sample stream and a carrier stream flow in layers, one on top of
the other, and one or more reagents are introduced to the bottom of
the carrier stream through either a fluid or a solid reagent inlet.
The reagent contains reagent particles which, in the presence of
the analyte, have a detectable change in a property. The analyte
diffuses into the carrier stream where it interacts with reagent
particles and is detected by optical, electrochemical or other
means.
Inventors: |
Yager , Paul; ( Seattle,
Washington) ; Weigl , Bernhard H; ( Seattle,
Washington) |
Correspondence
Address: |
Greenlee, Winner and Sullivan
Ellen Winner
5370 Manhattan Circle
Suite 201
Boulder
Colorado
80303
US
winner@greenwin.com
(303)499-8080
(303)499-8089
|
Assignee: |
The University of
Washington
1107 N.E. 45th Street Suite 200
Seattle
98105
Washington
|
Family ID: |
25470879 |
Appl. No.: |
09/863835 |
Filed: |
May 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09863835 |
May 22, 2001 |
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09/441,572 |
11, 199 |
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09/441,572 |
11, 199 |
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08/938,093 |
w�;w�;w�;�������� 92, 199 |
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6,007,775 |
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Current U.S.
Class: |
422/400 |
Current CPC
Class: |
G01N 30/0005 20130101;
Y10T 436/117497 20150115; Y10T 436/2575 20150115; Y10T 436/25375
20150115; Y10T 436/118339 20150115; G01N 2030/8429 20130101; G01N
30/0005 20130101 |
Class at
Publication: |
422/57 ; 422/102;
422/58 |
International
Class: |
G01N 031/22 |
Claims
Claims
A diffusion based sensor, comprising: a laminar flow channel having
an upstream end and a downstream end;a sample inlet connected to
said laminar flow channel for conducting a sample fluid stream into
said laminar flow channel;a carrier inlet connected to said laminar
flow channel for conducting a carrier fluid stream into said
laminar flow channel in layered laminar flow contact with said
sample fluid;a first reagent inlet connecting with said laminar
channel downstream from said carrier inlet for introducing a
reagent into said laminar flow channel in contact with said carrier
fluid stream.
The sensor of claim 1 wherein said first reagent inlet is a reagent
stream inlet for conducting a first reagent stream into said
laminar flow channel in layered laminar flow contact with said
carrier stream.
The sensor of claim 1 wherein said first reagent inlet is a solid
reagent inlet.
The sensor of claim 3 wherein said solid reagent inlet is a cavity
within said flow channel positioned to allow solid reagent to
diffuse into said carrier stream.
The sensor of claim 1 also comprising an outlet connected to said
laminar flow channel downstream from said inlets.
The sensor of claim 1 not comprising an outlet to said laminar flow
channel.
The sensor of claim 1 comprising an absorbent material within said
laminar flow channel downstream from said inlets.
The sensor of claim 1 further comprising a second reagent inlet
connected to said laminar flow channel downstream from said carrier
inlet for introducing a second reagent into said laminar flow
channel in contact with said carrier fluid stream.
The sensor of claim 8 wherein said second reagent inlet is
positioned downstream from said first reagent inlet.
The sensor of claim 8 further comprising a third reagent inlet
connected to said laminar flow channel downstream from said carrier
inlet for introducing a third reagent into said laminar flow
channel in contact with said carrier fluid stream.
The sensor of claim 10 wherein said third reagent inlet is
positioned downstream from said second reagent inlet.
The sensor of claim 1 comprising a plurality of reagent inlets
connected to said laminar flow channel downstream from said carrier
inlet for introducing second and subsequent reagents into said
laminar flow channel in contact with said carrier fluid stream,
said reagent inlets being positioned in parallel with each
other.
The sensor of claim 12 also comprising multiple detectors
positioned with respect to said channel to detect interaction of
components of said sample stream with said reagents.
The sensor of claim 1 comprising a plurality of reagent inlets
connected to said laminar flow channel downstream from said carrier
inlet for introducing second and subsequent reagents into said
laminar flow channel in contact with said carrier fluid stream,
said reagent inlets being positioned in series with each other.
The sensor of claim 14 also comprising multiple detectors
positioned with respect to said channel to detect interaction of
components of said sample stream with said reagents.
The sensor of claim 1 wherein said channel is enlarged at said
carrier inlet to accommodate the carrier stream layer.
The sensor of claim 1 wherein said channel is a convoluted
channel.
The sensor of claim 1 wherein the length of said channel is between
about 5 mm and 50 mm.
The sensor of claim 1 wherein said sample inlet is preceded by a
filter.
The sensor of claim 19 wherein said filter is a diffusion based
microfilter.
The sensor of claim 1 for use with optical detection wherein at
least a portion of said channel is formed in a transparent
material.
The sensor of claim 1 for use with electrochemical detection
further comprising an electrode positioned in said channel
downstream from said reagent inlet.
The sensor of claim 22 further comprising one or more additional
electrodes positioned in said channel downstream from said reagent
inlet.
The sensor of claim 23 wherein said electrodes are positioned in
series.
The sensor of claim 22 wherein said electrode is deposited on the
surface of said channel.
The sensor of claim 1 for use with both electrochemical and optical
detection, wherein at least a portion of said channel is formed in
a transparent material, and wherein said channel comprises an
electrode positioned in said channel downstream from said reagent
inlet.
Description
Cross Reference to Related Applications
[0001] This application is a continuation-in-part of co-pending
application serial no. 09/441,572 filed November 17, 1999, which is
a divisional of application serial no. 08/938,093 filed September
26, 1997, Patent No. 6007,775, both of which are incorporated
herein by reference to the extent not inconsistent herewith.
Background of Invention
[0002] This invention relates to diffusion based microsensors and
methods for analyzing the presence and concentration of multiple
analytes in samples containing both these analytes and, optionally,
larger particles. The invention is particularly useful for
analyzing complex samples, such as blood, to detect the presence of
small particles, such as ions or proteins, in a stream containing
larger particles, such as cells.
[0003] 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.
[0004] Using tools developed by the semiconductor industry to
miniaturize electronics, 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.
[0005] A process called "field-flow fractionation" (FFF) has been
used to separate and analyze components of a single input stream in
a system not made on the microscale, but having channels small
enough to produce laminar flow. Various fields, including
concentration gradients, are used to produce a force perpendicular
to the direction of flow to cause separation of particles in the
input stream (see, e.g. Giddings, J.C., U.S. Patent 4,147,621;
Caldwell, K.D. et al., U.S. Patent 5,240,618; Wada, Y., et al.,
U.S. Patent 5,465,849). None of these references disclose the use
of a separate input stream to receive particles diffused from a
particle-containing input stream.
[0006] A related method for particle fractionation is the "Split
Flow Thin Cell" (SPLITT) process (see, e.g., Williams, P.S., et al.
(1992), Ind. Eng. Chem. Res. 31:2172-2181; and J.C. Giddings U.S.
Patent 5,039,426). These publications disclose channel cells with
channels small enough to produce laminar flow, but again only
provide for one inlet stream. A further U.S. patent to J.C.
Giddings, U.S. Patent No. 4,737,268, discloses a SPLITT flow cell
having two inlet streams. Giddings U.S. Patent 4,894,146 also
discloses a SPLITT flow cell having two input streams. All these
SPLITT flow methods require the presence of more than one output
stream for separating various particle fractions.
[0007] None of the foregoing publications describe a channel system
capable of analyzing small particles in very small quantities of
sample containing larger particles, particularly larger particles
capable of affecting the indicator used for the analysis. No
devices or methods provide simultaneous measurement of more than
one analyte.
Summary of Invention
[0008] The present invention provides a microfabricated sensor
capable of rapid simultaneous measurement of multiple analytes in a
fluid sample. Reagents can be loaded into the sensor during
fabrication and only the sample fluid needs to be introduced for
measurement, making it ideal for use outside of the laboratory. The
detection apparatus can be as simple as a human eye, a camera, or a
voltmeter, which also supports field use. The sensor is
sufficiently simple and inexpensive to manufacture that it is
practical for disposable use. Multiple analytes can be
simultaneously detected with only microliters of sample, a
particular advantage with precious fluids such as blood.
[0009] A previously-provided channel cell system for detecting the
presence of analyte particles in a sample stream also comprising
larger particles is described in U.S. Patents 5,972,710 and
5,716,852, both of which are incorporated by reference herein to
the extent not inconsistent herewith. The previous system comprised
a laminar flow channel having at least two inlets for conducting
fluids into the laminar flow channel. The inlets contained (1) a
sample stream containing analyte particles and also containing
larger particles and (2) an indicator stream having a substance
which indicates the presence of the analyte particles by a
detectable change in property. The two streams flow side by side in
the channel without turbulent mixing. The analyte particles diffuse
into the indicator stream to the substantial exclusion of the
larger particles, and their presence is detected by reaction with
the indicator substance.
[0010] The present invention provides an apparatus and a method
which utilize diffusion between layered laminar streams rather than
side by side streams. This allows multiple side by side reagent
inlets for simultaneous detection of multiple analytes.
Additionally, utilizing diffusional separation, the sensor can
tolerate fluid samples also containing larger particles.
[0011] Specifically, a diffusion based sensor is provided
comprising a laminar flow channel having an upstream end and a
downstream end; a sample inlet connected to said laminar flow
channel for conducting a sample fluid stream into said laminar flow
channel; a carrier inlet connected to said laminar flow channel for
conducting a carrier fluid stream into said laminar flow channel in
layered laminar flow contact with said sample fluid; and a first
reagent inlet connecting with said laminar channel downstream from
said carrier inlet for introducing a reagent into said laminar flow
channel in contact with said carrier fluid stream. The reagent
inlet can be a solid reagent inlet, such as a cavity in the channel
over which the carrier fluid stream layer passes, so that the solid
reagent diffuses into the carrier fluid stream. The reagent inlet
may also be a reagent stream inlet for conducting a reagent stream
into the laminar flow channel in layered laminar flow contact with
the carrier fluid stream.
[0012] The laminar flow channel of the sensor may be enlarged at
the carrier inlet to accommodate the carrier stream layer.
[0013] The laminar flow channel may or may not comprise an outlet.
The channel may expand into a chamber containing an absorbent
material, or the channel may simply terminate without an outlet,
and may have disposed within the terminal end an absorbent
material. The absorbent material may be any material which absorbs
liquid, preferably all the liquid in the sensor, such as a tissue,
a filter, a hydrogel, or a material that absorbs nonaqueous fluids
such as activated charcoal. The absorbent material may be in the
form of a pad, and can be of any size sufficient to absorb at least
a portion of the fluid in the channel. In a preferred embodiment,
the sensor is about the height and width of a playing card, and
comprises absorbent pads about 2 cm in diameter and about 3 mm
thick made of paper. These pads are capable of absorbing about 1 ml
of fluid.
[0014] The channels may be equipped with second and, optionally,
subsequent, reagent inlets downstream from the carrier inlet. These
reagent inlets may be arranged in parallel, i.e. side by side on a
line orthogonal to the flow direction and parallel to the plane of
the interface between the sample fluid and carrier fluid layers, or
they may be arranged in series, downstream from the carrier inlet
and in a line parallel to the flow direction, or in a staggered
line parallel to the flow direction.
[0015] Sensors containing the various sample, carrier and reagent
streams are also provided by this invention.
[0016] The laminar flow channels may be straight or convoluted.
[0017] The sensors of this invention may be combined with art-known
filters, detectors, pumps and other attachments, as well as other
microflow devices known to the art such as those described in
Patent Nos. 5,932,100, 5,716,852, 5,726,751, 5,726,404 5,922,210,
5,747,349, 5,748,827, 5,971,158, 6,007,775, 5,974,867 5,948,684,
and 6,067,157, all of which are incorporated by reference to the
extent not inconsistent herewith.
[0018] The sensors of this invention may also comprise a detector,
such as an optical or electrochemical detector, or multiple
detectors positioned with respect to the channel to detect
interaction of components of the sample stream with the reagents.
When optical detectors are used, at least a portion of the channel
should be formed in a transparent material, i.e. the channel should
be visible in its entirety or through windows in the channel walls.
For electrochemical detection, an electrode or electrodes may be
positioned in the channel or in communication with the channel.
Electrodes can be coated on the channel walls. Detectors may be
positioned in parallel or in series.
Brief Description of Drawings
[0019] FIG. 1, comprising FIGS. 1a-b, is (a) a cross section and
(b) a plan view of a reagent inlet and of the change in reagent
particles on interaction with analyte particles.
[0020] FIG. 2, comprising FIGS. 2a-b, is (a) a cross section and
(b) a plan view of a diffusion based sensor having three reagent
inlets in parallel.
[0021] FIG. 3 is a cross section of a solid reagent inlet with
reagent diffusing into a carrier fluid.
[0022] FIG. 4 is a cross section of a sensor having two reagent
inlets in series and having a preceding diffusion based
microfilter.
[0023] FIG 5 is a plan view of a portion of a laminar flow channel
of a sensor having a chamber containing an absorbent pad.
[0024] FIG. 6 is a plan view of a portion of a laminar flow channel
of a sensor having a terminal end containing an absorbent pad
rather than an outlet.
Detailed Description
[0025] In this invention, a sample stream and a carrier stream flow
in layers, one on top of the other, rather than side by side. A
reagent is introduced to the bottom of the carrier stream through
either a fluid or a solid reagent inlet. The reagent contains
reagent particles which, in the presence of the analyte, have a
detectable change in a property. The sample stream can contain
larger particles such as cells as well as the analytes of interest.
The term "particles" refers to any species, including dissolved and
particulate species such as molecules, cells, suspended and
dissolved particles, ions and atoms. The analyte diffuses into the
carrier stream where it interacts with reagent particles and is
detected by optical, electrochemical or other means.
[0026] The sample stream may also contain larger particles, which
may also be sensitive to the reagent. Because these do not diffuse
into the carrier stream, they do not interfere with detection of
the analyte. By diffusion of the analyte but not the larger
particles, crossof 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, crossto pH or ionic strength can be suppressed by using
strongly buffered carrier solutions.
[0027] A reagent inlet joins the laminar flow channel on the
surface abutting the carrier stream. For multiple analyte
detection, each inlet is narrower than the flow channel. For single
analyte detection the inlet can be as wide as the flow channel. A
fluid reagent inlet is a fluid channel. The fluid reagent forms a
stream in layered laminar flow with the carrier and sample streams,
but it is a relatively thin and narrow layer since the inlet is
narrower than the channel and the reagent fluid volume is small
relative to the sample and carrier fluids. A solid reagent inlet is
a cavity in the laminar flow channel on the side containing the
carrier stream, or other means by which a solid or viscous reagent
can be immobilized. Flow of the carrier fluid over the solid
reagent dissolves or suspends the reagent particles in the carrier
stream.
[0028] Because the sample and carrier streams are layered rather
than side by side, multiple reagent inlets can be positioned side
by side in parallel to allow simultaneous detection of multiple
analytes. A layered flow of sample and carrier streams is
established and reagents are introduced into the carrier stream
using parallel inlets which are narrow compared to the width of the
carrier stream. The reagent inlets are spaced sufficiently far
apart that there need be no undesired interdiffusion between the
reagents. Optical measurement of analyte concentrations can be made
by multi-wave two-dimensional imaging. Multiple analytes can be
detected simultaneously using equipment as simple as a camera.
Electrochemical detection can utilize a plurality of electrodes
placed in the flow channel.
[0029] A second reagent inlet can be positioned downstream of and
in series with the first reagent inlet for sequential addition of
reagents. The first and second inlets can be used, for example, to
admit first and second antibodies for sandwich assay of an antigen.
In another example, the first inlet admits a reagent which reacts
with the analyte to form a product, and the second inlet admits an
indicator for the product. For example, to detect glucose the first
reagent can be glucose oxidase and the second a pH sensor.
[0030] The preferred embodiments of this invention utilize liquid
streams, although the methods and devices are also suitable for use
with gaseous streams.
[0031] The channel cell system of this invention can be used with
external detecting means for detecting changes in reagent particles
as a result of contact with analyte particles. Detection is done by
optical, electrical, chemical, electrochemical, radioactive 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. The reagent can be a chemical indicator
which changes color or other properties when exposed to the
analyte. The reagent can comprise substrate particles such as
polymers or beads having a reagent particle immobilized thereon, as
described in U.S. Patent Application 08/621,170, filed March 20,
1996, now U.S. Patent No. 5,747,349, which is incorporated by
reference herein in its entirety.
[0032] The term "detection" as used herein means determination that
a particular substance is present. Typically, the concentration of
a particular substance is also determined. The concentration of the
analyte particles in the sample stream is determined by detecting
the position within the laminar flow channel at which a detectable
change in the reagent particles is caused. Generally there is not a
distinct boundary in the channel at which a detectable change
occurs, but rather a gradient in the detected property. The
detection gradient can be used to provide information about flow
speed and/or sample concentration.
[0033] The detection gradient for a given analyte stays the same
over time as long as the flow speed is constant and the sample
unchanged. The gradient can be varied by varying flow rate, sample
concentration, and/or reagent concentration so as to optimize the
signal for detection. For example, the system can be adjusted so
that the gradient falls in the central portion of the device. If
the flow rates and reagent concentration are known, the analyte
concentration can be determined from the gradient.
[0034] The sample stream may be any stream containing an analyte
and, optionally, 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
particle in the sample stream which is capable of diffusing into
the carrier stream in the device. The term "small particle" is used
herein for any particle sufficiently diffusive to diffuse between
streams within the length of the flow channel. 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. Preferably the
analyte particles are no larger than about 3 micrometers, more
preferably no larger than about 0.5 micrometers, or are no larger
than about 1,000,000 MW, and more preferably no larger than about
50,000 MW.
[0035] The carrier can be any fluid capable of accepting particles
diffusing from the sample stream and reagent inlet. Preferred
carrier streams comprise water and isotonic solutions such as salt
water, or organic solvents like acetone, isopropyl alcohol,
ethanol, or any other liquid convenient which does not interfere
with the effect of the analyte on the reagent, or interfere with
the detection means.
[0036] 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. Substrate plate as used herein refers to the piece of
material in which the channel system of this invention is formed,
e.g., silicon wafer and plastics. The channel cell may be
fabricated by microfabrication methods known to the art, e.g. by
forming channels in a silicon microchip and placing a glass cover
over the surface, or precision injection molding plastic.
[0037] For optical detection, such as absorption, luminescence or
fluorescence detection, at least a portion of the channel in the
analyte detection area is transparent. Optionally other parts of
the channel cell system are also transparent. Analyte detection
area as used herein refers to that portion of a flow channel where
a detectable change in the analyte or reagent particles is
measured.
[0038] For electrochemical detection, one or more electrodes is
positioned in the detection area. For turgid sample fluids, the
electrodes are preferably placed on the carrier fluid side to
prevent fouling of the electrodes. For multiple analytes, multiple
electrodes are placed in parallel. To measure the detection
gradient for each analyte, multiple electrodes can be positioned in
series. The position of the electrodes can be used to distinguish
between analytes having similar redox potentials but different
diffusion coefficients. The electrodes can be deposited on the
channel surface, on either or both plates.
[0039] The method of this invention is 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.
[0040] The laminar flow 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 a few minutes.
[0041] To allow greater time for reaction between the analyte
particles and the reagent particles, the length of the product
stream channel can be increased. The length of the flow channel
depends on its geometry. The flow channel can be straight or
convoluted in any of a number of ways. In one embodiment, the flow
channel can include a series of turns, making a stairstep or square
wave geometry. Convoluted channels provide longer distances for
diffusion 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. The
diffusion coefficient of the analyte, which is usually inversely
proportional to the size of the analyte, affects the desired flow
channel length. For a given flow speed, particles with smaller
diffusion coefficients require a longer flow channel to have time
to diffuse into the carrier stream. In preferred embodiments of
this invention the channel length of a straight flow channel is
between about 5 mm and about 50 mm. In embodiments of this
invention wherein the flow channel is convoluted, the length of the
flow channel is defined or limited only by the size of the
microchip or other material into which the channel is etched or
otherwise formed.
[0042] As an alternative to increasing channel length to allow more
time for diffusion to occur, the flow rate can be decreased.
However, several factors limit the minimum flow rate and therefore
make a longer flow channel desirable in some cases. 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 particles with small
diffusion coefficients. Second, if the flow rate is slow enough and
some particles are of significantly different density from the
surrounding fluid streams, particles denser than the surrounding
fluid streams may sink to the bottom of the flow channel and
particles less dense than the surrounding fluid streams 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. 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. For systems wherein sedimentation is not a
problem, the flow can be stopped to allow greater time for
diffusion and reaction and then resumed. Preferably the flow rate
for both the sample and carrier streams is the same.
[0043] The channel depth (diffusion direction) is preferably
between about 5 and 500 m. The channel depth is small enough to
allow laminar flow of two layered streams, preferably no greater
than about 200 micrometers and more preferably between about 50
micrometers and about 100 micrometers. However, the channel can be
made as shallow as possible while avoiding clogging the channel
with the particles being used. Decreasing the depth of the channel
makes diffusion of analytes into the carrier stream occur more
rapidly, and thus detection can be done more rapidly.
[0044] The channel width can be as large as desired, since the
laminar flow is relatively insensitive to width. The preferred
width depends primarily on the number of parallel reagent inlets.
Typically the width is between 0.1 and 1 cm. A 1 cm wide channel
can accommodate 10 parallel reagent inlets, for 10 analytes.
[0045] For detecting analytes optically in turbid and strongly
colored solutions such as blood, the sample fluid is preferably
filtered before measurement. The filter can be any filter known in
the art. It can also be a diffusion based microfilter device
comprising microchannels in the shape of an "H", as disclosed in
U.S. Patent Application 08/663,916, filed June 14, 1996, Patent No.
5,932,100, which is incorporated by reference in its entirety
herein. In the microfilter, the unfiltered-sample stream, which is
a mixture of particles suspended in a fluid, and an extraction
stream join in the central channel (the crossbar of the "H") and
flow together in the central channel. Due to the small size of the
channels, the flow is laminar and the streams do not mix. The
residual-sample stream and the extraction stream split at the other
end of the channel. While the streams are in laminar flow contact
in the central channel, particles having a greater diffusion
coefficient (smaller particles) have time to diffuse into the
extraction stream, while the larger particles (e.g. blood cells)
remain in the sample stream. Particles in the exiting extraction
stream (now called the filtered sample stream) may be analyzed
without interference from the larger particles. In the present
invention the microfilter can be formed in the plane perpendicular
to the wafer surface rather than in the plane of the surface.
[0046] Information useful for determining the concentration of the
analyte particles in the sample stream can be obtained by providing
means for conducting specimen streams from the carrier stream at
successive intervals along the length of the laminar flow channel,
as described in U.S. Patent Applications 08/829,679, filed March
31, 1997, Patent No. 6,159,739 and 08/625,808, filed March 29,
1996, Patent No. 5,716,852.
[0047] Dual detection embodiments of the device of the present
invention which allow for detection of both undissolved and
dissolved analytes are also provided. Detection of both undissolved
and dissolved analytes can be achieved in one dual detection device
with branching flow channels in fluid connection with the flow
channel of the diffusion based sensor. Undissolved particles are
preferably detected with a flow cytometer. The fluid streams can
flow first through a diffusion based sensor flow channel for
detection of dissolved particles, and then through a branching
channel to a flow cytometer for detection of undissolved particles.
Alternatively, the fluid stream can flow first through a flow
cytometer and then through a diffusion based sensor flow
channel.
[0048] 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 08/534,515, filed September 27, 1995, Patent No.
5,726,751, 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.
[0049] An alternative means of achieving single file particle flow
through a flow channel is the sheath flow module disclosed in U.S.
Patent Application 08/823,747, filed March 26, 1997, Patent No.
6,159,739 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.
[0050] Inlets, which comprise the inlet channels, can also include
other means such as tubes, syringes, pumps, and the like which
provide means for injecting fluid into the device. Outlets can
include collection ports, and/or means for removing fluid from the
outlet, including receptacles for the fluid, means for inducing
flow, such as by capillary action, pressure, gravity, and other
means known to the art. Such receptacles may be coupled to an
analytical or detection device.
[0051] The method can be conducted by a continuous flow-through of
sample and carrier streams. The steadynature of this method makes
longer signal integration times possible.
[0052] Additionally, a method is provided for determining kinetic
rate constants as a function of the detection gradient. Generally,
kinetic measurements are made by plotting a physical property
versus time, i.e., time of reaction. The method provided herein for
making kinetic measurements as a function of distance traveled by
the sample and reagent, rather than time, is advantageous in that
it allows for integrating the data from detection over time,
thereby increasing the accuracy of the data collected.
[0053] The operation of the diffusion based sensor of this
invention is shown in FIG. 1 in (a) cross section and (b) plan
view. Flow channels and inlets and outlets are formed in substrate
plate 10, covered by transparent cover plate 15. Laminar flows of
sample stream 130 and carrier stream 140 are established in channel
20. Due to the low Reynolds number in the small flow channel, no
turbulence-induced mixing occurs and the two streams flow parallel
to each other without mixing. However, because of the short
distances involved, diffusion does act perpendicular to the flow
direction, so sample components (analyte particles) diffuse from
the sample stream to the carrier stream. The smaller sample
components diffuse more rapidly and equilibrate close to the
reagent inlet, whereas larger components equilibrate farther up in
flow channel 20. The diffusion of analyte particles is indicated by
dashed line 131.
[0054] Reagent 150 enters the flow channel through fluid inlet 50,
and reagent particles diffuse into the carrier stream, as indicated
by dashed line 151. The interaction of the analyte particles with
the reagent particles changes a detectable property of the reagent
particles. In this example, the reagent turns from green before
reaction (150) to red after reaction (155).
[0055] The interaction is viewed from above, as indicated by
detection axis 100, and as shown in FIG. 1b. At the inlet the
reagent is green. As analyte particles contact reagent particles,
when viewed from above the cell the fluid in the channel begins to
appear somewhat red in addition to green. The change in color
becomes observable at the start 101 of the detection gradient.
Moving downstream, the green fades out and the red increases in
intensity until only red is observed at the end 102 of the
detection gradient. Although this is a flow system, the physical
location of the detection gradient in the flow channel stays the
same over time as long as the flows are constant and the sample
unchanged. The presence of analyte is detected by a change in a
property, in this case absorbance. The concentration of the analyte
can be determined from the distance it takes to change the
property, and in particular from the detection gradient.
[0056] In the illustrated example, the absorbance of the reagent
particle changes and the sensor is monitored in transmission.
Plates 10 and 15 are made of an optically transparent material such
as glass or plastic. The 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 or even a human eye. Alternatively the
fluorescence of analyte particles can change in response to the
analyte, in which case the fluorescence can be monitored. In
another embodiment the reaction of the analyte and reagent makes a
luminescent product. For reflection measurements plate 10 need not
be transparent, and is preferably made of a reflective material
such as silicon.
[0057] Although the sensor is illustrated with optical detection,
other detection means can be employed. In particular, the sensor is
suited to electrochemical detection.
[0058] The structure of an embodiment of the sensor is shown in
FIG. 2 in (a) cross section and (b) plan view. Sample inlet 30
passes through substrate plate 10 into channel 20. Carrier inlet 40
joins the bottom of channel 20. The terms bottom and top as used
herein refer to the substrate plate and the cover plate sides of
the channel, respectively, although the cell need not necessarily
be oriented in use with the substrate plate on the bottom. In fact,
because particles such as blood cells sediment in fluids such as
plasma, it is preferred that the sensor be oriented with the sample
stream below the carrier stream if the sample stream is turbid. If
the cell is fabricated in a way which does not utilize substrate
and cover plates, the top is the side with the sample fluid and the
bottom is the side with the carrier fluid. In a preferred
embodiment the depth of the channel increases at the carrier inlet
to accommodate the carrier stream. In the illustrated embodiment a
plurality of reagent inlets, 50, 52 and 54, positioned in parallel,
join the flow channel at the bottom, between carrier inlet 40 and
outlet 60. Parallel inlets need not be lined up as illustrated, but
are laterally positioned in different parts of the stream.
[0059] Preferably the depth does not increase at the reagent
inlets. The flowing layers are not perturbed because the reagent
inlets are narrower than the channel and because the reagent volume
is small relative to the sample and carrier stream volumes. In this
embodiment, all fluid streams exit through outlet 60.
Alternatively, specimen channels can branch from the side or bottom
of the channel before the outlet.
[0060] Each reagent inlet can be used for a different reagent, and
multiple analytes can be simultaneously detected. In one
embodiment, the spacing of the different reagent inlets is far
enough apart so that there is no significant mixing or interaction
between reagents. Alternatively, to avoid crosstalk partitions can
be placed between separate flow streams. In a second embodiment,
the reagent inlets are spaced so that the reagent plumes overlap,
and the interaction region can be monitored.
[0061] The sensor has been illustrated with fluid reagent inlet 50.
The reagent particles can be in solution or can be immobilized on a
bead carried by a fluid. In lieu of a fluid inlet, a solid reagent
inlet can be employed, as illustrated in FIG. 3. Plate 10 has
cavity 58 which can be filled with a solid pellet of reagent 158.
For the case of multiple reagents there is a series of cavities.
The term pellet is used herein for a solid or viscous mass of
reagent which can be immobilized on the sensor plate. It can be
recessed in a cavity, as shown, or can sit on the surface of the
plate. Carrier stream 140 flows over the reagent pellet and carries
the dissolved or suspended reagent downstream, as shown by dashed
line 151.
[0062] The reagent pellet is preferably soluble in the carrier
fluid. The pellet can utilize a soluble reagent carrier, for
example a sugar such as trehalose. The reagent pellet is placed
into the sensor before use. Preformed pieces of dry reagent can be
inserted or, more simply, a solution of the reagent can be injected
into the cavity followed by immobilizing the reagent by a process
such as drying or gelation. After insertion of the reagent pellet,
the sensor is assembled for use. This embodiment, with solid
reagent pellets, is particularly suited for disposable use.
[0063] Because the flow channel operates in low Reynolds number
conditions, the flow of the carrier across the solid pellet
maintains a smooth flow. The flow velocity immediately over the
pellet varies as the pellet shrinks, and, for a cavity placed
pellet, the cavity deepens. The dissolution rate of the analyte
varies over time as the pellet shrinks. Reproducibilitycan be
improved by making the measurements at a specific point during the
dissolution process. The measurement point can be determined, in
one embodiment, by the rate of dissolution of a reference
substance. An adjacent cavity contains a fluorescent or otherwise
easily traceable marker immobilized in a pellet with similar
dispersal characteristics to the reagent pellet. Alternatively, the
system can be calibrated with a calibrant solution before and/or
after sample measurements. In another embodiment, the rate of
dissolution is made relatively constant by modifying the shape of
the cavity or by layering different reagent/reagent-carrier
mixtures.
[0064] Reporter beads as the reagent can be introduced through a
liquid or solid reagent inlet. For cases when the reagent is
immobilized on a bead, the reagent does not appreciably diffuse
into the carrier stream, and the analyte must diffuse through the
carrier stream to the reagent. Reporter beads can be used to
measure pH, oxygen saturation, ion content, alcohols, pesticides,
organic salts such as lactate, sugars such as glucose, heavy
metals, and drugs such as salicylic acid, halothane and narcotics.
Each reporter bead comprises a bead having a plurality of at least
one type of fluorescent reporter molecules immobilized thereon. A
fluorescent or absorption property of the reporter bead is
sensitive to a corresponding analyte. Reporter beads can be added
at the reagent inlet and the analyte concentration can be
determined by observing the sensor flow channel from above or by
removing beads and analyte through a specimen channel and measuring
individual beads, for example in a flow cytometer. When removed in
a specimen channel, 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.
[0065] Magnetic beads can be used as the reporter beads. In this
embodiment, the beads are pulled into the sample stream by a
magnetic field. They can be immobilized to allow longer contact
time with the analyte. Optionally, they can be pulled back into the
carrier stream, for example for contact with a second analyte.
[0066] When the sample is a turbid sample, such as blood, it is
useful to filter the sample before measurement, especially for
optical measurement. While any filter known in the art can be
employed, a preferred filter is a diffusion based microfilter, as
described in U.S. Patent Application 08/663,916, Patent No.
5,932,100. The filter can lie in the plane of the substrate or a
plane perpendicular to the substrate. The perpendicular filter is
shown in combination with a sensor in Fig. 4. Unfiltered-sample
fluid 180 enters through inlet 80 and extraction fluid 170 enters
through inlet 70. The two fluids flow in adjacent laminar streams
in the filter's laminar flow channel, and analyte particles from
the sample fluid diffuse into the extraction stream. The remaining
turbid material 190, such as cells, termed herein the residual
sample, is discharged through outlet 90. Because particles such as
blood cells sediment in fluids such as plasma, it is preferred that
the filter be oriented with the turbid sample stream below the
extraction stream. The extraction stream of the filter is the
sample stream for the sensor, and enters through sample inlet 30.
In the sensor, carrier fluid 140 joins the sample stream through
inlet 40. Fluid reagent 150 enters through inlet 50, and reagent
particles diffuse through the carrier stream. Changes in optical
properties on interaction with the analyte are not illustrated in
this figure.
[0067] A second reagent 156 can enter downstream of the first
reagent through port 56 positioned in series with the first reagent
inlet. Electrodes 51 and 57 useful for electrochemical detection
may be placed between the reagent inlets 50 and 56 and the outlet.
Sequential addition of reagents is used, for example, in sandwich
assays. For example, when the analyte is an antigen the first
reagent can be a bead with a selective antibody and the second
reagent can be a secondary antibody with an attached flourophor. In
another example of an embodiment using multiple reagents in series,
the sample stream is blood, the first reagent is glucose oxidase,
and the second reagent contains a pH sensitive dye. As glucose
particles from the blood diffuse into the carrier stream they are
changed to gluconic acid which is detected by the pH-sensitive
dye.
[0068] 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 feed inlets and/or the outlet (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.
Rather than a pressure source, a flow source can be used at the
inlets, for example a syringe.
[0069] Figure 5 shows a portion of a laminar flow channel 20
terminating in a chamber 75 containing an absorbent pad 76. The
absorbent pad is a piece of filter paper designed to absorb about 1
ml of fluid, the required volume of fluid required to perform the
assay for which the sensor is devised. Figure 6 shows a portion of
a laminar flow channel 20 terminating in a wall or barrier, and
containing an absorbent pad 76.
[0070] The channel cells of this invention and the channels therein
can be sized as determined by the size of the particles desired to
be detected. As is known in the art, the diffusion coefficient for
the analyte particles is generally inversely related to the size of
the particle. Once the diffusion coefficient for the particles
desired to be detected is known, the contact time of the two
streams, size of the central channel, relative volumes of the
streams, pressure and velocities of the streams can be adjusted to
achieve the desired diffusion pattern.
[0071] Fluid dynamic behavior is directly related to the Reynolds
number of the flow. As the Reynolds number is reduced, flow
patterns depend more on viscous effects and less on inertial
effects. Below a certain Reynolds number, e.g., 0.1, inertial
effects can essentially be ignored. The microfluidic devices of
this invention do not require inertial effects to perform their
tasks, and therefore have no inherent limit on their
miniaturization due to Reynolds number effects. The devices of this
invention require laminar, non-turbulent flow and are designed
according to the foregoing principles to produce flow having low
Reynolds numbers, i.e. Reynolds numbers below about 1.
[0072] The devices of the preferred embodiment of this invention
are capable of analyzing a sample of a size between about 0.01
microliters and about 20 microliters within a few seconds, e.g.
within about three seconds. For fluid reagent inlets, the devices
may be reused. Clogging is minimized and reversible.
[0073] 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 carrier stream, the size of the particles remaining
in the sample stream and diffusing into the carrier stream can be
controlled.
[0074] For some analytes with relatively small diffusion
coefficients, a straight channel does not provide a long enough
flow channel for diffusion to occur adequately. Detection of
analytes with relatively small diffusion coefficients, e.g.
relatively large analytes or non-spherical analytes, preferably
employs a convoluted flow channel.
[0075] The channel cell system of this invention can be used to
measure concentration of an analyte as a function of distance (from
the reagent inlet) rather than time. An increment of distance is
proportional to an increment of time. With laminar flow and a known
flow speed, an increment of distance can be converted to an
increment of time. The rate of, or rate constant for, a reaction
can be determined using the diffusion based sensor of this
invention. If the analyte concentration is known, the rate of
reaction with the reagent can be obtained from the detection
gradient. A rate constant for a reaction can be determined with
only one measurement.
[0076] This invention has been described in a few preferred
embodiments. Many variations falling within the spirit and scope of
the invention will be readily apparent to those skilled in the art.
Application of the channel system of this invention for use as a
sensor has been described. It can also be used to synthesize
multiple products as opposed to sensing multiple analytes. In the
reactor embodiment, the "sample" stream contains a first reagent,
which reacts with a reagent from the reagent inlet to form a
product. In this embodiment the product is collected rather than
detected. Since the structure is the same, the device is herein
termed a sensor, though it may also be used as a reactor.
[0077] A few specific chemical reactions have been recited; any
reaction or interaction which produces a detectable change in
either the reagent or analyte can be used. A few detection means
have been described; other analytical means can be employed. The
sensor can be coupled with pretreatment and post treatment
apparatuses, such as fillers, separators, reactors and detectors.
The device can include additional inlets and outlets. The scope is
limited only by the following claims and their equivalents.
* * * * *