U.S. patent application number 10/684707 was filed with the patent office on 2004-05-13 for devices and methods for using centripetal acceleration to drive fluid movement in a microfluidics system for performing biological fluid assays.
Invention is credited to Jensen, Mona D., Kellogg, Gregory, Kieffer-Higgins, Stephen G., Kob, Mikayla, Lin, Hsin Chiang, Morneau, Keith, Ommert, Shari, Pierce, Andrea.
Application Number | 20040089616 10/684707 |
Document ID | / |
Family ID | 28789722 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040089616 |
Kind Code |
A1 |
Kellogg, Gregory ; et
al. |
May 13, 2004 |
Devices and methods for using centripetal acceleration to drive
fluid movement in a microfluidics system for performing biological
fluid assays
Abstract
This invention provides methods and apparatus for performing
microanalytic and microsynthetic analyses and procedures.
Specifically, the invention provides a microsystem platform for use
with a micromanipulation device to manipulate the platform by
rotation, thereby utilizing the centripetal force resulting from
rotation of the platform to motivate fluid movement through
microchannels embedded in the microplatform. The microsystem
platforms of the invention are also provided having microfluidics
components, resistive heating elements, temperature sensing
elements, mixing structures, capillary and sacrificial valves, and
methods for using these microsystems platforms for performing
biological, enzymatic, immunological and chemical assays.
Inventors: |
Kellogg, Gregory;
(Somerville, MA) ; Kieffer-Higgins, Stephen G.;
(Dorchester, MA) ; Jensen, Mona D.; (Hampstead,
NH) ; Ommert, Shari; (Medford, MA) ; Kob,
Mikayla; (Allston, MA) ; Pierce, Andrea;
(Hollis, NH) ; Morneau, Keith; (Boston, MA)
; Lin, Hsin Chiang; (Cambridge, MA) |
Correspondence
Address: |
McDonnell Boehnen Hulbert & Berghoff
32nd Floor
300 S. Wacker Drive
Chicago
IL
60606
US
|
Family ID: |
28789722 |
Appl. No.: |
10/684707 |
Filed: |
October 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10684707 |
Oct 14, 2003 |
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09315114 |
May 19, 1999 |
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6632399 |
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09315114 |
May 19, 1999 |
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09083678 |
May 22, 1998 |
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6063589 |
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60047488 |
May 23, 1997 |
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Current U.S.
Class: |
210/749 ;
210/198.1; 210/322; 210/600 |
Current CPC
Class: |
B01L 2300/0887 20130101;
B01L 3/502738 20130101; B01L 2400/0406 20130101; B01L 2200/0605
20130101; Y10T 436/25375 20150115; B01L 2300/0806 20130101; B01F
2101/23 20220101; B01L 2300/087 20130101; B01L 3/50273 20130101;
B01L 2400/0409 20130101; G01N 35/00069 20130101; B01F 35/71725
20220101; B01L 2400/0677 20130101; B01L 2200/10 20130101; B01L
2300/0867 20130101; H01R 39/64 20130101; B01F 2101/44 20220101;
F16K 31/002 20130101; Y10T 436/111666 20150115; B01F 33/30
20220101; B01L 3/5025 20130101; B01L 2400/0688 20130101; B01L
2300/1827 20130101; B01F 35/7172 20220101; G01N 21/07 20130101;
H01C 17/06586 20130101 |
Class at
Publication: |
210/749 ;
210/600; 210/198.1; 210/322 |
International
Class: |
B01D 015/00; B01J
020/00; C02F 001/00 |
Claims
We claim:
1. A microsystem platform for separating an analyte from a fluid
sample, comprising a) a rotatable platform, comprising a substrate
having a first flat, planar surface and a second flat, planar
surface opposite thereto, each surface comprising a center about
which the platform is rotated, wherein the first surface comprises
in combination b) an entry port comprising a depression in the
first surface having a volumetric capacity of about 1 to about 150
.mu.L, that is fluidly connected by a first microchannel with c) a
mixing chamber positioned on the platform farther from the center
of rotation than the entry port; the platform further comprising i)
a reagent reservoir comprising liquid reagents for preparing the
fluid sample for the analyte separation assay, wherein the reagent
reservoir if fluidly connected to the mixing chamber by a second
microchannel, the platform further comprising d) a secondary
metering chamber comprising a first metering portion and a second
metering portion each defining a volume of the fluid and separated
by a septum that extends from a position in the chamber farthest
from the center of rotation to a position just short of a chamber
wall closest to the center of rotation, wherein the end of the
septum and the chamber wall define a fluid connection between the
first and second metering portions, the metering chamber further
comprising an overflow portion that is separated from the second
metering portion by a septum that extends from a position in the
chamber farthest from the center of rotation to a position just
short of a chamber wall closest to the center of rotation, and
wherein the first portion of the metering chamber is fluidly
connected by a third microchannel to e) an analyte separation assay
chamber further comprising i) an analyte binding matrix, wherein
the analyte specifically binds to the matrix and is retained in the
separation chamber thereby, wherein the analyte separation assay
chamber is further fluidly connected by a fourth microchannel with
a separation matrix preparation buffer reservoir containing a
preparation buffer and the analyte separation assay chamber is
further fluidly connected by a fifth microchannel with a separation
matrix wash buffer reservoir containing a separation matrix wash
buffer, wherein each of the preparation buffer and wash buffer
reservoirs are positioned closer to the center of rotation than the
analyte separation assay chamber; and wherein the second metering
portion of the secondary metering chamber is fluidly connected by a
sixth microchannel with f) a read window that is further fluidly
connected to the analyte separation assay chamber by a seventh
microchannel and is further fluidly connected by a eighth
microchannel to g) a waste reservoir wherein rotation of the
platform motivates the fluid sample from the entry port into the
mixing chamber and the reagents from the reagent reservoir into the
mixing chamber to provide a sample reagent mixture, and wherein
rotation of the platform motivates the sample reagent mixture from
the mixing chamber into the secondary metering chamber, wherein the
first metering portion is filled before the second metering portion
and the second metering portion is filled before the overflow
portion; and wherein rotation of the platform motivates separation
matrix preparation buffer through the analyte separation assay
chamber, through the read chamber and into the waste reservoir, and
wherein rotation of the platform motivates the volume of the sample
reagent mixture from the first metering portion of the secondary
metering chamber through the analyte separation assay chamber
whereby analyte in the sample reagent mixture binds to the analyte
binding matrix; and wherein rotation of the platform motivates
analyte separation matrix wash buffer through the analyte
separation assay chamber, thereby displacing the sample reagent
mixture through the read window and into the waste reservoir; and
wherein rotation of the platform motivates the volume of the sample
reagent mixture in the second metering portion of the secondary
metering chamber into the read window and displaces the eluate from
the analyte separation assay chamber from the read window.
2. The microsystem platform of claim 1 wherein the analyte binding
matrix is an inositol phosphate-derived membrane.
3. The microsystem platform of claim 1 further comprising e) a
metering capillary and an overflow capillary, each being fluidly
connected with the entry port, wherein each capillary defines a
cross-sectional area of about 0.02 mm to about 1 mm in diameter,
and wherein each capillary extends radially from the center of the
platform and defines a first end proximally arrayed towards the
center of the platform and a second end distally arrayed from the
center of the platform, wherein the proximal end of each capillary
defines a curved opening; wherein the metering capillary defines a
volume of the fluid and wherein the metering capillary is fluidly
connected with the mixing chamber and wherein the overflow
capillary is fluidly connected with f) an overflow chamber having a
depth equal to or greater than the overflow capillary and
positioned radially more distant from the center of the platform
than the mixing chamber and the entry port, wherein a capillary
junction is formed at the junction of the metering capillary and
the mixing chamber and at the junction of the overflow capillary
and the overflow chamber, whereby fluid placed in the entry port
flows by capillary action to the junction of the metering capillary
and the mixing chamber, and excess fluid flows by capillary action
to the junction of the overflow capillary and the overflow chamber;
and wherein rotation of the platform at a first rotation speed
motivates fluid displacement in the overflow capillary into the
overflow chamber but not fluid displacement in the metering
capillary, whereby rotation of the platform at the first rotational
speed drains the fluid from the entry port into the overflow
chamber; and wherein rotation of the platform at a second rotation
speed that is greater than the first rotational speed motivates
fluid displacement of the volume of the fluid in the metering
capillary into the mixing chamber; and wherein each of the assay
chamber and overflow chamber also comprise air displacement
channels whereby air displaced by fluid movement is vented to the
surface of the platform.
4. The microsystem platform of claim 1, further comprising j) a
sacrificial valve in the fourth, fifth, sixth, or seventh
microchannels, wherein release of the sacrificial valve permits
fluid flow through the microchannel when the platform is rotated at
a non-zero rotational speed.
5. The microsystem platform of claim 4 wherein the sacrificial
valve is a solid, semi-solid or viscous liquid hydrocarbon, or a
plastic.
6. The microsystem platform of claim 5 further comprising a heating
element in the platform in thermal contact with the sacrificial
valve, wherein heating the heating element releases the sacrificial
valve.
7. The microsystem platform of claim 1, wherein the read chamber
comprises a top surface that is translucent.
8. The microsystem platform of claim 1 wherein the fluid sample is
blood.
9. A microsystem platform for separating an analyte from a fluid
sample, comprising a) a rotatable platform, comprising a substrate
having a first flat, planar surface and a second flat, planar
surface opposite thereto, each surface comprising a center about
which the platform is rotated, wherein the first surface comprises
in combination b) an entry port comprising a depression in the
first surface having a volumetric capacity of about 1 to about 150
.mu.L, that is fluidly connected by a first microchannel with c) a
mixing chamber positioned on the platform farther from the center
of rotation than the entry port and comprising liquid reagents for
preparing the fluid sample for the analyte separation assay,
wherein the mixing chamber is fluidly connected by a second
microchannel with d) a secondary metering chamber comprising a
first metering portion and a second overflow portion wherein the
first metering portion defines a volume of the sample reagent
mixture, wherein the first metering portion and the second overflow
portion are separated by a septum that extends from a position in
the chamber farthest from the center of rotation to a position just
short of a chamber wall closest to the center of rotation, wherein
the end of the septum and the chamber wall define a fluid
connection between the first metering portion and the overflow
portion, and wherein the fluid connection between the first
metering portion and the overflow portion is fluidly connected by a
third microchannel to a read chamber positioned radially more
distant from the center of rotation than the secondary metering
chamber, and wherein the first metering portion of the secondary
metering chamber is fluidly connected by a fourth microchannel to
e) an analyte separation assay chamber further comprising i) an
analyte binding matrix, wherein the analyte specifically binds to
the matrix and is retained in the separation chamber thereby,
wherein the analyte separation assay chamber is further fluidly
connected by a fifth microchannel with a separation matrix buffer
reservoir containing a buffer, wherein the matrix buffer reservoir
is positioned closer to the center of rotation than the analyte
separation assay chamber; and wherein the analyte separation assay
chamber is further fluidly connected by a sixth microchannel with
f) a read window manifold comprising a series of chambers separated
by septa and arranged linearly and adjacently on the surface of the
platform away from the position of the fluid connection of the
manifold with the fifth microchannel; wherein rotation of the
platform motivates the fluid sample from the entry port into the
mixing chamber to a sample reagent mixture, and wherein rotation of
the platform motivates the sample reagent mixture from the mixing
chamber into the secondary metering chamber, wherein the first
metering portion is filled before the overflow portion and the read
chamber is filled by fluid flow through the second microchannel
before the overflow portion is filled; and wherein rotation of the
platform motivates separation matrix preparation buffer through the
analyte separation assay chamber and into the read chamber
manifold, and wherein rotation of the platform motivates the volume
of the sample reagent mixture from the first metering portion of
the secondary metering chamber through the analyte separation assay
chamber whereby analyte in the sample reagent mixture binds to the
analyte binding matrix; and wherein rotation of the platform
motivates analyte separation matrix wash buffer through the analyte
separation assay chamber, thereby displacing the sample reagent
mixture into the read manifold.
10. The microsystem platform of claim 9 further comprising g)
metering capillary and an overflow capillary, each being fluidly
connected with the entry port, wherein each capillary defines a
cross-sectional area of about 0.02 mm to about 1 mm in diameter,
and wherein each capillary extends radially from the center of the
platform and defines a first end proximally arrayed towards the
center of the platform and a second end distally arrayed from the
center of the platform, wherein the proximal end of each capillary
defines a curved opening; wherein the metering capillary defines a
volume of the fluid and wherein the metering capillary is fluidly
connected with the mixing chamber and wherein the overflow
capillary is fluidly connected with h) overflow chamber having a
depth in the platform equal to or greater than the overflow
capillary and positioned radially more distant from the center of
the platform than the mixing chamber and the entry port, wherein a
capillary junction is formed at the junction of the metering
capillary and the mixing chamber and at the junction of the
overflow capillary and the overflow chamber, whereby fluid placed
in the entry port flows by capillary action to the junction of the
metering capillary and the mixing chamber, and excess fluid flows
by capillary action to the junction of the overflow capillary and
the overflow chamber; and wherein rotation of the platform at a
first rotation speed motivates fluid displacement in the overflow
capillary into the overflow chamber but not fluid displacement in
the metering capillary, whereby rotation of the platform at the
first rotational speed drains the fluid from the entry port into
the overflow chamber; and wherein rotation of the platform at a
second rotation speed that is greater than the first rotational
speed motivates fluid displacement of the volume of the fluid in
the metering capillary into the mixing chamber; and wherein each of
the assay chamber and overflow chamber also comprise air
displacement channels whereby air displaced by fluid movement is
vented to the surface of the platform.
11. The microsystem platform of claim 9, further comprising j) a
sacrificial valve in the third, fourth, fifth, or sixth
microchannels, wherein release of the sacrificial valve permits
fluid flow through the microchannel when the platform is rotated at
a non-zero rotational speed.
12. The microsystem platform of claim 11 wherein the sacrificial
valve is a solid, semi-solid or viscous liquid hydrocarbon, or a
plastic.
13. The microsystem platform of claim 12 further comprising a
heating element in the platform in thermal contact with the
sacrificial valve, wherein heating the heating element releases the
sacrificial valve.
14. The microsystem platform of claim 9, wherein the read chamber
comprises a top surface that is translucent.
15. The microsystem platform of claim 9 wherein the fluid sample is
blood.
16. A microsystem platform for separating an analyte from a fluid
sample, comprising a) a rotatable platform, comprising a substrate
having a first flat, planar surface and a second flat, planar
surface opposite thereto, each surface comprising a center about
which the platform is rotated, wherein the first surface comprises
in combination b) an entry port comprising a depression in the
first surface having a volumetric capacity of about 1 to about 150
.mu.L, that is fluidly connected by a first microchannel with c) an
assay chamber fluidly connected with the entry port, the reaction
chamber further comprising i) a porous matrix comprising reagents
for performing an analyte detection assay wherein a fluid sample
applied to the entry port is delivered to the assay chamber through
the capillary microchannel by rotation of the platform, and wherein
delivery of the fluid sample to the assay chamber initiates the
analyte detection assay; wherein the entry port is further fluidly
connected with d) a mixing chamber positioned on the platform
farther from the center of rotation than the entry port and
comprising liquid reagents for preparing the fluid sample for the
analyte separation assay, wherein the mixing chamber is fluidly
connected by a second microchannel with e) a secondary metering
chamber comprising a first metering portion and a second overflow
portion wherein the first metering portion defines a volume of the
sample reagent mixture, wherein the first metering portion and the
second overflow portion are separated by a septum that extends from
a position in the chamber farthest from the center of rotation to a
position just short of a chamber wall closest to the center of
rotation, wherein the end of the septum and the chamber wall define
a fluid connection between the first metering portion and the
overflow portion, and wherein the fluid connection between the
first metering portion and the overflow portion is fluidly
connected by a third microchannel to a read chamber positioned
radially more distant from the center of rotation than the
secondary metering chamber, and wherein the first metering portion
of the secondary metering chamber is fluidly connected by a fourth
microchannel to f) an analyte separation assay chamber further
comprising i) an analyte binding matrix, wherein the analyte
specifically binds to the matrix and is retained in the separation
chamber thereby, wherein the analyte separation assay chamber is
further fluidly connected by a fifth microchannel with a separation
matrix buffer reservoir containing a buffer, wherein the matrix
buffer reservoir is positioned closer to the center of rotation
than the analyte separation assay chamber; and wherein the analyte
separation assay chamber is further fluidly connected by a sixth
microchannel with g) a read window manifold comprising a series of
chambers separated by septa and arranged linearly and adjacently on
the platform away from the position of the fluid connection of the
manifold with the fifth microchannel; wherein rotation of the
platform motivates the fluid sample from the entry port into the
mixing chamber to a sample reagent mixture, and wherein rotation of
the platform motivates the sample reagent mixture from the mixing
chamber into the secondary metering chamber, wherein the first
metering portion is filled before the overflow portion and the read
chamber is filled by fluid flow through the second microchannel
before the overflow portion is filled; and wherein rotation of the
platform motivates separation matrix preparation buffer through the
analyte separation assay chamber and into the read chamber
manifold, and wherein rotation of the platform motivates the volume
of the sample reagent mixture from the first metering portion of
the secondary metering chamber through the analyte separation assay
chamber whereby analyte in the sample reagent mixture binds to the
analyte binding matrix; and wherein rotation of the platform
motivates analyte separation matrix wash buffer through the analyte
separation assay chamber, thereby displacing the sample reagent
mixture into the read manifold.
17. The microsystem platform of claim 16 wherein the analyte
binding matrix is an inositol phosphate-derived membrane.
18. The microsystem platform of claim 16, further comprising h) a
sacrificial valve in the fourth, fifth, sixth, or seventh
microchannels, wherein release of the sacrificial valve permits
fluid flow through the microchannel when the platform is rotated at
a non-zero rotational speed.
19. The microsystem platform of claim 18 wherein the sacrificial
valve is a solid, semi-solid or viscous liquid hydrocarbon, or a
plastic.
20. The microsystem platform of claim 19 further comprising a
heating element in the platform in thermal contact with the
sacrificial valve, wherein heating the heating element releases the
sacrificial valve.
21. The microsystem platform of claim 16, wherein the read chamber
comprises a top surface that is translucent.
22. The microsystem platform of claim 16 wherein the fluid sample
is blood.
Description
[0001] This application claim priority to U.S. Ser. No. 09/083,678,
filed May 22, 1998. This application is also related to U.S. Ser.
No. 08/995,056, filed Dec. 19, 1997, U.S. Ser. No. 08/910,726,
filed Aug. 12, 1997, U.S. Ser. No. 08/768,990, filed Dec. 18, 1996
and U.S. Ser. No. 08/761,063, filed Dec. 5, 1996, the disclosures
of every one of which are explicitly incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to methods and apparatus for
performing microanalytic analyses and procedures on fluid samples.
In particular, the invention relates to microminiaturization of
genetic, biochemical and chemical processes related to analysis,
synthesis and purification. Specifically, the invention provides a
microsystem platform that is rotationally manipulated by a
micromanipulation device, thereby utilizing the centripetal forces
resulting from rotation of the platform to motivate fluid movement
through microchannels embedded in the microplatform. The
microsystem platforms of the invention are provided having
microfluidics components, chambers and reservoirs, resistive
heating elements, temperature sensing elements, mixing structures,
capillary and sacrificial valves, and methods for using these
microsystems platforms for performing biological, enzymatic,
immunological and chemical assays.
[0004] 2. Summary of the Related Art
[0005] Assays for detecting analytes in fluid samples, particularly
complex fluids such as biological fluid samples, are used for a
variety of diagnostic, environmental, synthetic and analytical
purposes in the medical, biological, chemical, biochemical and
environmental arts.
[0006] Certain analytes are important for diagnosis and monitoring
of acute and chronic disease in humans. For example, diabetes is
the fourth major cause of morbidity and mortality in the U.S., even
though the biochemical basis for the disease has been known for at
least a century and drugs (primarily insulin) and methods for
managing the disease are robust and widely used. It has been
recognized that sensitive monitoring of blood sugar levels, either
directly or by detecting levels of metabolites and
disease-associated modifications (such as the relative fraction of
glycated hemoglobin in the bloodstream) is important in managing
the disease. However, fast and inexpensive ways of performing the
assays necessary for efficient management of multiple indicators of
the disease state are not currently available.
[0007] In the field of medical, biological and chemical assays,
mechanical and automated fluid handling systems and instruments are
known in the prior art.
[0008] U.S. Pat. No. 4,279,862, issued Jul. 21, 1981 to Bertaudiere
et al. disclose a centrifugal photometric analyzer.
[0009] U.S. Pat. No. 4,381,291, issued Apr. 26, 1983 to Ekins teach
analytic measurement of free ligands.
[0010] U.S. Pat. No. 4,515,889, issued May 7, 1985 to Klose et al.
teach automated mixing and incubating reagents to perform
analytical determinations.
[0011] U.S. Pat. No. 4,676,952, issued Jun. 30, 1987 to Edelmann et
al. teach a photometric analysis apparatus.
[0012] U.S. Pat. No. 4,745,072, issued May 17, 1998 to Ekins
discloses immunoassay in biological fluids.
[0013] U.S. Pat. No. 5,061,381, issued Oct. 29, 1991 to Burd
discloses a centrifugal rotor for performing blood analyses.
[0014] U.S. Pat. No. 5,122,284, issued Jun. 16, 1992 to Braynin et
al. discloses a centrifugal rotor comprising a plurality of
peripheral cuvettes.
[0015] U.S. Pat. No. 5,160,702, issued Nov. 3, 1993 to Kopf-Sill
and Zuk discloses rotational frequency-dependent valves using
capillary forces and siphons, dependent on wettablility" of liquids
used to prime said siphon.
[0016] U.S. Pat. No. 5,171,695, issued Dec. 15, 1992 to Ekins
discloses determination of analyte concentration using two labeling
markers.
[0017] U.S. Pat. No. 5,173,193, issued Dec. 22, 1992 to Schembri
discloses a centrifugal rotor for delivering a metered amount of a
fluid to a receiving chamber on the rotor.
[0018] U.S. Pat. No. 5,242,803, issued Sep. 7,1993 to Burtis et al.
disclose a rotor assembly for carrying out an assay.
[0019] U.S. Pat. No. 5,409,665, issued Apr. 25, 1995 to Burd
discloses a cuvette filling in a centrifuge rotor.
[0020] U.S. Pat. No. 5,413,009, issued Jul. 11, 1995 to Ekins
discloses a method for analyzing analytes in a liquid.
[0021] U.S. Pat. No. 5,472,603, issued Dec. 5, 1995 to Schembri
discloses an analytical rotor comprising a capillary passage having
an exit duct wherein capillary forces prevent fluid flow at a given
rotational speed and permit flow at a higher rotational speed.
[0022] Anderson, 1968, Anal. Biochem. 28: 545-562 teach a multiple
cuvette rotor for cell fractionation.
[0023] Renoe et al., 1974 Clin. Chem. 20: 955-960 teach a minidisc
module for a centrifugal analyzer.
[0024] Burtis et al., 1975, Clin. Chem. 20: 932-941 teach a method
for a dynamic introduction of liquids into a centrifugal
analyzer.
[0025] Fritsche et al., 1975, Clin. Biochem. 8: 240-246 teach
enzymatic analysis of blood sugar levels using a centrifugal
analyzer.
[0026] Burtis et al., 1975, Clin Chem. 21: 1225-1233 teach a
multipurpose optical system for use with a centrifugal
analyzer.
[0027] Hadjiioannou et al., 1976, Clin. Chem. 22: 802-805 teach
automated enzymatic ethanol determination in biological fluids
using a miniature centrifugal analyzer.
[0028] Lee et al., 1978, Clin. Chem. 24: 1361-1365 teach a
automated blood fractionation system.
[0029] Cho et al., 1982, Clin. Chem. 28: 1956-1961 teach a
multichannel electrochemical centrifugal analyzer.
[0030] Bertrand et al., 1982, Clinica Chimica Acta 119: 275-284
teach automated determination of serum 5'-nucleotidase using a
centrifugal analyzer.
[0031] Schembri et al., 1992, Clin Chem. 38: 1665-1670 teach a
portable whole blood analyzer.
[0032] Walters et al., 1995, Basic Medical Laboratory Technologies,
3rd ed., Delmar Publishers: Boston teach a variety of automated
medical laboratory analytic techniques.
[0033] Recently, microanalytical devices for performing select
reaction pathways have been developed.
[0034] U.S. Pat. No. 5,006,749, issued Apr. 9, 1991 to White
disclose methods apparatus for using ultrasonic energy to move
microminiature elements.
[0035] U.S. Pat. No. 5,252,294, issued Oct. 12, 1993 to Kroy et al.
teach a micromechanical structure for performing certain chemical
microanalyses.
[0036] U.S. Pat. No. 5,304,487, issued Apr. 19, 1994 to Wilding et
al. teach fluid handling on microscale analytical devices.
[0037] U.S. Pat. No. 5,368,704, issued Nov. 29, 1994 to Madou et
al. teach microelectrochemical valves.
[0038] International Application, Publication No. WO93/22053,
published 11 Nov. 1993 to University of Pennsylvania disclose
microfabricated detection structures.
[0039] International Application, Publication No. WO93/22058,
published 11 Nov. 1993 to University of Pennsylvania disclose
microfabricated structures for performing polynucleotide
amplification.
[0040] Columbus et al., 1987, Clin. Chem. 33: 1531-1537 teach fluid
management of biological fluids.
[0041] Ekins et al., 1994 Ann. Biol. Clin. 50: 337-353 teach a
multianalytic microspot immunoassay.
[0042] Wilding et al., 1994, Clin. Chem. 40: 43-47 disclose
manipulation of fluids on straight channels micromachined into
silicon.
[0043] One drawback in the prior art microanalytical methods and
apparati has been the difficulty in designing systems for moving
fluids on microchips through channels and reservoirs having
diameters in the 10-500 .mu.m range. Microfluidic systems require
precise and accurate control of fluid flow and valving to control
chemical reactions and analyte detection. Conventional pumping and
valving mechanisms have been difficult to incorporate into
microscale structures due to inherent conflicts-of-scale. These
conflicts of scale arise in part due to the fact that molecular
interactions arising out of mechanical components of such
components, which are negligible in large (macroscopic) scale
devices, become very significant for devices built on a microscopic
scale.
[0044] While devices and pumping and valving mechanisms have been
developed which overcome some of these conflict-of-scale
difficulties, there are other inherent problems with these systems.
A number of microanalytical platforms have been developed which use
electrokinetic forces for fluid pumping: electroosmotic flow
devices; electrohydrodynamic devices; and electrophoretic devices.
An inherent drawback in these systems is that they rely on precise
control of pH and free charges in the fluid being pumped. This
makes them incapable of pumping most raw biological samples, such
as blood and urine, and creates difficulties in pumping organic
solvents. In cases where these systems may be used,
pre-conditioning of the fluid to enhance electrokinetic effects is
usually required.
[0045] Systems that use centripetal force to effect fluid movement
in microstructures address the need for a pumping mechanism to
effect fluid flow, but cannot alone solve these scale-related
drawbacks of conventional fluidics reduced to microfluidics scale.
There remains a need for a simple, flexible, reliable, rapid and
economical microanalytic and microsynthetic reaction platform for
performing biological, biochemical and chemical analyses and
syntheses that can move fluids within the structural components of
a microsystems platform. Such a platform should be able to move
nanoliter-to microliter amounts of fluid, including reagents and
reactants, at rapid rates to effect the proper mixing of reaction
components, removal of reaction side products, and isolation of
desired reaction products and intermediates. There remains a need
in the art for centripetally-motivated microfluidics platforms
capable of precise and accurate control of flow and metering of
fluids in both microchip-based and centrifugal microplatform-based
technologies.
SUMMARY OF THE INVENTION
[0046] This invention provides microsystems platforms as disclosed
in co-owned and co-pending U.S. Ser. No. 08/761,063, filed Dec. 5,
1996 and U.S. Ser. No. 09/083,678, filed May 22, 1998, each of
which is incorporated by reference herein. Specifically, this
invention provides microfluidics platforms for performing
biological, enzymatic, immunological and chemical assays.
[0047] It is an advantage of the centrifugal rotors and
Microsystems platforms of the invention that an imprecise amount of
a fluid comprising a biological sample can be applied to the rotor
or platform and a precise volumetric amount of the biological
sample is delivered to a fluid reservoir comprising a reaction
vessel or other component of the rotor or platform for performing
chemical, biochemical, immunological or other analyses. It is an
advantage of the centrifugal rotors and Microsystems platforms of
the invention that metering of said precise amount of a biological
fluid sample, for example, a drop of blood, is provided as an
intrinsic property of the metering capillary channel of the rotor
or platform, thereby avoiding variability introduced by centripetal
metering of the sample into a reaction reservoir. It is a further
advantage of the centrifugal rotors and Microsystems platforms of
the invention that an operator can avoid having to precisely
measure an amount of a fluid comprising a biological sample for
application to the rotor or microsystem platform, thereby
permitting end-users, including consumers, having a lower level of
sophistication to use a medically diagnostic or other embodiment of
the rotor or microsystem platform of the invention.
[0048] It is an advantage of the centrifugal rotors and
Microsystems platforms of the invention that fluid movement into
and out of fluid reservoirs on the rotor or platform is precisely
determined by displacement of a first fluid, such as biological
sample, from a fluid reservoir by a second fluid contained in a
second reservoir on the rotor or platform. It is also an advantage
of the centrifugal rotors and Microsystems platforms of the
invention that approximately complete replacement of the volumetric
capacity of a first reservoir can be achieved by using fluid
displacement as disclosed herein, thereby providing for maximum
recovery of a first fluid sample upon displacement by a second
fluid, or maximum delivery and replacement of the first fluid by
the second fluid. This aspect of the invention is advantageous for
providing sequential chemical or biochemical reaction steps wherein
mixing of the reagents is not desired.
[0049] It is also an advantage of the centrifugal rotors and
Microsystems platforms of the invention that these platforms
provide an integrated microfluidics system containing components
and structures for performing microanalytic assays whereby fluid
flow on the platform is motivated by centripetal force and
controlled by capillary and/or sacrificial valves. The invention
provides such integrated platforms whereby an operator is required
simply to apply a sample, most preferably an imprecise volume of a
fluid sample, to an entry port on the disk surface, and a complex
series of analytical steps are performed without further operator
manipulation on the platform. Movement of fluids on the disk, and
the sequence of analytical reaction steps performed thereupon, is a
consequence of changes in rotor speed and/or the opening of
sacrificial valves as directed by an instruction set contained in a
program contained on the disk itself or in the memory of the
micromanipulation apparatus that controls disk rotation and
performance.
[0050] The Microsystems platforms also provide disks that can
perform a multiplicity of analytical reactions on either several
samples or a particular sample, whereby the reactions are performed
sequentially or individually. In addition, a wide variety of
analytic reactions can be performed on the Microsystems platforms
of the invention, as further described below.
[0051] Specific preferred embodiments of the present invention will
become evident from the following more detailed description of
certain preferred embodiments and the claims.
DESCRIPTION OF THE DRAWINGS
[0052] FIGS. 1 through 8 are schematic representations of
microfluidics arrays and components for performing direct analyte
detection assays using the Microsystems platforms of the
invention.
[0053] FIGS. 9 through 11 are schematic representations of
microfluidics arrays and components for performing separations of
analyte from a fluid sample using the Microsystems platforms of the
invention.
[0054] FIGS. 12 and 13 are schematic representations of
microfluidics arrays and components for performing both direct
analyte detection assays and analyte separations using the
Microsystems platforms of the invention.
[0055] FIGS. 14A and 14B illustrate the results of the assays
disclosed in Example 4 FIG. 15 is a photograph showing DNA recovery
using the assays described in Example 4.
[0056] FIG. 16 is a schematic diagram of a snap-in glucose assay
component of a Microsystems platform of the invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0057] The present invention provides centrifugal rotors and
Microsystems platforms for providing centripetally-motivated fluid
micromanipulation.
[0058] For the purposes of this invention, the term "sample" will
be understood to encompass any fluid, solution or mixture, either
isolated or detected as a constituent of a more complex mixture, or
synthesized from precursor species.
[0059] For the purposes of this invention, the term "in fluid
communication" or "fluidly connected" is intended to define
components that are operably interconnected to allow fluid flow
between components. In preferred embodiments, the platform
comprises a rotatable platform, more preferably a disk, whereby
fluid movement on the disk is motivated by centripetal force upon
rotation of the disk.
[0060] For the purposes of this invention, the term "a
centripetally motivated fluid micromanipulation apparatus" is
intended to include analytical centrifuges and rotors, microscale
centrifugal separation apparati, and most particularly the
microsystems platforms and disk handling apparati of International
Application No. WO97/21090, incorporated by reference.
[0061] For the purposes of this invention, the term "Microsystems
platform" is intended to include centripetally-motivated
microfluidics arrays as disclosed in International Application No.
WO97/21090.
[0062] For the purposes of this invention, the terms "capillary",
"microcapillary and "microchannel" will be understood to be
interchangeable and to be constructed of either wetting or
non-wetting materials where appropriate.
[0063] For the purposes of this invention, the term "fluid chamber"
will be understood to mean a defined volume on a rotor or
Microsystems platform of the invention comprising a fluid.
[0064] For the purposes of this invention, the term "entry port"
will be understood to mean a undefined volume on a rotor or
microsystems platform of the invention comprising a means for
applying a fluid to the rotor or platform.
[0065] For the purposes of this invention, the term "capillary
junction" will be understood to mean a junction of two components
wherein one or both of the lateral dimensions of the junction are
larger than the corresponding dimensions of the capillary. In
wetting or wettable systems, such junctions are where capillary
valving occurs, because fluid flow through the capillaries is
stopped at such junctions. In non-wetting or non-wettable
junctions, the exit from the chamber or reservoir is where the
capillary junction occurs. In general, it will be understood that
capillary junctions are formed when the dimensions of the
components change from a small diameter (such as a capillary) to a
larger diameter (such as a chamber) in wetting systems, in contrast
to non-wettable systems, where capillary junctions form when the
dimensions of the components change from a larger diameter (such as
a chamber) to a small diameter (such as a capillary).
[0066] For the purposes of this invention, the term "biological
sample" or "biological fluid sample" will be understood to mean any
biologically-derived analytical sample, including but not limited
to blood, plasma, serum, lymph, saliva, tears, cerebrospinal fluid,
urine, sweat, plant and vegetable extracts, semen, cell culture
fluids, cellular lysate aqueous or non-aqueous fractions of the
above and ascites fluid.
[0067] For the purposes of this invention, the term "air
displacement channels" will be understood to include ports in the
surface of the platform that are contiguous with the components
(such as chambers and reservoirs) on the platform, and that
comprise vents and microchannels that permit displacement of air
from components of the platforms and rotors by fluid movement.
[0068] For the purposes of this invention, the term "capillary
action" will be understood to mean fluid flow in the absence of
rotational motion or centripetal force applied to a fluid on a
rotor or platform of the invention.
[0069] For the purposes of this invention, the term "capillary
microvalve" will be understood to mean a capillary comprising a
capillary junction whereby fluid flow is impeded and can be
motivated by the application of pressure on a fluid, typically by
centripetal force created by rotation of the rotor or platform of
the invention.
[0070] The microplatforms of the invention (preferably and
hereinafter collectively referred to as discs or disks for the
purposes of this invention), the terms "microplatform",
"Microsystems platform" and "disc" or "disk" are considered to be
interchangeable, and are provided to comprise one or a multiplicity
of microsynthetic or microanalytic systems. Such microsynthetic or
microanalytic systems in turn comprise combinations of related
components as described in further detail herein that are operably
interconnected to allow fluid flow between components upon rotation
of the disk. These components can be fabricated as described below
either integral to the disk or as modules attached to, placed upon,
in contact with or embedded in the disk. The invention also
comprises a micromanipulation device for manipulating the disks of
the invention, wherein the disk is rotated within the device to
provide centripetal force to effect fluid flow on the disk.
Accordingly, the device provides means for rotating the disk at a
controlled rotational velocity, for stopping and starting disk
rotation, and advantageously for changing the direction of rotation
of the disk. Both electromechanical means and control means, as
further described herein, are provided as components of the devices
of the invention. User interface means (such as a keypad and a
display) are also provided, as further described in International
Application WO97/21090.
[0071] Fluid (including reagents, samples and other liquid
components) movement is controlled by centripetal acceleration due
to rotation of the platform. The magnitude of centripetal
acceleration required for fluid to flow at a rate and under a
pressure appropriate for a particular microsystem is determined by
factors including but not limited to the effective radius of the
platform, the position angle of the structures on the platform with
respect to the direction of rotation and the speed of rotation of
the platform.
[0072] The capillary junctions and microvalves of the invention are
based on the use of rotationally-induced fluid pressure to overcome
capillary forces. Fluids which completely or partially wet the
material of the microchannels (or reservoirs, reaction chambers,
detection chambers, etc.) which contain them experience a
resistance to flow when moving from a microchannel of narrow
cross-section to one of larger cross-section, while those fluids
which do not wet these materials resist flowing from microchannels
(or reservoirs, reaction chambers, detection chambers, etc.) of
large cross-section to those with smaller cross-section. This
capillary pressure varies inversely with the sizes of the two
microchannels (or reservoirs, reaction chambers, detection
chambers, etc., or combinations thereof), the surface tension of
the fluid, and the contact angle of the fluid on the material of
the microchannels (or reservoirs, reaction chambers, detection
chambers, etc.) Generally, the details of the cross-sectional shape
are not important, but the dependence on cross-sectional dimension
results in microchannels of dimension less than 500 .mu.m exhibit
significant capillary pressure. By varying the intersection shapes,
materials and cross-sectional areas of the components of the
Microsystems platform of the invention, valves are fashioned that
require the application of a particular pressure on the fluid to
induce fluid flow. This pressure is applied in the disks of the
invention by rotation of the disk (which has been shown above to
vary with the square of the rotational frequency, with the radial
position and with the extent of the fluid in the radial direction).
By varying capillary valve cross-sectional dimensions as well as
the position and extent along the radial direction of the fluid
handling components of the microsystem platforms of the invention,
capillary valves are formed to release fluid flow in a
rotation-dependent manner, using rotation rates of from 100 rpm to
several thousand rpm. This arrangement allows complex, multistep
fluid processes to be carried out using a pre-determined, monotonic
increase in rotational rate. The theoretical principles underlying
the use of capillary junctions and microvalves are disclosed in
International Patent Application, Publication No. WO98/07019,
incorporated by reference.
[0073] The instant invention provides Microsystems platforms
comprising microfluidics components, heating elements, temperature
sensing elements, capillary valves, sacrificial valves and a rotor
design for transmitting electrical signals to and from the
Microsystems platforms of the invention. The invention provides
fluidics components for capillary metering of precise amounts of a
volume of a fluid sample from the application of a less precise
volume of a fluid sample at an entry port on the microsystem
platform. These embodiments of the invention provide for delivery
of precise amounts of a sample such as a biological fluid sample
without requiring a high degree of precision or accuracy by the
operator or end-user in applying the fluid to the platform, and is
advantageous in embodiments of the Microsystems platforms of the
invention that are used by consumers and other relatively
unsophisticated users. The invention also provides laminar
flow-dependent replacement of a fluid in a first chamber by a
second displacement fluid in a second chamber on the platform.
These embodiments of the invention provide approximately complete
replacement of a fluid in one chamber on the platform with fluid
from another, and thereby provide means for practicing sequential
chemical reactions and other sequential processes on the platform
under conditions wherein mixing of the two fluids is
disadvantageous. The invention also provides turbulent flow mixing
components, which permit thorough mixing of different fluid
components on the platform, and in particular, the invention
provides mixing chambers fluidly connected with fluid reservoirs
containing equal amounts of two or more different fluids or unequal
amounts of two or more different fluids. In addition, the invention
provides fluid reservoirs fluidly connected with mixing chamber of
the invention and shaped to determine the relative rate of flow of
each of the different fluids into the mixing chamber. In these
embodiments, gradients of two fluids differing in viscosity, solute
concentration or concentration of suspended particulates can be
produced using the mixing chambers of the invention, as disclosed;
U.S. Ser. No. 09/083,678 incorporated by reference. Such gradients
can be transferred to reservoirs on the platform for further
analytical manipulations, and can form the basis for controlled
testing of concentration-dependent effects of various catalysts,
drugs, toxins or other biological or chemical agents.
[0074] Platforms of the invention such as disks and the components
comprising such platforms are advantageously provided having a
variety of composition and surface coatings appropriate for a
particular application. Platform composition will be a function of
structural requirements, manufacturing processes, and reagent
compatibility/chemical resistance properties. Specifically,
platforms are provided that are made from inorganic crystalline or
amorphous materials, e.g. Silicon, silica, quartz, inert metals, or
from organic materials such as plastics, for example, poly(methyl
methacrylate) (PMMA), acetonitrile-butadiene-styrene (ABS),
polycarbonate, polyethylene, polystyrene, polyolefins,
polypropylene and metallocene. These may be used with unmodified or
modified surfaces. Surface properties of these materials may be
modified for specific applications. Surface modification can be
achieved by silanization, ion implantation and chemical treatment
with inert-gas plasmas (i.e., gases through which electrical
currents are passed to create ionization). Also provided by the
invention are platforms made of composites or combinations of these
materials, for example, platforms manufactures of a plastic
material having embedded therein an optically transparent glass
surface comprising for example the detection chamber of the
platform. Microplatform disks of the invention are preferably
fabricated from thermoplastics such as teflon, polyethylene,
polypropylene, methylmethacrylates and polycarbonates, among
others, due to their ease of molding, stamping and milling.
Alternatively, the disks can be made of silica, glass, quartz or
inert metal. A fluid handling system is built by sequential
application of one or more of these materials laid down in stepwise
fashion onto the thermoplastic substrate. Alternatively, the entire
disc can be injection molded, embossed or stamped. Disks of the
invention are fabricated with an injection molded, optically-clear
base layer having optical pits in the manner of a conventional
compact disk (CD). The optical pits provide means for encoding
instrument control programming, user interface information,
graphics, data analysis, and sound specific to the application and
driver configuration. The driver configuration depends on whether
the micromanipulation device is a handheld, benchtop or floor
model, and also on the details of external communication and other
specifics of the hardware configuration. This layer is then
overlaid with a reflective surface, with appropriate windows for
external detectors, specifically optical detectors, being left
clear on the disk. Other layers of polycarbonate of varying
thickness are laid down on the disk in the form of channels,
reservoirs, reaction chambers and other structures, including
provisions on the disk for valves and other control elements. These
layers can be pre-fabricated and cut with the appropriate
geometries for a given application and assembled on the disk.
Layers comprising materials other than polycarbonate can also be
incorporated into the disk. The composition of the layers on the
disk depend in large part on the specific application and the
requirements of chemical compatibility with the reagents to be used
with the disk. Electrical layers can be incorporated in disks
requiring electric circuits, such as electrophoresis applications
and electrically-controlled valves. Control devices, such as
integrated circuits, laser diodes, photodiodes and resistive
networks that can form selective heating areas or flexible logic
structures can be incorporated into appropriately wired recesses,
either by direct fabrication of modular installation onto the disk.
Reagents that can be stored dry can be introduced into appropriate
open chambers by spraying into reservoirs using means similar to
inkjet printing heads, and then dried on the disk. A top layer
comprising access ports and air vents, ports or shafts is then
applied. Liquid reagents are then injected into the appropriate
reservoirs, followed by application of a protective cover layer
comprising a thin plastic film.
[0075] The platforms of the invention are preferably provided with
a multiplicity of components, either fabricated directly onto the
platform, or placed on the platform as prefabricated modules. In
addition to the integral components, certain devices and elements
can be located external to the platform, optimally positioned on a
device of the invention in relation to the platform, or placed in
contact with the platform either while rotating or when at rest.
Components optimally comprising the platforms of the invention or a
controlling device in combination therewith include detection
chambers, reservoirs, valving mechanisms, detectors, sensors,
temperature control elements, filters, mixing elements, and control
systems.
[0076] This invention provides microsystems platforms comprising
the following components.
1. Fluidics Components
[0077] The platforms of the invention are provided comprising
microfluidics handling structures in fluidic contract with one
another. In preferred embodiments, fluidic contact is provided by
capillary or microchannels comprising the surface of the platforms
of the invention. Microchannel sizes are optimally determined by
specific applications and by the amount of delivery rates required
for each particular embodiment of the platforms and methods of the
invention. Microchannel sizes can range from 0.02 mm to a value
close to the thickness of the platform. Microchannel shapes can be
trapezoid, circular or other geometric shapes as required.
Microchannels preferably are embedded in a platform having a
thickness of about 0.1 to 100 mm, wherein the cross-sectional
dimension of the microchannels across the thickness dimension of
the platform is less than 500 .mu.m-800 .mu.m and from 1 to 90
percent of said cross-sectional dimension of the platform. In these
embodiments, which is based on the use of rotationally-induced
fluid pressure to overcome capillary forces, it is recognized that
fluid flow is dependent on the orientation of the surfaces of the
components. Fluids which completely or partially wet the material
of the microchannels, reservoirs, detection chambers, etc. (i.e.,
the components) of the platforms of the invention which contain
them experience a resistance to flow when moving from a component
of narrow cross-section to one of larger cross-section, while those
fluids which do not wet these materials resist flowing from
components of the platforms of the invention of large cross-section
to those with smaller cross-section. This capillary pressure varies
inversely with the sizes of the two components, or combinations
thereof, the surface tension of the fluid, and the contact angle of
the fluid on the material of the components. Generally, the details
of the cross-sectional shape are not important, but the dependence
on cross-sectional dimension results in microchannels of dimension
less than 500 .mu.m exhibit significant capillary pressure. By
varying the intersection shapes, materials and cross-sectional
areas of the components of the platform of the invention, "valves"
are fashioned that require the application of a particular pressure
on the fluid to induce fluid flow. This pressure is applied in the
disks of the invention by rotation of the disk (which has been
shown above to vary with the square of the rotational frequency,
with the radial position and with the extent of the fluid in the
radial direction). By varying capillary valve cross-sectional
dimensions as well as the position and extent along the radial
direction of the fluid handling components of the platforms of the
invention, capillary valves are formed to release fluid flow in a
rotation-dependent manner, using rotation rates of from 100 rpm to
several thousand rpm. This arrangement allows complex, multistep
fluid processes to be carried out using a predetermined, monotonic
increase in rotational rate.
[0078] A first example of the microfluidics arrays provided by this
invention is shown in FIGS. 1A through 1D. A Microsystems platform
is provided by the invention that is specifically designed for
performing an assay for detecting a chemical species in a complex
mixture, preferably an aqueous mixture. These Figures illustrate an
array advantageously used for any such assay; detection of blood
glucose concentration is illustrated herein. It will be understood
that in this description, the use of the words "cell" and
"particulate" will be interchangeable, and that cells are a
particular example of a specific particulate species comprising the
fluid samples, and most preferably the biological fluid samples,
analyzed using the present invention.
[0079] A Microsystems platform provided by the invention and
specifically designed for performing blood glucose assay is
illustrated in FIG. 1A. Disk embodiments of the platforms of the
invention were fashioned, for example, from machined acrylic or
injection-molded polycarbonate, polystyrene, polypropylene,
acetonitrile-butadiene-styrene, or high-density polyethylene
(HDPE). The overall disc dimensions include an outer radius of from
about 1 cm to about 15 cm and an inner radius of from about 0.1 cm
to about 1 cm, wherein the disk was mounted on the spindle of a
rotary device. The thickness of the disc ranged from about 0.2 mm
to about 1.5 cm. All surfaces coming into contact with blood on the
platform may be advantageously treated with heparin, EDTA or other
anticoagulants to facilitate fluid flow thereupon
[0080] The components of the blood glucose assay were prepared as
follows. Fluid sample entry port 101 having a depth in the platform
surface from about 0.15 cm to about 3 mm and lateral dimensions of
from about 0.1 cm to about 2.5 cm were constructed on the platform,
and designed to accommodate a volume of from about 5 to about 200
.mu.L. This entry port was fluidly connected with a metering
capillary 102 having a square cross-sectional diameter of from
about 0.02 mm to about 2 cm and proximal ends rounded with respect
to entry port 101; the length of this metering chamber array was
sufficient to contain a total volume of from about 15 to about 150
.mu.L. The entry port was also constructed to be fluidly connected
with an overflow capillary 121 having a cross-sectional diameter of
from about 0.02 mm to about 2 mm and proximal ends rounded with
respect to entry port 101. The overflow capillary was fluidly
connected with an overflow chamber 122 having a depth in the
platform surface of from about 0.02 mm to about 9 mm, greater than
the depth of the overflow capillary 121. Each of the chambers on
the platform were also connected with air ports or air channels,
such as 114, that have dimensions of from about 0.02 mm to about 2
mm deep and permitted venting of air displaced by fluid movement on
the platform. A capillary junction 115 that is from about 0.03 mm
to about 2.2 mm deep is present in the air channel to prevent fluid
flow into the air channel.
[0081] Entry port 101 was positioned on the platform from about 1
cm to about 12 cm from the center of rotation. Metering chamber 102
extended from about 0.25 cm to about 2 cm from entry port 101. The
extent of the length of overflow capillary 121 was greater than the
extent of the length of metering capillary 102. The position of
overflow chamber 122 was, for example, from about 1.2 cm to about
14 cm from the axis of rotation.
[0082] In an alternative embodiment of the fluid metering
structures of the invention, shown in FIG. 1B, fluid sample entry
port 101 is comprised of a funnel having a depth in the platform
surface from about 0.75 mm to 5 mm, lateral dimensions of from
about 2 mm to 2 cm in the long dimension and from about 2 mm to 2
cm in the short dimension and positioned from about 0.75 cm to
about 5 cm from the center of rotation, and designed to accommodate
a volume of about 5 to about 200 .mu.L. The bottom surface of the
entry port consists of a slot 120 fluidly connected to
microfluidics structures formed in the other (under) side of the
microfluidics surface of the platform. Entry port 101 is connected
to entry passageway 102 having a rectangular cross-sectional
diameter of from about 0.1 mm to 5 mm wide and from about 0.1 mm to
5 mm deep and extending from about 0.1 cm to about 3 cm from entry
port 101. The entry passageway 102 is fluidly connected to blood
entry chamber 103. Blood entry chamber 103 has a depth of from
about 0.1 mm to 5 mm, lateral dimensions of from about 1 mm to
about 4 cm, and is positioned from about 0.75 cm to about 2.5 cm
from the center of rotation. Blood entry chamber 103 further
comprises blood metering volume 104 designed to accommodate a
volume of from about 1 .mu.L to about 50 .mu.L and an overflow
passageway 105. The overflow passageway was fluidly connected with
an overflow capillary 106 having a depth in the platform surface of
from about 0.1 mm to about 1 mm. Overflow capillary 121 is further
fluidly connected with overflow chamber 122 which is comprised of
two parts, a shallow outer portion 108 having a depth from about
0.05 mm to about 0.5 mm and a deeper inner portion 109 having a
depth of from about 0.1 mm to 5 mm. Overflow chamber 122 is
positioned from about 4 cm to about 5.8 cm from the axis of
rotation. The distal end of the blood overflow capillary 121 is
chosen to be farther from the center of rotation than the distal
end of blood chamber 104. Each of the chambers on the platform were
also connected with air ports or air channels 114, that are from
about 0.1 mm to about 5 mm deep and permit venting of air displaced
by fluid movement on the platform. A capillary junction 115 that is
from about 0.03 mm to about 2.2 mm deep is present in the air
channel to prevent fluid flow into the air channel. In alternative
embodiments, these vents may be multiply connected to one another
through a manifold such that fluid flow merely displaces air within
the structure, rather than forcing it through vents in the platform
surface.
[0083] Alternatively, an unmetered amount of a blood sample is
placed directly in blood fluid chamber 104, which in this
embodiment is open to the surface of the disk to accept blood
application. In these embodiments, the amount of blood fluid to be
assayed is controlled by the capacity of assay chamber 107 or the
matrix 106 contained therein as described below.
[0084] As described herein for performing blood glucose assays (and
as understood in the art that essentially the same microfluidics
structures can be used for a multiplicity of blood analyte assays
or, more generally, for analyte assays in any fluid sample, most
preferably a biological fluid sample), a capillary barrier prevents
movement of the fluid sample directly into the assay chamber 107.
In the metering structure shown in FIG. 1A, fluid (or more properly
for this illustrative example, blood) metering capillary 102 acted
as a capillary barrier that prevented blood fluid flow from
metering capillary 102 at a first, non-zero rotational speed
f.sub.1, ranging from about 200 rpm to about 450 rpm and sufficient
to permit fluid flow comprising overflow from the entry port 101
through overflow capillary 121 and into overflow chamber 122. This
capillary boundary was constructed to be overcome at a second
rotational speed f.sub.2, ranging from about 250 rpm to about 900
rpm (where f.sub.2>f.sub.1). In the alternative embodiments
shown in FIGS. 1B and 1C, blood fluid chamber 104 acted as a
capillary barrier that was maintained during rotation at a
rotational speed sufficient to motivated excess fluid sample from
the entry port 101 to the overflow chamber 122, and was overcome at
a second rotational speed greater than the first rotational speed
to permit fluid sample flow into assay chamber 107.
[0085] Blood metering capillary 102 and blood fluid chamber 104
were in different alternatives of the microsystems platforms of the
invention fluidly connected to capillary 110 that was from about
0.02 mm to about 2 mm deep and had a cross-sectional diameter of
from about 0.02 mm to about 2 mm and was connected to capillary or
sacrificial valve 111. Sacrificial or capillary valve 111 was
further fluidly connected with capillary 112 that was from about
0.02 mm to about 2 mm deep and had a cross-sectional diameter of
from about 0.02 mm to about 2 mm, and further to assay chamber 107.
In capillary valve embodiments, the junction between capillary 110
and capillary 112 creates capillary valve 111, wherein said
capillary valve 111 was from about 0.03 mm to about 2.2 mm deep and
had a cross-sectional diameter of from about 0.03 mm to about 2.2
mm. In order to function as a capillary valve, the junction between
capillary 110 and capillary 112 must have a depth and/or cross
sectional area greater than that of capillary 110 in a disc
fabricated from hydrophilic materials such as acrylic. In
sacrificial valve embodiments, intermediate melting temperature
materials (including, for example, waxes as described above) are
placed in the lumen of capillary 110 forming a fluid-tight seal. In
these embodiments, chamber 111 is fluidly connected to the
sacrificial valve so that melted wax from the release sacrificial
valve is sequestered in the chamber.
[0086] Rectangular assay chamber 107 was constructed in the surface
of the platform to have a depth of from about 0.2 mm to about 3 mm,
most preferably comprising a circular or rectangular depression 113
connected to capillary 112. Depression 113 was constructed to have
a volumetric capacity of from half to twice the assay volume. Assay
chamber 107 also comprised a pad or matrix 106 of a hydrophilic
substance. Materials used to prepare said matrices include but are
not limited to derivatized nylons, nitrocellulose, fiberglass and
polyesters, most preferably having a pore size of 0.2-2.0 .mu.m,
most preferably comprising a positively-charged nylon matrix having
a pore size of about 0.8 .mu.m. The upper limit on pore size of
matrix 106 is chosen to inhibit or prevent blood cell entry into
the matrix. The matrix is positioned in assay chamber 107 to be in
fluidic contact with depression 113, more preferably covering
depression 113, and most preferably having a surface area greater
than the surface area of depression 113. The matrix was further
impregnated with immobilized reagents which produce a detectable
product proportional to the amount or concentration of glucose in a
blood sample. Most preferably, the detectable product is a colored
product, i.e., a product absorbing light at a detectable, most
preferably a visible, wavelength.
[0087] In preparing matrices according to the invention, reagents
advantageously used to detect and more preferably quantitate an
amount of a component of a biological fluid sample are impreganted
into the matrix. As a non-limiting example, glucose is detected
according to the invention using a glucose oxidase assays system,
as described in additional detail below. Matrices for performing
such assays using the microfluidics platforms of the invention are
prepared by saturating the matrix membrane with an 8 mL solution of
distilled water containing 0.12 g 2,5-ferandione polymer with
methoxylene (CAS: 9011-16-9), 10 mg EDTA, 200 mg Polypep.RTM. Low
Viscosity (Sigma Chemical Company, St. Louis, Mo.), 668 mg sodium
citrate, 28.75 mg glucose oxidase and 27.3 mg peroxidase. The
saturated membrane is then dried and then saturated with 5 mL
acetonitrile and 5 mL distilled water containing 40 mg
3-methyl-2-benzothiazolinone hydrazone and 80 mg
3-dimethylaminobenzoic acid. The saturated membrane is dried and
applied to the microfluidics disk of the invention for use.
[0088] In an alternative embodiment shown in FIG. 15, assay chamber
107 is comprised of a rectangular cavity in the surface of the
platform having a depth of from about 0.2 mm to about 3 mm which is
fluidly connected at its end proximal to the axis of rotation to
capillary 112 and to air displacement channels 114 and ports 115.
The second member of the assay chamber is a rectangular piece made
from the same material as the platform or other material and
designed to snap into the cavity forming liquid-tight seals around
all edges. The snap-in piece has two faces, an A face and a B face.
The A face consists of a fluid entry channel 112 connected to
depression 113; depression 113 is further connected to air
displacement channels 114. Depression 113 is from about 0.05 mm to
about 5 mm deep and has a dimensions from about 0.5 mm by 4 mm,
having a volumetric capacity of from about half to about twice the
assay volume applied to the disc. A pad or matrix 106 is attached
to the A face of the snap-in piece, comprising a hydrophilic
substance possessing a pore size of 0.2-2.0 .mu.m, most preferably
comprising a positively-charged nylon matrix having a pore size of
about 0.8 .mu.m. The upper limit on pore size of matrix 106 is
chosen to inhibit or prevent blood cell entry into the matrix.. The
matrix is positioned in assay chamber 107 to be in fluidic contact
with depression 113, more preferably covering depression 113, and
most preferably having a surface area greater than the surface area
of depression 113. The matrix is further impregnated with
immobilized reagents which produce a detectable product
proportional to the amount or concentration of glucose in a blood
sample. Most preferably, the detectable product is a colored
product, i.e., a product absorbing light at a detectable, most
preferably a visible, wavelength.
[0089] As illustrated in FIGS. 2A through 2F, in the use of this
platform an imprecise volume (ranging from 20-150 .mu.L) of blood
was applied to the entry port 101. In embodiments of the platform
comprising air displacement channels, the fluid wicked into air
channel 114 and was stopped by capillary junction 115. Fluid also
wicked into metering capillary 102 and overflow capillary 121.
Fluid flowed through the metering capillary 102 and overflow
capillary 121 at no rotational speed until the fluid reached
capillary junctions at the junction between metering capillary 102
and capillary 110 and overflow capillary 121 and overflow chamber
122. Metering capillary 102 was constructed to define a precise
volume from about 15 to about 60 .mu.L of fluid between entry port
101 and capillary junction 111, which was designed to be at least
the amount of the fluid placed by the user in entry port 101.
[0090] After sample loading by a user and filling of metering
capillary 102 and overflow capillary 121 at no rotational speed,
the platform was spun at a first rotational speed f.sub.1, ranging
from about 50 rpm to about 600 rpm, which was sufficient to
motivate fluid flow through the overflow capillary 121 in this
microfluidics array having an entry port 101 with a depth of from
about 0.2 mm to about 3 mm, metering capillary 102 with dimensions
of about 0.5 mm.times.0.5 mm in cross-section and a length of about
2.2-3.8 cm from the center of rotation and an overflow capillary
121 with dimensions of about 0.5 mm.times.0.5 mm in cross-section
and a length of about 5.4 cm from the center of rotation.
[0091] Due to the greater distance of the end of overflow capillary
121 from the center of rotation than the end of metering capillary
102, at rotational speed f.sub.1 fluid flowed through overflow
capillary 121 into overflow chamber 122. The platform was spun
until all excess fluid is evacuated from entry port 101 and into
overflow chamber 122, except the fluid contained in metering
chamber 102.
[0092] At a second rotational speed f.sub.2 of from about 100 rpm
to about 1000 rpm, the precise amount of fluid contained in
metering capillary 102 was delivered into assay chamber 107. In
embodiments comprising a sacrificial valve 111 in-line with
capillary 110 at a position between capillary 110 and 112 shown in
FIG. 2B, release of the sacrificial valve resulted in fluid flow
through capillary 112 and into assay chamber 107. In said
embodiments, fluid flow is achieved at rotational speed f.sub.2
with removal of the sacrificial valve. In embodiments of the
platforms of the invention comprising capillary valve 111 at a
position between capillary 110 and 112 shown in FIG. 2B, capillary
110 preferably filled along with filling of metering capillary 102
until blood reached capillary junction 111 at the junction between
capillary 110 and capillary 112; in such embodiments, the capillary
junction had a depth of from about 0.03 mm to about 2.2 mm, or at
least greater than the depth of capillary 110.
[0093] Blood flowing into assay chamber 107 is preferentially
directed to depression 113 in the assay chamber; the dimensions of
depression 113 are conveniently chosen to be able to contain
substantially all of the blood fluid of the sample metered through
metering capillary 102 into assay chamber 107 (FIG. 2C). Displaced
air flows through air channel 114, and may be vented to the surface
of the disc or in communication with blood fluid chamber 104.
[0094] As blood flows into depression 113, the fluid component of
the blood is driven by pressure and hydrophilic forces into matrix
106; the pore size of the matrix is chosen to prevent the cellular
components of the blood from entering the matrix (FIG. 1.4). In
preferred embodiment, the cellular component of the blood is
retained in depression 113 and the fluid component is efficiently
distributed by wicking and by centripetal force into matrix 106. In
an alternative embodiment, alternative matrix 118 comprises at
least two distinct elements that are compressed or adhered to one
another in the assays chamber. The first element is similar to the
reagent-containing matrix 106 described above; however, this
embodiment of the matrix has a pore size that is not limited by the
size of cellular components of the blood, and can be any pore size
deemed optimal on experimental, economic, manufacturing or
availability grounds. The second element comprises a filtering
layer having a pore size that prevents cells and cellular debris
from entering this portion of the matrix. In a preferred
embodiment, the two elements and rearranged in assay chamber 107 so
that second matrix element is in contact with depression 113
wherein the blood aliquot is first contacted with the second matrix
component. Blood fluid flows into and through the second matrix
element and into the first matrix element, whereby cellular
components of blood are prevented from entering assay chamber 107
by the pore size of second matrix element. Preferably, the
dimensions of the matrix element is about 1 cm by about 0.75 cm and
has a thickness of about 0.05 cm.
[0095] As the fluid component of the blood wicks into matrix 106,
dried reagents are solubilized and the reaction of the blood
component catalyzed by said reagents proceeds. The timescale over
which these chemical reactions take place is chosen to be long
compared with the time it takes for the fluid component of blood to
saturate the matrix 106. The time it takes for the blood fluid to
saturate the matrix is dependent on the capacity of the matrix 106
to absorb the fluid and the delivery speed of the blood fluid to
the assay chamber 107, which in turn is dependent on the rotational
velocity and the cross-sectional dimensions of capillary 110. In
preferred embodiments, the reaction(s) goes to completion within
about 1 min. Reaction of the blood component(s) with the reagents
produce colored product which is then detected (FIG. 2E). In
preferred embodiments, detection is performed
spectrophotometrically, including absorbance, transmittance,
reflection, fluorescence, and chemiluminescence, although visual
inspection is also contemplated in alternative embodiments of the
invention.
[0096] In an alternative embodiment, assay chamber 107 further
comprises a detection cell 117 that is laterally adjacent to the
portion of assay chamber 107 comprising depression 113 (shown in
FIGS. 3A through 3E). This embodiment uses a variation of
alternative matrix 118 wherein the matrix comprises a blood
separation element and a blood fluid wicking element. These
elements are arranged in assay chamber 107 so that the blood
separation element is in contact with depression 113 and the blood
fluid wicking element is compressed or attached to the blood
separation element along its entire extent. A blood fluid wicking
element having dimensions as described above and sufficient to
encompass the entire surface extent of the blood separation element
and to further extend throughout the complete extent of assay
chamber 107, including detection cell 117. Reagents are only
present in the portion of the blood fluid wicking element in that
portion of the element comprising detection cell 117; this
arrangement is shown in detail in FIG. 3A.
[0097] In the practice of this embodiment of the invention, blood
delivered to depression 113 wicks through the blood separation
element and into the blood fluid wicking element (FIGS. 3B and 3C).
As the region of blood fluid wicking element above depression 113
saturates with blood plasma, the blood fluid wicks laterally into
the portion of the matrix 118 comprising detection cell 117 (FIG.
3D). Wetting of this portion of the matrix, which comprises the
immobilized reagents and initiates the glucose detection
reaction(s) as described in Example 1, with the production of a
colored product (FIG. 3E). The amount of colored product produced
is detected and the amount of glucose in the blood sample
determined thereby.
[0098] Another alternative embodiment of a blood glucose assay
Microsystems platform is shown in FIGS. 4A through 4E. Construction
of the disk embodiments of the platforms of the invention were as
described above.
[0099] The blood application and metering components and their
dimensions and relationships to one another are identical to those
described above, comprising sample entry port chamber 201, metering
capillary 202, overflow capillary 203, and overflow chamber 205. As
in Example 1, each of the overflow and fluid chambers is also
connected with air ports or air channels, such as 214, and
capillary junction(s) 215, that permit venting of air displaced by
fluid movement on the platform.
[0100] Metering capillary 202 is fluidly connected to capillary 210
that was from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm and is
connected to capillary or sacrificial valve 211. Sacrificial or
capillary valve 211 is further fluidly connected with capillary 212
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm;
capillary 212 is further fluidly connected to assay chamber 207.
Where capillary 210 is connected with capillary valve 211, said
capillary valve 211 is from about 0.03 mm to about 2.2 mm deep and
has a cross-sectional diameter of from about 0.03 mm to about 2.2
mm.
[0101] Assay chamber 207 comprises a depression in the surface of
the platform having a depth of from about 0.2 mm to about 3 mm
preferably comprising a circular or rectangular depression 213
connected to capillary 212 and most preferably wherein a metered or
otherwise controlled amount of blood can be contained in the assay
chamber. Assay chamber 207 also comprises a pad or matrix 206 of a
hydrophilic substance possessing a pore size of from about 0.2 to
about 2 .mu.m, most preferably about 0.8 .mu.m. The upper limit on
pore size of matrix 206 is chosen to inhibit or prevent blood cell
entry into the matrix, and to promote entry or wicking of the fluid
component of blood (plasma or serum) to enter the body of the
matrix. The matrix is positioned in assay chamber 207 to be in
fluidic contact with depression 213, more preferably covering
depression 213, and most preferably having a surface area greater
than the surface area of depression 213; in embodiments not having
depression 213 as a component of assay chamber 207, the matrix
substantially fills the volumetric extent of the assay chamber. The
matrix is further impregnated with immobilized reagents which
produce a detectable product proportional to the amount or
concentration of glucose in a blood sample. Most preferably, the
detectable product is a colored product, i.e., a product absorbing
light at a detectable, most preferably a visible, wavelength.
[0102] In an alternative embodiment, assay chamber 207 comprises a
surface wherein reagents 219 are directly dried or otherwise
immobilized on the surface of the chamber, and in such embodiments
the assay chamber does not comprise matrix 206.
[0103] Assay chamber 206 is further fluidly connected with
capillary 220 at a position preferably most distal to the axis of
rotation. Capillary 220 is from about 0.02 mm to about 2 mm deep
and has a cross-sectional diameter of from about 0.02 mm to about 2
mm, and is further fluidly connected with waste reservoir 221.
Waste reservoir 221 is positioned from about 1.2 cm to about 14 cm
from the axis of rotation, and has a depth in the platform of from
about 0.2 mm to about 3 mm. Assay chamber 206 is also fluidly
connected with capillary 222 at a position preferably most proximal
to the axis of rotation. Capillary 222 is from about 0.02 mm to
about 2 mm deep and has a cross-sectional diameter of from about
0.02 mm to about 2 mm, and is further fluidly connected with wash
buffer reservoir 223. Wash buffer reservoir 223 has a depth in the
platform of from about 0.2 mm to about 3 mm and is positioned from
about 1.2 cm to about 14 cm from the axis of rotation, and in any
event more proximal to the axis than assay chamber 206. Fluid flow
from wash buffer reservoir 223 through capillary 222 is controlled
by either capillary valve 224 or sacrificial valve 225. Where
capillary 222 was connected with capillary valve 224, said
capillary valve 224 is from about 0.03 mm to about 2.2 mm deep and
has a cross-sectional diameter of from about 0.03 mm to about 2.2
mm.
[0104] As illustrated in FIGS. 4A through 4D, in the use of this
platform an imprecise volume (ranging from 20-150 .mu.L of fluid)
of blood is applied to the entry port 201. Application of blood to
the platform and delivering a metered amount of blood to metering
capillary 202 was achieved as described above. Alternatively, an
unmetered amount of blood is introduced onto the platform directly
into assay chamber 207. Preferably, from about 15 .mu.L to about
150 .mu.L of blood is delivered to assay chamber 207.
[0105] At a rotational speed f.sub.2 of about 100-1000 rpm, the
precise amount of fluid contained in metering capillary 202 is
delivered into assay chamber 207. In embodiments comprising a
sacrificial valve 211 in-line with capillary 210 at a position
between capillary 210 and assay chamber 207 as shown in FIG. 3.1,
release of the sacrificial valve results in fluid flow through
capillary 212 and into assay chamber 207. In said embodiments,
fluid flow is achieved at rotational speed f.sub.2 with removal of
the sacrificial valve. In embodiments of the platforms of the
invention comprising capillary valve 211 at a position between
capillary 210 and 212 as shown in FIG. 3B, capillary 210 preferably
fills along with filling of blood metering capillary 202 until
blood reaches capillary junction 211 at the junction between
capillary 210 and capillary 212; in such embodiments, the capillary
junction had a depth of from about 0.03 mm to about 2.2 mm. At a
rotational speed f.sub.2 of about 100-1000 rpm, the fluid contained
in blood metering capillary 202 is delivered into assay chamber 207
(FIG. 3C).
[0106] Blood flowing into assay chamber 207 is directed to matrix
206 in the assay chamber; the dimensions of matrix 206 are
conveniently chosen to be able to contain substantially all of the
blood fluid of the sample metered through metering capillary 202
into assay chamber 207. Displaced air flows through air channel
214, and may be vented to the surface of the disc or in
communication with blood metering capillary 202 or entry port 201.
Alternatively, in embodiments wherein the blood sample is
unmetered, the capacity of the matrix 206 is sufficient to absorb a
controlled amount of blood. In further alternative embodiments,
wherein assay chamber does not comprise matrix 206, the assay
chamber is provided having a capacity for a controlled volume of
blood.
[0107] As blood flows into assay chamber 207, the fluid component
of the blood wicks into matrix 206; the pore size of the matrix is
chosen to prevent the cellular components of the blood from
entering the matrix. In preferred embodiment, the cellular
component of the blood is retained in assay chamber 207 and the
fluid component is efficiently wicked uniformly throughout matrix
206.
[0108] As the fluid component of the blood wicks into matrix 206,
dried reagents are solubilized and the reaction of the blood
component catalyzed by said reagents proceeds. The timescale over
which these reactions take place is chosen to be long compared with
the time it takes for the fluid component of blood to saturate the
matrix 206; however, in preferred embodiments, the reaction(s) goes
to completion within about 0.5 to about 5 min. Reaction of the
blood component(s) with reagents produce colored product which is
then detected. In preferred embodiments, detection is performed
spectrophotometrically, although visual inspection is also
contemplated in alternative embodiments of the invention.
[0109] After a time sufficient to produce a detectable amount of a
colored product, the wash buffer is released from wash buffer
reservoir 223 through capillary 222 and into assay chamber 207. In
embodiments comprising a sacrificial valve 225 in-line with
capillary 222 at a position between capillary 222 and assay chamber
207 shown in FIG. 3.3, release of the sacrificial valve results in
fluid flow through capillary 222 and into assay chamber 207. In
said embodiments, fluid flow is achieved at rotational speed
f.sub.3 of about 250 rpm to about 1500 rpm with removal of the
sacrificial valve. In embodiments of the platforms of the invention
comprising capillary valve 224 at a position between capillary 222
and assay chamber 207 shown in FIG. 3D, capillary 222 preferably
fills along with filling of blood fluid chamber 204 until blood
reached capillary junction 224; in such embodiments, the capillary
junction had a depth of from about 0.03 mm to about 2.2 mm. At a
higher rotational speed f.sub.4 of about 400-2000 rpm, the fluid
contained in wash reservoir 223 is delivered into assay chamber 207
(FIG. 3D). Because the fluid flow of wash buffer into the assay
chamber and fluid from the assay chamber to the waste chamber is
laminar, there is very little mixing of the washing fluid with the
fluid initially in the assay chamber. The wash fluid displaces the
fluid sample in the assay by pushing it into the waste chamber. The
exit of capillary 220 into chamber 221 is at a radial position such
that assay chamber 207 must remain filled with fluid during this
washing process. The quality of fluid removal is such that no more
than 1 part in 1000 of the fluid in the chamber 207 (which has not
been imbibed into matrix 206) remains. The fluid which has wicked
into matrix 206 is not removed during this wash because of the
small pore size of the matrix which resists fluid flow;
furthermore, color reagents do not diffuse out of matrix 206 if the
wash time is relatively short (less than a few hundred seconds). As
a result, interfering blood fluid components such as hemoglobin are
removed from chamber 207 while substantially leaving behind color
reagents in matrix 206. These are then interrogated
spectrophotometrically in assay chamber 207 (FIG. 4E).
[0110] Another alternative embodiment of a blood glucose assay
Microsystems platform is shown in FIGS. 5A through 5E. Construction
of the disc embodiments of the platforms of the invention were as
described above.
[0111] The blood application and metering components and their
dimensions and relationships to one another are identical to those
described above, comprising sample entry port chamber 301, metering
capillary 302, overflow capillary 303, and overflow chamber 305. As
in Example 1, each of the overflow and fluid chambers is also
connected with air ports or air channels, such as 314, and
capillary junction(s) 315, that permit venting of air displaced by
fluid movement on the platform.
[0112] Metering capillary 302 is fluidly connected to capillary 310
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm and is
connected to capillary or sacrificial valve 311. Sacrificial or
capillary valve 311 is further fluidly connected with capillary 312
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm, and
is further fluidly connected to assay chamber 307. Where capillary
310 is connected with capillary valve 311, said capillary valve 311
is from about 0.03 mm to about 2.2 mm deep and has a
cross-sectional diameter of from about 0.03 mm to about 2.2 mm.
[0113] Assay chamber 307 comprises a pad or matrix 306 of a
hydrophilic substance possessing a pore size of from about 0.2
.mu.m to about 2 .mu.m, most preferably about 0.8 .mu.m. The upper
limit on pore size of matrix 306 is chosen to inhibit or prevent
blood cell entry into the matrix. The matrix is further impregnated
with immobilized reagents which produce a detectable product
proportional to the amount or concentration of glucose in a blood
sample. Most preferably, the detectable product is a colored
product, i.e., a product absorbing light at a detectable, most
preferably a visible, wavelength.
[0114] As illustrated in FIGS. 5A through 5E, in the use of this
platform an imprecise volume (ranging from about 15 .mu.L to about
150 .mu.L of fluid) of blood is applied to the entry port 301.
Application of blood to the platform and delivering a metered
amount of blood to blood metering capillary 302 was achieved as
described above. Alternatively, an unmetered amount of blood is
introduced onto the platform directly into assay chamber 307,
preferably, from about 15 .mu.L to about 150 .mu.L.
[0115] At a rotational speed f.sub.1 of 100-1000 rpm the precise
amount of fluid contained in metering chamber 302 is delivered into
assay chamber 307. In embodiments comprising a sacrificial valve
311 in-line with capillary 310 at a position between capillary 310
and 312 shown in FIG. 5A, release of the sacrificial valve results
in fluid flow through capillary 312 and into assay chamber 307. In
said embodiments, fluid flow is achieved at rotational speed
f.sub.1 with removal of the sacrificial valve. In embodiments of
the platforms of the invention comprising capillary valve 311 at a
position between capillary 310 and 312 shown in FIG. 5B, capillary
310 preferably fills along with filling of blood metering capillary
302 until blood reaches capillary junction 311 at the junction
between capillary 310 and capillary 312; in such embodiments, the
capillary junction has a depth of from about 0.03 mm to about 2.2
mm. At a higher rotational speed f.sub.2 of about 250 rpm to about
1500 rpm, the fluid contained in blood metering capillary 302 is
delivered into assay chamber 307 (FIG. 5C).
[0116] Blood flows into assay chamber 307 with displaced air
flowing through air channel 314, and may be vented to the surface
of the disc or in communication with blood metering capillary 302
or entry port 301 (FIG. 5C). As blood flows into assay chamber 307,
the fluid component of the blood wicks into matrix 306; the pore
size of the matrix is chosen to prevent the cellular components of
the blood from entering the matrix. In an alternative embodiment,
alternative matrix 318 comprises at least two distinct elements
that are compressed or adhered to one another in the assays
chamber. The first element is similar to the reagent-containing
matrix 306 described above; however, this embodiment of the matrix
has a pore size that is not limited by the size of cellular
components of the blood, and can be any pore size deemed optimal on
experimental, economic, manufacturing or availability grounds. The
general dimensions of the matrix are equivalent to the dimensions
disclosed above, so that the matrices are advantageously
standardized and interchangeable on the microsystems platforms of
the invention. The second element comprises a filtering layer
having a pore size that prevents cellular components from entering
this portion of the matrix. In a preferred embodiment, these
elements are arranged in assay chamber 307 so that the second
matrix element is in contact with the surface of assay chamber 307
wherein the blood aliquot is first contacted with the second matrix
component. Blood fluid wicks into and through second matrix element
and into the first matrix element, whereby cellular components of
blood are prevented from entering assay chamber 307 by the pore
size of second matrix element.
[0117] As the fluid component of the blood wicks into matrix 306,
dried reagents are solubilized and the reaction of the blood
component catalyzed by said reagents proceeds as described below.
The timescale over which these reactions take place is chosen to be
long compared with the time it takes for the fluid component of
blood to saturate the matrix 306; however, in preferred
embodiments, the reaction(s) goes to completion within about 0.5 to
about 5 min. Reaction of the blood component(s) with reagents
produce colored product (FIG. 5D). After sufficient time for the
reaction to proceed, the platform is centrifuged at a speed of from
about 800 rpm to about 200 rpm, wherein said speed pellets the
cellular component of the blood, particularly the red blood cell
component thereof, to the "bottom" or most axially distal portion
of assay chamber 307. Detection of the colored product of the
glucose detecting reaction in matrix 306 is performed at a position
in assay chamber 307 radially more proximal to the axis of rotation
than the position to which the cellular fraction has been pelleted
(FIG. 5E). In preferred embodiments, detection is performed
spectrophotometrically, although visual inspection is also
contemplated in alternative embodiments of the invention. The
amount of colored product produced is detected and the amount of
glucose in the blood sample determined thereby.
[0118] Another alternative embodiment of a blood glucose assay
Microsystems platform is shown in FIGS. 6A through 6E. Construction
of the disk embodiments of the platforms of the invention were as
described above.
[0119] The blood application and metering components and their
dimensions and relationships to one another are identical to those
described above, comprising sample entry port chamber 401, metering
capillary 402, overflow capillary 403, and overflow chamber 405. As
in Example 1, each of the overflow and fluid chambers is also
connected with air ports or air channels, such as 414, and
capillary junction(s) 415, that permit venting of air displaced by
fluid movement on the platform.
[0120] Metering capillary 402 is fluidly connected to capillary 410
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm and is
connected to capillary or sacrificial valve 411. Sacrificial or
capillary valve 411 is further fluidly connected with capillary 412
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm.
Capillary 412 is further fluidly connected to cell separation
chamber 407. Where capillary 410 is connected with capillary valve
411, said capillary valve 411 is from about 0.02 mm to about 2 mm
deep and has a cross-sectional diameter of from about 0.02 mm to
about 2 mm.
[0121] Cell separation chamber 407 comprised a depression in the
surface of the platform having a depth of from about 0.02 mm to
about 3 cm, most preferably comprising a circular or rectangular
depression 413 connected to capillary 412. Cell separation chamber
also comprises a filter 406 consisting of a porous material whose
pores are sized (from about 0.2 .mu.m to about 2 .mu.m) to filter
red blood cells. Filter 406 is in contact with and more preferably
adhered to the surface of cell separation chamber 407 and most
extends over depression 413. Blood flowing into cell separation
chamber 407 is directed to depression 413 by capillary 412. As
depression 413 fills, serum is both wicked and driven by
rotation-induced pressure through the filter 406 to the upper
surface of cell separation chamber 407, leaving red blood cells and
other cellular components trapped beneath filter 406 in depression
413. The volume of blood fluid at the upper surface of cell
separation chamber 407 ranges from about 15 .mu.L to about 150
.mu.L.
[0122] Cell separation chamber 407 is fluidly connected at a radial
position distal from the axis of rotation to capillary 420 that is
from about 0.02 mm to about 2 mm deep and has a cross-sectional
diameter of from about 0.02 mm to about 2 mm. Capillary 420 is
further fluidly connected with assay chamber 421 that is from about
0.02 mm to about 3 cm deep and has a cross-sectional diameter of
from about 0.02 mm to about 10 cm. Assay chamber 421 comprises a
pad or matrix 422 of a hydrophilic substance possessing any
convenient pore size, for example, a pore size of 0.2-2.0 .mu.m,
such as a pore size of about 0.8 .mu.m. The matrix is further
impregnated with immobilized reagents which produce a detectable
product proportional to the amount or concentration of glucose in a
blood sample. Most preferably, the detectable product is a colored
product, i.e., a product absorbing light at a detectable, most
preferably a visible, wavelength.
[0123] As illustrated in FIGS. 6A through 6E, in the use of this
platform an imprecise volume (ranging from 15 to about 150 .mu.L of
fluid) of blood is applied to the entry port 401. Application of
blood to the platform and delivering a metered amount of blood to
metering capillary 402 was achieved as described in Example 1
above. At a rotational speed f.sub.1 of 100-1000 rpm, the precise
amount of fluid contained in metering capillary 402 is delivered
into assay chamber 407. Alternatively, an unmetered amount of blood
is introduced onto the platform directly into assay chamber 407,
preferably, from about 15 .mu.L to about 150 .mu.L.
[0124] Fluid movement into metering capillary 402 is accompanied by
filling of capillary 410. In embodiments comprising a sacrificial
valve 411 in-line with capillary 410 at a position between
capillary 410 and 412 shown in FIG. 6A, release of the sacrificial
valve results in blood fluid flow through capillary 412 and into
cell separation chamber 407. In said embodiments, fluid flow is
achieved at rotational speed f.sub.1 with removal of the
sacrificial valve. In embodiments of the platforms of the invention
comprising capillary valve 411 at a position between capillary 410
and 412 shown in FIG. 6A, capillary 410 preferably fills along with
filling of metering capillary 402 until blood reaches capillary
junction 411 at the junction between capillary 410 and capillary
412. In such embodiments, the capillary junction has a depth of
from about 0.03 mm to about 2.2 mm. In these embodiments, the fluid
contained in metering capillary 402 is delivered into cell
separation chamber 407 by rotation of the disc at a higher
rotational speed f.sub.2 of from about 250 rpm to about 1500 rpm
(FIG. 6B).
[0125] Blood flows into cell separation chamber 407 with displaced
air flowing through air channel 414, and may be vented to the
surface of the disc or in communication with metering capillary 402
or entry port 401 (FIG. 6C). As blood flows into cell separation
chamber 407, the fluid component of the blood wicks into matrix
406; the pore size of the matrix is chosen to prevent the cellular
components of the blood from entering the matrix. In a preferred
embodiment, matrix 406 is arranged in cell separation chamber 407
so that the matrix is in contact with or more preferably adhered to
the lower surface of cell separation chamber 407. Blood fluid, such
as plasma or serum, traverses matrix 406 by wicking and under
rotation-induced pressure, saturating the matrix and filling a
space formed between the top surface of the matrix and the top
surface of cell separation chamber 407 (FIG. 6C).
[0126] Blood fluid exits cell separation chamber 407 through
capillary 420 and flows into assay chamber 421 (FIG. 6D), with
displaced air flowing through air channel 414, and may be vented to
the surface of the disc or in communication with cell separation
chamber 407 (FIG. 5.4). Blood fluid flows into assay chamber 421
and wicks into matrix 422. As the blood fluid wicks into matrix
422, dried reagents are solubilized and the reaction of the blood
component catalyzed by said reagents proceeds as described below.
The timescale over which these reactions take place is chosen to be
long compared with the time it takes for the fluid component of
blood to saturate the matrix 422; however, in preferred
embodiments, the reaction(s) goes to completion within about 0.5
min to about 5 min. Reaction of the blood component(s) with
reagents produce a colored product (FIG. 6E). In preferred
embodiments, detection of colored product is performed
spectrophotometrically, although visual inspection is also
contemplated in alternative embodiments of the invention. The
amount of colored product produced is detected and the amount of
glucose in the blood sample determined thereby.
[0127] Another alternative embodiment of a blood glucose assay
microsystems platform is shown in FIGS. 7A-7D. Construction of the
disk embodiments of the platforms of the invention were as
described above.
[0128] The blood application and metering components and their
dimensions and relationships to one another are identical to those
described above, comprising sample entry port chamber 501, metering
capillary 502, overflow capillary 503, and overflow chamber 505. As
in Example 1, each of the overflow and fluid chambers is also
connected with air ports or air channels, such as 514, and
capillary junction(s) 515, that permit venting of air displaced by
fluid movement on the platform.
[0129] Metering capillary 502 is fluidly connected with capillary
510, having a cross-sectional diameter of from about 0.02 mm to
about 2 mm and extending from about 1 mm to about 5 cm from the
blood fluid chamber. Capillary 510 is also fluidly connected with
mixing chamber 515 that is from about 0.02 mm to about 3 cm deep
and having a cross-sectional diameter of from about 0.02 mm to
about 10 cm, and is positioned from about 1.2 cm to about 14 cm
from the center of rotation. Mixing chamber 515 is also fluidly
connected with capillary 520 having a cross-sectional diameter of
from about 0.02 mm to about 2 mm. Capillary 520 is further fluidly
connected with reagent chamber 521, wherein reagents 508 for
detecting blood glucose and determining the concentration thereof
are stored. Reagent chamber 521 is from about 0.02 mm to about 3 cm
deep and having a cross-sectional diameter of from about 0.02 mm to
about 10 cm, and is positioned from about 1.2 cm to about 14 cm
from the center of rotation. In certain embodiments, reagents 508
are stored on the disc in solution; in these embodiments, fluid
flow through capillary 520 is preferably controlled using
sacrificial valve 525. In alternative embodiments, reagents 508 are
stored in reagent chamber 521 in dry form. In these embodiments,
capillary 520 can preferably comprise sacrificial valve 525 or
capillary valve 525 positioned between mixing chamber 515 and
reagent chamber 521. In these embodiments, reagent chamber 521
further comprises means for a user to add an appropriate amount of
a reagent diluent 530, or the platform further comprises diluent
chamber 531 fluidly connected to reagent chamber 521 by way of
capillary 532. In these embodiments, reagent diluent chamber 531
that is from about 0.02 mm to about 3 cm deep and having a
cross-sectional diameter of from about 0.02 mm to about 10 cm, and
is positioned from about 1.2 cm to about 14 cm from the center of
rotation. In these embodiments, reagents 508 are solubilized in
reagent chamber 521 immediately prior to or during use.
[0130] Mixing chamber 515 is fluidly connected by capillary channel
536 having a cross-sectional diameter of from about 0.02 mm to
about 2 mm and extending from about 1 mm to about 5 cm from the
mixing chamber. Capillary channel 536 is further fluidly connected
with mixed fluid receiving chamber 537. Mixed fluid chamber 537 is
from about 0.02 mm to about 3 cm deep and having a cross-sectional
diameter of from about 0.02 mm to about 10 cm, and is positioned
from about 1.2 cm to about 14 cm from the center of rotation.
Alternatively, capillary 536 is further fluidly connected to second
mixing chamber 540. Second mixing chamber 540 is from about 0.02 mm
to about 3 cm deep and having a cross-sectional diameter of from
about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm
to about 14 cm from the center of rotation. Second mixing chamber
is fluidly connected by a capillary channel 546 which is further
connected with mixed fluid receiving chamber 537. In these
embodiments, capillary channel 546 has a cross-sectional diameter
of from about 0.02 mm to about 2 mm and extends from about 1 mm to
about 5 cm from the second mixing chamber.
[0131] Capillary channels 510 and 520, and capillary channels 536
and 546, may be offset in their connection with the mixing
chamber(s). As a consequence, fluid flowing through capillary
channels 536 and 546 is forced to encounter the opposite wall of
mixing chambers 515 and 540 before fluid flow can proceed through
further capillary channels. The fluid streams entering a small
channel flow in a laminar fashion and therefore mix only by
diffusion; the mixing chamber allows the fluids to move in a
turbulent fashion and thus mix more effectively. This results in
the creation of turbulence in the mixed laminar fluid stream in
capillary channels 536 and 546 caused by the conjoint flow of fluid
from the input capillaries without appreciable mixing. The
turbulence created by the structure of mixing chambers 515 and 540
is sufficient to disrupt laminar flow and cause fluid mixing in the
chamber prior to continued fluid flow through capillary channel 546
and into mixed fluid receiving chamber 537.
[0132] As illustrated in FIGS. 7A through 7D, in the use of this
platform a volume of blood is applied to metering capillary 502,
either directly or using the metering components of the platform
described above. Fluid enters the each of the capillaries 510 and
520 and stops at capillary junction(s) or sacrificial valve(s)
525.
[0133] At a rotational speed f.sub.1 of 100 to 1000 rpm, the fluids
from each capillary flow past capillary junction 525 and through
mixing chamber 515 (FIG. 7B). Alternatively, fluid flow is
activated by release of sacrificial valves 525. Fluid flow within
mixing chamber 515 is turbulent, in contrast to fluid flow through
capillaries 510 and 520, which is primarily laminar, so that mixing
occurs predominantly in mixing chamber 515. Fluid flow proceeds
through channel 536 and then either through second mixing chamber
540 or directly through capillary 546 into mixed fluid receiving
chamber 537 (FIG. 7C).
[0134] Glucose detection reagents mixed with blood reacts in mixed
fluid receiving chamber 537 (FIG. 7C). The timescale over which
these reactions take place preferably goes to completion within
about 0.5 min to about 5 min. Reaction of the blood component(s)
with reagents 508 produce a colored product (FIG. 7D). After
sufficient time for the reaction to proceed, the platform is
centrifuged at a speed of about 500 rpm to about 3000 rpm, wherein
said speed pellets the cellular component of the blood,
particularly the red blood cell component thereof, to the "bottom"
or most axially distal portion of mixed fluid receiving chamber
537. Detection of the colored product of the glucose detecting
reaction is performed at a position in mixed fluid receiving
chamber 537 radially more proximal to the axis of rotation than the
position to which the cellular fraction has been pelleted (FIG.
7D). In preferred embodiments, detection is performed
spectrophotometrically, although visual inspection is also
contemplated in alternative embodiments of the invention. The
amount of colored product produced is detected and the amount of
glucose in the blood sample determined thereby.
[0135] Another alternative embodiment of a blood glucose assay
Microsystems platform is shown in FIGS. 8A through 8D. Construction
of the disk embodiments of the platforms of the invention were as
described above.
[0136] The blood application and metering components and their
dimensions and relationships to one another are identical to those
described above, comprising sample entry port chamber 601, metering
capillary 602, overflow capillary 603, and overflow chamber 605. As
in Example 1, each of the overflow and fluid chambers is also
connected with air ports or air channels, such as 634, and
capillary junction(s) 635, that permit venting of air displaced by
fluid movement on the platform.
[0137] Metering capillary 602 is fluidly connected to capillary 610
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm and is
connected to capillary or sacrificial valve 611. Sacrificial or
capillary valve 611 is further fluidly connected with capillary 612
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm.
Capillary 612 is further fluidly connected to cell separation
chamber 607. Cell separation chamber 607 is from about 0.02 mm to
about 3 cm deep, has a cross-sectional diameter of from about 002
mm to about 3 cm, and is positioned from about 1.2 cm to about 14
cm from the center of rotation. Where capillary 610 is connected
with capillary valve 611, said capillary valve 611 is from about
0.03 mm to about 2.2 mm deep and has a cross-sectional diameter of
from about 0.03 mm to about 2.2 mm.
[0138] Cell separation chamber 607 comprises a depression in the
surface of the platform having a depth of from about 0.02 mm to
about 3 cm, most preferably comprising a circular or concave
depression 613 connected to capillary 612. Depression 613 has a
depth of from about 0.02 mm to about 1 cm and a volume of from 15
.mu.L to about 150 .mu.L. Cell separation chamber 607 also
comprises a filter 606 consisting of a porous material whose pores
are sized to filter red blood cells, ranging from about 0.2 .mu.m
to about 2 .mu.m. Filter 606 is in contact with and more preferably
adhered to the surface of cell separation chamber 607 and most
preferably extends over depression 613. Blood flowing into cell
separation chamber 607 is directed to depression 613 by capillary
612. As depression 613 fills, serum is both wicked and driven by
rotation-induced pressure through the filter 606 to the upper
surface of cell separation chamber 607, leaving red blood cells and
other cellular components trapped beneath filter 606 in depression
613. The volume of blood fluid at the upper surface of cell
separation chamber 607 is from about 15 .mu.L to about 150
.mu.L.
[0139] Cell separation chamber 607 is fluidly connected at a radial
position distal from the axis of rotation to capillary 620 that is
from about 0.02 mm to about 2 mm deep, has a cross-sectional
diameter of from about 0.02 mm to about 2 mm and extends from about
1 mm to about 5 cm from cell separation chamber 607. Capillary 620
is further fluidly connected with mixing chamber 615 that is from
about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of
from about 0.02 mm to about 10 cm, and is positioned from about 1.2
cm to about 14 cm from the center of rotation. Capillary 620 is
further fluidly connected with reagent chamber 621, wherein
reagents 608 for detecting blood glucose and determining the
concentration thereof is stored. Reagent chamber 621 is from about
0.02 mm to about 3 cm deep, has a cross-sectional diameter of from
about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm
to about 14 cm from the center of rotation. In certain embodiments,
reagents 608 are stored on the disc in solution; in these
embodiments, fluid flow through capillary 620 is preferably
controlled using sacrificial valve 625. In alternative embodiments,
reagents 608 are stored in reagent chamber 621 in dry form. In
these embodiments, the platform further comprises diluent chamber
631 fluidly connected to reagent chamber 621 by way of capillary
632. Diluent chamber 631 is from about 0.02 mm to about 3 cm deep,
has a cross-sectional diameter of from about 0.02 mm to about 10
cm, and is positioned from about 1.2 cm to about 14 cm from the
center of rotation. Capillary 632 has a cross-sectional diameter of
from about 0.02 mm to about 2 mm, and is positioned from about 1.2
cm to about 14 cm from the center of rotation. In these
embodiments, reagents 608 are solubilized in reagent chamber 621
immediately prior to or during use.
[0140] Mixing chamber 615 is fluidly connected by capillary channel
636 having a cross-sectional diameter of from about 0.02 mm to
about 2 mm and extending from about 1 mm to about 5 cm from the
mixing chamber. Capillary channel 636 is further fluidly connected
with mixed fluid receiving chamber 637. Mixed fluid chamber 637 is
from about 0.02 mm to about 2 mm deep, has a cross-sectional
diameter of from about 0.02 mm to about 2 mm, and is positioned
from about 1.2 cm to about 14 cm from the center of rotation.
[0141] Alternatively, capillary 636 is further fluidly connected to
second mixing chamber 640. Second mixing chamber 640 is from about
0.02 mm to about 3 cm deep, has a cross-sectional diameter of from
about 0.02 mm to about 10 cm, and is positioned from about 1.2 cm
to about 10 cm from the center of rotation. Second mixing chamber
is fluidly connected by a capillary channel 646 which is further
connected with mixed fluid receiving chamber 637. Capillary channel
646 has a cross-sectional diameter of from about 0.02 mm to about2
mm and extends from about 1 mm to about 5 cm from the second mixing
chamber.
[0142] Capillary channels 610 and 620, and capillary channels 636
and 646, may be offset in their connection with the mixing
chamber(s). As a consequence, fluid flowing through capillary
channels 636 and 646 are forced to encounter the opposite wall of
mixing chambers 615 and 640 before fluid flow can proceed through
further capillary channels. This results in the creation of
turbulence in the mixed laminar fluid stream in capillary channels
636 and 646 caused by the conjoint flow of fluid from the input
capillaries without appreciable mixing. The turbulence created by
the structure of mixing chambers 615 and 640 is sufficient to
disrupt laminar flow and cause fluid mixing in the chamber prior to
continued fluid flow through capillary channel 646 and into mixed
fluid receiving chamber 637. Mixed fluid receiving chamber 637 is
from about 0.02 mm to about 3 cm deep, has a cross-sectional
diameter of from about 0.02 mm to about 10 cm, and is positioned
from about 1.2 cm to about 14 cm from the center of rotation.
[0143] As illustrated in FIGS. 8A through 8D, in the use of this
platform a volume of blood is applied to metering capillary 602,
either directly or using the metering components of the platform
described above. Blood flows into cell separation chamber 607 with
displaced air flowing through air channel 654, and may be vented to
the surface of the disc or in communication with blood fluid
chamber 604 (FIG. 8B). As blood flows into cell separation chamber
607, the fluid component of the blood wicks into matrix 606; the
pore size of the matrix (from about 0.2 mm to about 2 .mu.m) is
chosen to prevent the cellular components of the blood from
entering the matrix. In a preferred embodiment, matrix 606 is
arranged in cell separation chamber 607 so that the matrix is in
contact with or more preferably adhered to the lower surface of
cell separation chamber 607. Blood fluid, such as plasma or serum,
traverses matrix 606 by wicking and under rotation-induced
pressure, saturating the matrix and filling a space formed between
the top surface of the matrix and the top surface of cell
separation chamber 607 (FIG. 8C).
[0144] Blood fluid exits cell separation chamber 607 through
capillary 610 and flows into mixing chamber 615. Similarly, at a
rotational speed of from about 200 rpm to about 200 rpm sufficient
to overcome capillary valve 625, or upon release of sacrificial
valve 625, solubilized reagents 608 flow through capillary 620 and
into mixing chamber 615 (FIG. 8C). Fluid flow within mixing chamber
615 is turbulent, in contrast to fluid flow through capillaries 610
and 620, which is primarily laminar, so that mixing occurs
predominantly in mixing chamber 615. Fluid flow proceeds through
channel 636 and then either through second mixing chamber 640 or
directly through capillary 646 into mixed fluid receiving chamber
637 (FIG. 8D).
[0145] Glucose detection reagents mixed with blood in mixed fluid
receiving chamber 637 (FIG. 8D). The timescale over which these
reactions take place preferably goes to completion within about 0.5
min. to about 5 min. Reaction of the blood component(s) with
reagents 608 produce a colored product (FIG. 8D). Detection of the
colored product of the glucose detecting reaction is performed in
mixed fluid receiving chamber 637. In preferred embodiments,
detection is performed spectrophotometrically, although visual
inspection is also contemplated in alternative embodiments of the
invention. The amount of colored product produced is detected and
the amount of glucose in the blood sample determined thereby.
[0146] The microfluidics structures of the invention are used to
detect the amount or concentration of a chemical species in a
solution or complex mixture, most preferably an aqueous solution or
mixture. In certain preferred embodiments, the chemical species to
be detected in glucose in blood. In one embodiment, blood glucose
in blood is detected using a hexokinase assay.
[0147] In this assay, hexokinase converts glucose to
glucose-6-phosphate (G-6-P) in the presence of magnesium cation.
G-6-P is then converted to 6-phosphogluconate (6-PG) by
glucose-6-phosphate dehydrogenase in the presence of NADP producing
a stoichiometric amount of NADPH. NADPH is oxidized by reaction
with phenazine methosulfate (PMS) as an intermediate, which is then
oxidized by reaction with indotetrotazolium chloride, which forms a
colored product that absorbs visible light at 520 nm. Optimum
reagent component concentrations vary according to the specifics of
the application of this chemical assay, as well understood and
practiced by one versed in the art. This reaction scheme is
illustrated as follows: 1
[0148] The amount of glucose in the blood fluid is directly related
to the amount of reduced indotetrotazolium chloride, and the
concentration of reduced indotetrotazolium chloride was related to
glucose concentration using light spectroscopy as described below,
as understood by those with skill in the art. All of these reagents
are preferably provided as dried reagents 108 applied to the disc,
for example, comprising matrix 106 shown in FIGS. 1 through 8.
These reagents can be lyophilized or air dried directly onto the
surface of the disk, for example by "ink-jet" methods, or can be
applied as dried beads or other particulate components.
[0149] In a variation of this reaction scheme, reduction of NADP is
assayed directly without the use of PMS or indotetrazolium
chloride. In this embodiment, the concentration of oxidized NADP is
detected spectrophotometrically by illuminating the sample with
light at 340 nm, and detecting absorbance. The amount of glucose in
the sample is inversely proportional to the amount of oxidized NADP
present in the sample after reaction. In these embodiments, the
amount of NADP must be precisely controlled to be certain that the
inverse proportionality between oxidized NADP and glucose is
maintained.
[0150] Detection is performed by transmission, reflection or
reflectance spectroscopy. In transmission spectroscopy, light at a
wavelength of 520 nm produced by a narrow band light source, most
preferably using a light emitting diode (LED) with a filter, enters
one face of an assay chamber, more preferably at a position in the
chamber that comprises a detection cell, and the light transmitted
through the detection cell is detected using a photomultiplier
tube, photodiode, photodiode array, or avalanche photodiode. The
photomultiplier tube is calibrated so that the amount of
transmitted light detected is interpreted according to Beer's law
to determine optical density and hence concentration of glucose in
the sample by the well-known linear relation between the logarithm
of the incident intensity/transmitted intensity and the
concentration of colored (absorbing) product.
[0151] Alternatively, production of colored reaction products is
detected by reflection spectroscopy. Light at a wavelength of 520
nm is produced by a narrow band light source, most preferably a
further combination with a monochromator, grating, or filter.
Monochromatic light sources such as lasers or rare gas lamps as
well as quasi-monochromatic light sources such as LEDs may also be
used and enters one face of an assay chamber, more preferably at a
position in the chamber that comprises a detection cell. In
preferred embodiments, the face of the assay chamber or detection
cell opposite to the face illuminated by the light source comprises
a reflective surface, preferably formed using a reflective
material, including but not limited to an aluminum layer, a
metallized glass, a mirror, and high gloss paint, or a diffusely
reflecting surface such as TiO.sub.2, underneath the colored fluid,
which advantageously decreases the contribution of scratches or
rotor wobble. Illuminated light is reflected back through the
detection cell at a direct or through an oblique angle and is
detected using a photomultiplier tube photo diode, photodiode
array, or avalanche photodiode.
[0152] For embodiments of the platforms of the invention wherein
the assay chamber comprises a solid or porous matrix, production of
colored reaction products is most preferably detected by a
variation on reflection spectroscopy described above. Light at a
wavelength of 520 nm is produced by a narrow band light source,
most preferably in further combination with a monochromator is
produced that enters one face of an assay chamber, more preferably
at a position in the chamber that comprises a detection cell. Light
is absorbed from the colored reaction products comprising the
matrix and is scattered from the matrix which comprises a
diffusely-scattering material. The diffusely scattered, reflected
light is detected.
[0153] In an alternative reaction protocol, glucose oxidase is used
to produce hydrogen peroxide by oxidation of glucose in the blood
sample. In this reaction scheme, glucose oxidase converts glucose
to gluconic acid and hydrogen peroxide; a twice-stoichiometric
amount of hydrogen peroxide is produced relative to the amount of
glucose present in the blood sample. The hydrogen peroxide then
oxidizes a dye precursor present in the assay chamber or preferably
within a detection cell, yielding a colored product. A variety of
dye precursors are useful in the practice of this aspect of the
invention, including but not limited to O-dianisidine, O-toluidine,
O-tolidine, benzidine, 2,2'-azinodi-(3-ethylbenzthiazoline sulfonic
acid), 3-methyl-2-benzthiazolinone hydrazone plus
N,N-dimethylaniline, phenyl plus 4-aminophenzanone, sulfonated
2,4-dichlorophenol plus 4-aminophenzanone,
3-methyl-2-benzothiazolinone hydrazone plus 3-(dimethylamino)
benzoic acid, 2-methoxy-4-allyl phenol and
4-aminoantipyrene-dimethylaniline. Optimum reagent component
concentration vary according to the specifics of the application of
this chemical assay, as well understood and practiced by one versed
in the art. This reaction scheme is illustrated as follows: 2
[0154] The amount of glucose in the blood fluid is directly related
to the amount of oxidized dye produced. The concentration of
oxidized dye produced is determined by Beers law and light
spectroscopy measurements. The total amount of oxidized dye
produced is then used to calculate the sample glucose
concentration. Alternatively and preferably, the rate of oxidized
dye production is measured and that rate related to the sample
glucose concentration. Calibration, to relate glucose concentration
to optical measurements calculated by either method above, are well
understood by one versed in this art. All of these reagents are
preferably provided as dried reagents 108 applied to the disc, for
example, comprising matrix 106 shown in FIGS. 1 through 8. These
reagents can be applied, by for example methods, including but not
limited to, filling, spraying, dipping, rolling and stamping a
solution containing reagent components followed by lyophilization
or air drying. Several applications may be made sequentially, with
different components.
[0155] The invention also provides microsystem platforms for
performing separations of particular components of a solution or
complex mixture. In particular, the invention provides disc
embodiments of the platforms of the invention comprising separation
chambers containing components or matrices that specifically bind
and retain particular chemical species comprising a chemical
solution or complex mixture. This aspect of the invention is
illustrated by a microfluidics array for separating glycated
hemoglobin from a blood sample, as shown in FIG. 9.
[0156] Construction of the disk embodiments of the platforms of the
invention were as described above. The blood application and
metering components and their dimensions and relationships to one
another are identical to those described above, comprising sample
entry port chamber 701, metering capillary 702, overflow capillary
703, and overflow chamber 705. As in Example 1, each of the
overflow and fluid chambers is also connected with air ports or air
channels, such as 754, and capillary junction(s) 755, that permit
venting of air displaced by fluid movement on the platform.
[0157] Metering capillary 702 is fluidly connected to capillary 710
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm.
Capillary 710 is further fluidly connected to mixing chamber 715
that is from about 0.02 mm to about 3 cm deep, has a
cross-sectional diameter of from about 0.02 mm to about 10 cm, and
is positioned from about 1.2 cm to about 14 cm from the center of
rotation. Lysis buffer chamber 716 containing blood lysis buffer is
from about 0.02 mm to about 3 cm deep, has a cross-sectional
diameter of from about 0.02 mm to about 10 cm, and is positioned
from about 1.2 cm to about 14 cm from the center of rotation. Lysis
buffer chamber 716 is positioned more proximally to the axis of
rotation that mixing chamber 715, and has a volumetric capacity of
from about 15 .mu.L to about 150 .mu.L of lysis buffer, composed of
0.1% Triton X100 in 50 mM Tris pH 9.5. Lysis buffer chamber 715 is
fluidly connected through capillary 718 to mixing chamber 715.
[0158] Mixing chamber 715 is fluidly connected to capillary 717
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm and is
connected to secondary metering structure 719. Secondary metering
structure 719 is from about 0.02 mm to about 3 cm deep, and is
positioned from about 1.2 cm to about 14 cm from the center of
rotation. Secondary metering structure 719 is constructed to
comprise three sections. A first metering section is arranged
proximal to the entry position of capillary 717 and is separated
from a second metering section by a septum that extends from the
distal wall of the structure to a position just short of the
proximal wall of the structure. This arrangement produces a fluid
connection between the first metering section having a volumetric
capacity of from about 5 .mu.L to about 15 .mu.L and second
metering section having a volumetric capacity of from about 5 .mu.L
to about 15 .mu.L. An overflow chamber is positioned adjacent to
the second metering section and separated by a septum that extends
from the distal wall of the structure to a position just short of
the proximal wall of the structure. This arrangement produces a
fluid connection between the second metering section and the
overflow sections of the secondary metering structure 719.
[0159] Capillary 721 is in fluid connection with secondary metering
structure 719 at the distal wall of the first metering section.
Capillary 721 is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm and is
fluidly connected to boronate affinity matrix chamber 722. Boronate
affinity matrix chamber 722 is from about 0.02 mm to about 0.3 cm
deep, has a cross-sectional diameter of from about 0.02 mm to about
10 cm and is positioned from about 1.2 cm to about 14 cm from the
axis of rotation. Boronate affinity matrix chamber 722 further
comprises 20-50 .mu.L boronate-functionalized agarose beads having
a mean diameter of about 60 .mu.m; the beads are maintained in the
chamber 722 using a porous frit 727. Fluid flow through capillary
721 is connected to capillary or sacrificial valve 723. Capillary
724 is in fluid connection with secondary metering structure 719 at
the distal wall of the second metering section. Capillary 724 is
from about 0.02 mm to about 2 mm deep and has a cross-sectional
diameter of from about 0.02 mm to about 2 mm and is connected to
read window 726. Read window 726 is from about 0.02 mm to about 3
cm deep, has a cross-sectional diameter of from about 0.02 mm to
about 10 cm and is positioned from about 1.2 cm to about 14 cm from
the axis of rotation. Read window is comprised of a material
transparent to light at a wavelength of about 430 nm. Fluid flow
through capillary 724 is connected to capillary or sacrificial
valve 725.
[0160] Boronate affinity matrix chamber 722 is further fluidly
connected to capillary 728. Capillary 728 is from about 0.02 mm to
about 2 mm deep and has a cross-sectional diameter of from about
0.02 mm to about 2 mm and is connected to column preparation buffer
reservoir 729. Column preparation buffer reservoir 729 is from
about 0.02 mm to about 3 cm deep and has a cross-sectional diameter
of from about 0.02 mm to about 10 cm and is positioned from about
1.2 cm to about 12 cm from the axis of rotation, more proximal than
boronate affinity matrix chamber 722. Column preparation buffer
reservoir 729 comprises from about 100 .mu.L to about 500 .mu.L of
column preparation buffer comprising magnesium chloride, taurine,
D,L-methionine, sodium hydroxide, antibiotics and stabilizers
(obtained from IsoLab as described in the Examples below). Fluid
flow through capillary 728 is connected to capillary or sacrificial
valve 735.
[0161] Boronate affinity matrix chamber 722 is further fluidly
connected to capillary 730. Capillary 730 is from about 0.02 mm to
about 2 mm deep and has a cross-sectional diameter of from about
0.02mm to about 2 mm and is connected to column wash buffer
reservoir 731. Column wash buffer reservoir 731 is from about 0.02
mm to about 3 cm deep and has a cross-sectional diameter of from
about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to
about 14 cm from the axis of rotation, more proximal than boronate
affinity matrix chamber 722. Column wash buffer reservoir 731
comprises from about 100 .mu.L to about 500 .mu.L of column
preparation buffer as described above. Fluid flow through capillary
730 is connected to capillary or sacrificial valve 736.
[0162] In alternative embodiments, column preparation buffer
reservoir 729 and column wash buffer reservoir 731 can be the same
reservoir, or can be fluidly connected as shown in FIG. 9A.
[0163] Boronate affinity matrix chamber 722 is further fluidly
connected to capillary 732. Capillary 732 is from about 0.02 mm to
about 2 mm deep and has a cross-sectional diameter of from about
0.02 mm to about 2 mm and is connected to read window 726.
[0164] Read window 726 is further fluidly connected to capillary
733. Capillary 733 is from about 0.02 mm to about 2 mm deep and has
a cross-sectional diameter of from about 0.02 mm to about 2 mm and
is connected to waste reservoir 734. Waste reservoir 734 is from
about 0.02 mm to about 3 cm deep and has a cross-sectional diameter
of from about 0.02 mm to about 10 cm and is positioned from about
1.2 cm to about 14 cm from the axis of rotation.
[0165] As illustrated in FIG. 9A, in the use of this platform a
volume of blood from about 15 .mu.L to about 150 .mu.L is applied
to metering capillary 702, either directly or using the metering
components of the platform described above. Blood flowing through
capillary 710 and lysis buffer flowing through capillary 718 are
mixed in mixing chamber 715 by overcoming capillary valve 711 or
release of sacrificial valve 711. A volume of lysis buffer from
about 25 .mu.L to about 90 .mu.L was mixed with the blood sample.
Fluid-flow within mixing chamber 715 is turbulent, in contrast to
fluid flow through capillaries 710 and 718, which is primarily
laminar, so that mixing occurs predominantly in mixing chamber 715.
Fluid flow proceeds through channel 717 and into secondary metering
structure 719.
[0166] The mixture of lysis buffer and blood, comprising a lysed
blood sample, flows at a rotational speed f.sub.2 from about 200
rpm to about 2000 rpm into secondary metering structure 719. The
lysed blood sample enters and fills the first section of secondary
metering structure 719. Continued lysed blood sample flow into
secondary metering structure 719 then fills the second section of
secondary metering structure 719. Any additional lysed blood sample
then empties into the overflow chamber of secondary metering
structure 719. Most preferably, a sufficient volume of lysis buffer
and blood sample is applied to the disc to fill at least the first
and second metered sections of secondary metering structure
719.
[0167] After the lysed blood sample is completely transferred to
secondary metering structure 719, capillary or sacrificial valve
735 is released, allowing from 100 .mu.L to about 500 .mu.L of
column preparation buffer to flow at rotational speed f.sub.2
through capillary 730 and into boronate affinity matrix 722.
Continued or discontinuous rotation motivates column preparation
buffer through boronate affinity matrix 722, capillary 732, read
window 726, capillary 733 and into waste reservoir 734.
[0168] After the column preparation buffer is applied to boronate
affinity matrix chamber 722, capillary or sacrificial valve 723 is
released, allowing the metered lysed blood sample from the first
metered section of secondary metering structure 719 through
capillary 721 and into boronate affinity matrix 722 and allowed to
incubate in the affinity matrix chamber for from about 0.5 to about
5 min. Capillary or sacrificial valve 736 is then released,
allowing from 100 .mu.L to about 500 .mu.L of column wash buffer to
flow at rotational speed f.sub.? through capillary 730 and into
boronate affinity matrix 722. Continued or discontinuous rotation
motivates column preparation buffer through boronate affinity
matrix 722, capillary 732 and into read window 726. During fluid
flow of wash buffer through the boronate affinity matrix chamber,
read window is preferably illuminated by light at a wavelength of
430 nm and the concentration of hemoglobin in the sample after
glycated hemoglobin has been removed by the boronate affinity
matrix is determined thereby.
[0169] Capillary or sacrificial valve 725 is released at rotational
speed f.sub.3 of from about 100-1000 rpm and the metered lysed
blood sample from the second metered section of secondary metering
structure 719 flows through capillary 724 and into read window 726.
Read window is then illuminated by light at a wavelength of 430 nm
and the concentration of hemoglobin in the sample determined
transmission, reflection, or reflectance spectroscopy. The amount
of glycated hemoglobin in the sample is determined by subtracting
the amount of hemoglobin obtained in the first reading from the
amount of hemoglobin obtained in the second reading.
[0170] An alternative embodiment of the glycated hemoglobin assay
microsystem platform of the invention is shown in FIG. 10.
Construction of the disk embodiments of the platforms of the
invention were as described above. The blood application and
metering components and their dimensions and relationships to one
another are identical to those described above, comprising sample
entry port chamber 801, metering chamber 802, overflow capillary
803, and overflow chamber 805. As in Example 1, each of the
overflow and fluid chambers is also connected with air ports or air
channels, such as 854, and capillary junction(s) 855, that permit
venting of air displaced by fluid movement on the platform.
[0171] Metering capillary 802 is fluidly connected to capillary 810
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm.
Capillary 810 is further fluidly connected to mixing chamber 815
that is from about 0.02 mm to about 3 cm deep, has a
cross-sectional diameter of from about 0.02 mm to about 10 cm, and
is positioned from about 1.2 cm to about 14 cm from the center of
rotation. Lysis buffer chamber 816 containing blood lysis buffer is
from about 0.02 mm to about 3 cm deep, has a cross-sectional
diameter of from about 0.02 mm to about 10 cm, and is positioned
from about 1.2 cm to about 14 cm from the center of rotation. Lysis
buffer chamber 816 is positioned more proximally to the axis of
rotation that mixing chamber 815, and has a volumetric capacity of
from about 15 .mu.L to about 150 .mu.L of lysis buffer, composed of
0.1% Triton X100 in 50 mM Tris pH 9.5. Lysis buffer chamber 815 is
fluidly connected through capillary 818 to mixing chamber 815.
[0172] Mixing chamber 815 is fluidly connected to capillary 817
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm and is
connected to secondary metering structure 819. Secondary metering
structure 819 is from about 0.02 mm to about 3 cm deep, and is
positioned from about 1.2 cm to about 14 cm from the center of
rotation. Secondary metering structure 819 is constructed to
comprise two sections. A metering section is arranged proximal to
the entry position of capillary 817 and is separated from an
overflow section by a septum that extends from the distal wall of
the structure to a position just short of the proximal wall of the
structure. This arrangement produces a fluid connection between the
metering section having a volumetric capacity of from about 5 .mu.L
to about 15 .mu.L and the overflow chamber.
[0173] Capillary 821 is in fluid connection with secondary metering
structure 819 at the distal wall of the first metering section.
Capillary 821 is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm and is
fluidly connected to boronate affinity matrix chamber 822. Boronate
affinity matrix chamber 822 is from about 0.02 mm to about 0.3 cm
deep, has a cross-sectional diameter of from about 0.02 mm to about
10 cm and is positioned from about 1.2 cm to about 14 cm from the
axis of rotation. Boronate affinity matrix chamber 822 further
comprises 20-50 .mu.L boronate-functionalized agarose beads having
a mean diameter of about 60 .mu.m; the beads are maintained in the
chamber 822 using a porous frit 827. Boronate affinity matrix
chamber 822 further comprises at least one surface that is
translucent to light of at least wavelength of about 430 nm,
permitting direct illumination and interrogation of the amount of
glycated hemoglobin that is bound thereto. Fluid flow through
capillary 821 is connected to capillary or sacrificial valve
23.
[0174] Boronate affinity matrix chamber 822 is further fluidly
connected to capillary 828. Capillary 828 is from about 0.02 mm to
about 2 mm deep and has a cross-sectional diameter of from about
0.02 mm to about 2 mm and is connected to column preparation buffer
reservoir 829. Column preparation buffer reservoir 829 is from
about 0.02 mm to about 3 cm deep and has a cross-sectional diameter
of from about 0.02 mm to about 10 cm and is positioned from about
1.2 cm to about 12 cm from the axis of rotation, more proximal than
boronate affinity matrix chamber 822. Column preparation buffer
reservoir 829 comprises from about 100 .mu.L to about 500 .mu.L of
column preparation buffer comprising magnesium chloride, taurine,
D,L-methionine, sodium hydroxide, antibiotics and stabilizers
(obtained from IsoLab as described in the Examples below). Fluid
flow through capillary 828 is connected to capillary or sacrificial
valve 835.
[0175] Boronate affinity matrix chamber 822 is further fluidly
connected to capillary 830. Capillary 830 is from about 0.02 mm to
about 2 mm deep and has a cross-sectional diameter of from about
0.02 mm to about 2 mm and is connected to column wash buffer
reservoir 831. Column wash buffer reservoir 831 is from about 0.02
mm to about 3 cm deep and has a cross-sectional diameter of from
about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to
about 14 cm from the axis of rotation, more proximal than boronate
affinity matrix chamber 822. Column wash buffer reservoir 831
comprises from about 100 .mu.L to about 500 .mu.L of column
preparation buffer as described above. Fluid flow through capillary
830 is connected to capillary or sacrificial valve 836.
[0176] In alternative embodiments, column preparation buffer
reservoir 829 and column wash buffer reservoir 831 can be the same
reservoir, or can be fluidly connected as shown in FIG. 10A.
[0177] Boronate affinity matrix chamber 822 is further fluidly
connected to capillary 832. Capillary 732 is from about 0.02 mm to
about 2 mm deep and has a cross-sectional diameter of from about
0.02 mm to about 2 mm. Capillary 832 is further fluidly connected
with waste reservoir 834. Waste reservoir 834 is from about 0.02 mm
to about 3 cm deep and has a cross-sectional diameter of from about
0.02 mm to about 10 cm and is positioned from about 1.2 cm to about
14 cm from the axis of rotation.
[0178] As illustrated in FIG. 10A, in the use of this platform a
volume of blood from about 15 .mu.L to about 150 .mu.L is applied
to metering capillary 802, either directly or using the metering
components of the platform described above. Blood flowing through
capillary 810 and lysis buffer flowing through capillary 818 are
mixed in mixing chamber 815 by overcoming capillary valve 811 or
release of sacrificial valve 811. A volume of lysis buffer from
about 25 .mu.L to about 90 .mu.L was mixed with the blood sample.
Fluid flow within mixing chamber 815 is turbulent, in contrast to
fluid flow through capillaries 810 and 818, which is primarily
laminar, so that mixing occurs predominantly in mixing chamber 815.
Fluid flow proceeds through channel 817 and into secondary metering
structure 819.
[0179] The mixture of lysis buffer and blood, comprising a lysed
blood sample, flows at a rotational speed f.sub.2 from about 200
rpm to about 2000 rpm into secondary metering structure 819. The
lysed blood sample enters and fills the metering section of
secondary metering structure 819. Any additional lysed blood sample
then empties into the overflow chamber of secondary metering
structure 819. Most preferably, a sufficient volume of lysis buffer
and blood sample is applied to the disc to fill at least the
metered sections of secondary metering structure 819.
[0180] After the lysed blood sample is completely transferred to
secondary metering structure 819, capillary or sacrificial valve
835 is released, allowing from 100 .mu.L to about 500 .mu.L of
column preparation buffer to flow at rotational speed f.sub.2
through capillary 830 and into boronate affinity matrix 822.
Continued or discontinuous rotation motivates column preparation
buffer through boronate affinity matrix 822, capillary 832, and
into waste reservoir 834.
[0181] After the column preparation buffer is applied to boronate
affinity matrix chamber 822, capillary or sacrificial valve 823 is
released, allowing the metered lysed blood sample from the first
metered section of secondary metering structure 819 through
capillary 821 and into boronate affinity matrix 822 and allowed to
incubate in the affinity matrix chamber for from about 0.5 to about
5 min. Capillary or sacrificial valve 836 is then released,
allowing from 100 .mu.L to about 500 .mu.L of column wash buffer to
flow at rotational speed f.sub.? through capillary 830 and into
boronate affinity matrix 822. Continued or discontinuous rotation
motivates column preparation buffer through boronate affinity
matrix 822, capillary 732 and into waste reservoir 834. During
fluid flow of wash buffer through the boronate affinity matrix
chamber, the chamber is preferably illuminated by light at a
wavelength of 430 nm through the translucent portion of the
chamber. The concentration of glycated hemoglobin in the sample is
determined thereby.
[0182] An alternative embodiment of the glycated hemoglobin assay
microsystem platform of the invention is shown in FIG. 11.
Construction of the disk embodiments of the platforms of the
invention were as described above. The blood application and
metering components and their dimensions and relationships to one
another are identical to those described above, comprising sample
entry port chamber 901, metering capillary 902, overflow capillary
903, and overflow chamber 905. As in Example 1, each of the
overflow and fluid chambers is also connected with air ports or air
channels, such as 954, and capillary junction(s) 955, that permit
venting of air displaced by fluid movement on the platform.
[0183] Metered capillary 902 is fluidly connected to capillary 910
that is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm and is
connected to sacrificial wax valve 911. Sacrificial wax valve 911
is further fluidly connected with capillary 912 that is from about
0.03 mm to about 2.2 mm. Capillary 912 is further fluidly connected
to blood lysis chamber 915 that is from about 0.02 mm to about 3 cm
deep, has a cross-sectional diameter of from about 0.02 mm to about
10 cm, is positioned from about 1.2 cm to about 14 cm from the
center of rotation, and contains from about 25 .mu.L to about 90
.mu.L of blood lysis solution (0.1% Triton-X100 in 50 mM Tris, pH
9.5). Blood lysis chamber 915 is further fluidly connected with
capillary 918 that is from about 0.02 mm to about 2 mm deep and has
a cross-sectional diameter of from about 0.02 mm to about 2 mm and
is connected to sacrificial wax valve 913. Sacrificial wax valve
913 is further fluidly connected with wax recrystallization chamber
914 that is from about 0.02 mm to about 2 mm deep and has a
volumetric capacity sufficient to sequester melted wax from a
released wax valve and prevent occlusion of the lumen of the
capillary 918 controlled by the valve.
[0184] Capillary 918 is further fluidly connected to secondary
metering structure 919. Secondary metering structure 919 is from
about 0.02 mm to about 3 cm deep, and is positioned from about 1.2
cm to about 14 cm from the center of rotation. Secondary metering
structure 919 is constructed to comprise three sections. A first
section comprises a throwaway section sample having a volumetric
capacity of from about 5 .mu.L to about 10 .mu.L because it is
thought that by taking the second section a more representative
would be obtained. This throwaway section is arranged proximal to
the entry position of capillary 918 and is separated from a
metering section by a septum that extends from the distal wall of
the structure to a position just short of the proximal wall of the
structure. This arrangement produces a fluid connection between the
throwaway section and the metering section. The metering section
has a volumetric capacity of from about 5 .mu.L to about 10 .mu.L
and is fluidly connected to an overflow section having an excess
volumetric capacity of from about 15 .mu.L to about 150 .mu.L. The
volumetric capacity of the overflow section is sufficient to
accommodate the largest blood fluid volume applied to the disk.
[0185] Capillary 921 is in fluid connection with secondary metering
structure 919 at the distal wall of the metering section. Capillary
921 is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm and is
connected to boronate affinity matrix chamber 922. Boronate
affinity matrix chamber 922 is from about 0.02 mm to about 3 cm
deep, has a cross-sectional diameter of from about 0.02 mm to about
10 cm and is positioned from about 1.2 cm to about 14 cm from the
axis of rotation. Boronate affinity matrix chamber 922 further
comprises 10-50 .mu.L of boronate-functionalized agarose beads
having a mean diameter of about 60 cm; the beads are maintained in
the chamber 922 using a porous frit 927. Fluid flow through
capillary 921 is connected to sacrificial valve 923. Sacrificial
wax valve 923 is further fluidly connected with wax
recrystallization chamber 924 that is from about 0.03 mm to about
2.2 mm deep and has a volumetric capacity sufficient to sequester
melted wax from a released wax valve and prevent occlusion of the
lumen of the capillary 923 controlled by the valve.
[0186] Boronate affinity matrix chamber 922 is further fluidly
connected to capillary 928. Capillary 928 is from about 0.02 mm to
about 2 mm deep and has a cross-sectional diameter of from about
0.02 mm to about 2 mm and is connected to column wash buffer
reservoir 929. Column wash buffer reservoir 929 is from about 0.02
mm to about 3 cm deep and has a cross-sectional diameter of from
about 0.02 mm to about 10 cm and is positioned from about 1.2 cm to
about 14 cm from the axis of rotation, more proximal than boronate
affinity matrix chamber 922. Column wash buffer reservoir 929
comprises from about 100 .mu.L to about 500 .mu.L of column wash
buffer as described above. Fluid flow through capillary 928 is
connected to sacrificial valve 936. Sacrificial wax valve 936 is
further fluidly connected with wax recrystallization chamber 937
that is from about 0.03 mm to about 2.2 mm deep and has a
volumetric capacity sufficient to sequester melted wax from a
released wax valve and prevent occlusion of the lumen of the
capillary 928 controlled by the valve.
[0187] Boronate affinity matrix chamber 922 is further fluidly
connected to capillary 932. Capillary 932 is from about 0.02 mm to
about 2 mm deep and has a cross-sectional diameter of from about
0.02 mm to about 10 cm and is connected to sample collection
cuvette array 934. Sample collection cuvette array 934 is from
about 0.02 mm to about 3 cm deep and has a cross-sectional diameter
of from about 0.02 mm to about 10 cm and is positioned from about
1.2 cm to about 14 cm from the axis of rotation. Sample collection
cuvette array 934 is separated into a multiplicity of individual
chambers, each separated from one another by septa that extend from
the distal wall of the cuvettes to a position adjacent to the
proximal wall of the cuvettes, so that a fluid passage 950 is
maintained between each of the cuvettes. The fluid passage 950 is
formed by the back (proximal wall) of the sample collection cuvette
array 934 and the row of septa separating each of the sections of
the sample collection cuvettes 934. Capillary 932 is fluidly
connected to sample collection cuvette array 934 at a position
adjacent to the proximal wall of the array and directed to the
cuvette most proximal to the boronate affinity matrix chamber 922.
Alternatively, the septa can be eliminated in sample collection
cuvette array 934, wherein the sample cuvette is a single
chamber.
[0188] Capillary 941 is fluidly connected to secondary metering
structure 919. Capillary 941 is from about 0.02 mm to about 2 mm
deep and has a cross-sectional diameter of from about 0.02 mm to
about 2 mm and is connected to secondary metering structure 919 at
a position between the metering section and the overflow section.
Capillary 941 is further fluidly connected with total hemoglobin
read chamber 942. Total hemoglobin read chamber 942 is from about
0.02 mm to about 3 cm deep, is positioned from about 1.2 cm to
about 14 cm from the center of rotation, and has a volumetric
capacity of from about 50 .mu.L to about 250 .mu.L. Total
hemoglobin read chamber 942 is positioned radially more distal from
the center of rotation than secondary metering structure 919, and
comprises a read window translucent to light having a wavelength of
from about 430 nm. In addition, there is no capillary or
sacrificial valving controlling fluid flow in capillary 941.
[0189] The platform also comprises control sample read cuvettes 943
and 944, advantageously positioned in proximity to total hemoglobin
read chamber 942. Control sample read cuvettes 943 and 944 are each
from about 0.02 mm to about 3 cm deep, positioned from about 1.2 cm
to about 14 cm from the center of rotation, and have a volumetric
capacity of from about 50 .mu.L to about 200 .mu.L. Control sample
read cuvettes 943 and 944 comprise a read window translucent to
light having a wavelength of from about 430 nm. Control sample read
cuvettes 943 and 944 are not fluidly connected to any other
structure on the platform and contain standards and/or calibration
reagents.
[0190] As illustrated in FIG. 11, in the use of this platform a
volume of blood from about 15 .mu.L to about 150 .mu.L is applied
to metering capillary 902, either directly or using the metering
components of the platform described above. Release of sacrificial
valve 911 and rotation of the platform at a rotational speed
f.sub.1 of from about 50 rpm to about 1000 rpm motivates blood flow
through capillary 910 and into blood lysis chamber 915. The mixture
of blood and blood lysis buffer in blood lysis chamber 915 is mixed
by agitation, wherein the platform is accelerated repeatedly from
about +2000 rpm/sec to -2000 rpm/sec (wherein "+" and "-" indicate
rotation in different directions) over a time period of about 30
sec to about 600 sec. Release of sacrificial valve 913 and rotation
of the platform at a rotational speed f.sub.2 of from about 200 rpm
to about 2000 rpm motivates the lysed blood sample to flow through
capillary 918 and into secondary metering structure 919. Continued
rotation motivates lysed blood solution to fill the metering
section of secondary metering structure 919; after filling of this
section of the structure, excess lysed blood sample flows through
capillary 941 and into total hemoglobin read chamber 942. After
filling of total hemoglobin read chamber 942, any excess lysed
blood sample is displaced into the overflow section of secondary
metering structure 919.
[0191] Release of sacrificial valve 923 and rotation of the
platform at a rotational speed f.sub.3 of from about 200 rpm to
about 2000 rpm motivates the metered lysed blood sample in the
metering section of secondary metering structure 922 to flow
through capillary 921 and into boronate affinity matrix chamber
922. After incubation of the metered lysed blood sample 956 in
boronate affinity matrix chamber 922, sacrificial valve 936 is
released and the platform is rotated at a rotational speed f.sub.4
of from about 200 rpm to about 3000 rpm. Column wash buffer flows
from column wash buffer reservoir 929 through capillary 921 and
into boronate affinity matrix chamber 922, displacing the unbound
hemoglobin fraction through capillary 932 and into sample
collection cuvette array 934. Continued rotation of the platform
displaces the collected sample sequentially into the separate
cuvettes radially away from the position of boronate affinity
matrix chamber 922 on the platform. Sample collection cuvette array
934 is then interrogated by illumination with light at a wavelength
of 430 nm, and the concentration of non-glycated hemoglobin in the
blood sample determined. Additionally, illumination of total
hemoglobin read chamber 942 with light at a wavelength at 415 nm is
performed to determine the concentration of total hemoglobin in the
blood sample. The amount of glycated hemoglobin is calculated by
subtracting the amount of non-glycated hemoglobin in the sample
from the total hemoglobin concentration in the sample. Control
sample read cuvettes 943 and 944 are used to calibrate the
spectrophotometric readings.
[0192] In alternative embodiments of these microfluidics systems,
the boronate affinity matrix is replaced by other substances
capable of differentially binding glycated or non-glycated
hemoglobin species. In a first embodiment of such an alternative,
m-aminophenylboronate polyacrylic acid is used to derivatize a
positively-charged nylon 66 membrane (such as Biodyne-B, Pall
Biosupport Division, Port Washington, N.Y.). This membrane is used
in substitution for the boronate-functionalized agarose beads in
the boronate affinity matrix chambers of the invention. In
preferred embodiments, the boronate affinity matrix chambers are
modified to contain the membrane in contact with or more preferably
adhered to the platform surface within the chamber, so that one
face of the membrane derivatized with m-aminophenylboronate
polyacrylic acid is in contact with the lysed blood sample. Binding
of glycated hemoglobin to the membrane is quantitated by visible
light reflectance spectroscopy at a wavelength of 415 nm.
[0193] A second alternative embodiment of the glycated hemoglobin
microsystems assays of the invention comprises inositol
hexaphosphate. In these embodiments, inositol hexaphosphate is
attached (covalently or by electrostatic interactions) to a solid
support, including but not limited to beads, membranes, pads, etc.
The lysed blood sample is treated with sodium dithionite to convert
it to the deoxy form. In the Microsystems platforms of the
invention, an effective amount of sodium dithionite is provided
with the lysis buffer, or as a component of the secondary metering
structures, most preferably as a dry powder coating on the walls of
one or both of the metering sections thereof. The deoxygenated
lysed blood sample is then placed in contact with the solid support
comprising inositol hexaphosphate, preferably comprising and in
substitution for the boronate affinity matrix chamber of the
glycated hemoglobin platforms of the invention. In these
embodiments, the portion of hemoglobin that does not bind to the
inositol phosphate-containing solid support is the glycated
fraction, which can be delivered to a reads chamber, cuvette or
other optically-appropriate component of the platform and the
amount of glycated hemoglobin determine directly by visible light
reflectance spectrophotometry at a wavelength of 415 nm.
[0194] The invention also provides microsystem platforms for
performing a multiplicity of reactions including identification of
chemical species from solutions or complex mixtures and separations
of particular components of a solution or complex mixture. This
aspect of the invention is illustrated by a microfluidics array for
determining glucose concentration and separating glycated
hemoglobin from a blood sample, as shown in FIGS. 12A through 12Q
and 13A through 13D.
[0195] Construction of the disk embodiments of the platforms of the
invention were as described above. FIG. 13B shows a detailed
description of the microfluidics components of the platform, which
are described in additional detail below. FIG. 13C shows the
geometry of a screen printed electrical lead layer deposited on a
mylar substrate. FIG. 13D shows the positions of screen printed
heaters activated by the electrical leads of the lead layer and
screen printed on mylar. FIG. 13E shows a overlay of these
components in the assembled disc.
[0196] Referring to the microfluidics components of the platform
shown in FIG. 13B, an entry port 1 is positioned on the top surface
of the disc and is open for the user to apply an unmetered sample.
Entry port 1 is from about 0.02 mm to about 3 cm deep, has a
cross-sectional diameter of from about 0.02 mm to about 10 cm, is
positioned from about 1.2 cm to about 14 cm from the center of
rotation, and is fluidly connected to capillary channel 1A that is
from about. 0.02 mm to about 2 mm deep and has a cross-sectional
diameter of from about 0.02 mm to about 2 mm. Capillary channel 1A
is fluidly connected to metering component 2, which comprises four
sections. The first section is rectangularly-shaped and extends in
a direction proximal to the axis of rotation away from its fluid
connection with capillary channel 1A. About half way up this
rectangular section is a lateral chamber 3, which empties into a
blood glucose metering chamber 4 and an overflow chamber 5. The
first section of the metering component is from about 0.02 mm to
about 3 cm deep, has a cross-sectional diameter of from about 0.02
mm to about 10 cm, is positioned from about 1.2 cm to about 14 cm
from the center of rotation, and has a volumetric capacity of from
about 15 .mu.L to about 150 .mu.L. The lateral chamber 3 is from
about 0.02 mm to about 3 cm deep, has a cross-sectional diameter of
from about 0.02 mm to about 10 cm, and is positioned from about 1.2
cm to about 14 cm from the center of rotation. Blood glucose
metering chamber 4 is from about 0.02 mm to about 3 cm deep, has a
cross-sectional diameter of from about 0.02 mm to about 10 cm, is
positioned from about 1.2 cm to about 14 cm from the center of
rotation and has a volumetric capacity of from about 1 .mu.L to
about 15 .mu.L. Overflow chamber 5 is from about 0.02 mm to about 3
cm deep, has a cross-sectional diameter of from about 0.02 mm to
about 10 cm, is positioned from about 1.2 cm to about 14 cm from
the center of rotation and has a volumetric capacity of from about
5 .mu.L to about 50 .mu.L.
[0197] Overflow chamber 5 is fluidly connected to overflow channel
8 that is from about 0.02 mm to about 3 cm deep, has a
cross-sectional diameter of from about 0.02 mm to about 10 cm and
extends from about 10 mm to about 5 cm from overflow chamber 5.
Overflow channel 5 is fluidly connected to short sample detection
cuvette 9 that is from about 0.02 mm to about 3 cm deep, has a
cross-sectional diameter of from about 0.02 mm to about 10 cm, is
positioned from about 1.2 cm to about 10 cm from the center of
rotation and has a volumetric capacity of from about 15 .mu.L to
about 150 .mu.L Blood glucose metering chamber 4 is fluidly
connected to capillary 15 that is from about 0.02 mm to about 3 cm
deep, has a cross-sectional diameter of from about 0.02 mm to about
10 cm and extends from about 1 mm to about 5 cm from blood glucose
metering chamber 4. Capillary 15 is connected to sacrificial wax
valve 6, which is further fluidly connected with wax
recrystallization chamber 6A that is from about 0.03 mm to about
2.2 mm deep and has a volumetric capacity sufficient to sequester
melted wax from a released wax valve and prevent occlusion of the
lumen of the capillary 15 controlled by the valve. Capillary 15 is
further fluidly connected with glucose assay chamber 11 that is
from about 0.02 mm to about 3 cm deep, has a cross-sectional
diameter of from about 0.02 mm to about 10 cm, is positioned from
about 1.2 cm to about 14 cm from the center of rotation and has a
volumetric capacity of from about 5 .mu.L to about 50 .mu.L.
Glucose assay chamber 11 comprises a depression 11A in the surface
of the platform having a depth of from about 0.02 mm to about 3 cm,
most preferably comprising a circular or concave depression
connected to capillary 15 so that blood flows into the chamber
through the bottom of depression 11A. Depression 11A is constructed
to have a volumetric capacity of from half to twice the assay
volume. Blood glucose assay chamber 11 also comprises a pad or
matrix 10 of a hydrophilic substance possessing a pore size of
0.2-2.0 .mu.m, most preferably comprising a positively-charged
nylon matrix having a pore size of about 0.8 .mu.m. The upper limit
on pore size of matrix 10 is chosen to inhibit or prevent blood
cell entry into the matrix. The matrix is positioned in blood
glucose assay chamber 11 to be in fluidic contact with depression
11A, more preferably covering depression 11A, and most preferably
having a surface area greater than the surface area of depression
11A. The matrix was further impregnated with immobilized reagents
11B which produce a detectable product proportional to the amount
or concentration of glucose in a blood sample. Most preferably, the
detectable product is a colored product 11C, i.e., a product
absorbing light at a detectable, most preferably a visible,
wavelength.
[0198] Lysis metering chamber 2 is fluidly connected to capillary 7
controlled by sacrificial valve 7A. Sacrificial wax valve 7A is
further fluidly connected with wax recrystallization chamber 7B
that is from about 0.03 mm to about 2.2 mm deep and has a
volumetric capacity sufficient to sequester melted wax from a
released wax valve and prevent occlusion of the lumen of the
capillary 7 controlled by the valve. Capillary 7 is from about 0.02
mm to about 2 mm deep, has a cross-sectional diameter of from about
0.02 mm to about 2 mm, extends from about 1 mm to about 5 cm from
lysis metering chamber 2 and is fluidly connected to blood lysis
chamber 16. Blood lysis chamber 16 is from about 0.02 mm to about 3
cm deep, has a cross-sectional diameter of from about 0.02 mm to
about 10 cm, is positioned from about 1.2 cm to about 14 cm from
the center of rotation, and contains from about 25 .mu.L to about
90 .mu.L of blood lysis solution (0.1% Triton-X100 in 50 mM Tris,
pH 9.5).
[0199] Blood lysis chamber 16 is fluidly connected at a distal
aspect to capillary 17 controlled by sacrificial valve 18.
Capillary 17 is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm
Sacrificial wax valve 18 is further fluidly connected with wax
recrystallization chamber 18A that is from about 0.03 mm to about
2.2 mm deep and has a volumetric capacity sufficient to sequester
melted wax from a released wax valve and prevent occlusion of the
lumen of the capillary 17 controlled by the valve. Capillary 17 is
fluidly connected to secondary metering structure 19. Secondary
metering structure 19 is from about 0.02 mm to about 3 cm deep, and
is positioned from about 1.2 cm to about 14 cm from the center of
rotation. Secondary metering structure 19 is constructed to
comprise three sections. A first section 20 comprises a throwaway
section having a volumetric capacity of from about 5 .mu.L to about
10 .mu.L because it is thought that a more representative sample
would be obtained thereby. Throwaway section 20 is arranged
proximal to the entry position of capillary 17 and is separated
from a metering section 21 by a septum that extends from the distal
wall of the structure to a position just short of the proximal wall
of the structure. This arrangement produces a fluid connection
between throwaway section 20 and the metering section 21. Metering
section has a volumetric capacity of from about 5 .mu.L to about 10
.mu.L and is fluidly connected to an overflow section 24 having an
excess volumetric capacity of from about 15 .mu.L to about 150
.mu.L. The volumetric capacity of the overflow section is
sufficient to accommodate the largest blood fluid volume applied to
the disk.
[0200] Capillary 25 is in fluid connection with secondary metering
structure 21 at the distal wall of the metering section. Capillary
25 is from about 0.02 mm to about 2 mm deep and has a
cross-sectional diameter of from about 0.02 mm to about 2 mm and is
connected to boronate affinity matrix chamber 28. Capillary 25 is
fluidly connected to sacrificial wax valve 26 that is further
fluidly connected with wax recrystallization chamber 26A Wax
recrystallization chamber 26A is from about 0.03 mm to about 2.2 mm
deep and has a volumetric capacity sufficient to sequester melted
wax from a released wax valve and prevent occlusion of the lumen of
the capillary 25 controlled by the valve.
[0201] Boronate affinity matrix chamber 28 is from about 0.02 mm to
about 3 cm deep, has a cross-sectional diameter of from about 0.02
mm to about 10 cm and is positioned from about 1.2 cm to about 14
cm from the axis of rotation. Boronate affinity matrix chamber 28
further comprises boronate-functionalized agarose beads having a
mean diameter of about 60 .mu.m; the beads are maintained in the
chamber 28 using a porous frit 29. Boronate affinity matrix chamber
28 is further fluidly connected to capillary 31. Capillary 31 is
from about 0.02 mm to about 2 mm deep and has a cross-sectional
diameter of from about 0.02 mm to about 2 mm and is connected to
column wash buffer reservoir 30. Column wash buffer reservoir 30 is
from about 0.02 mm to about 3 cm deep and has a cross-sectional
diameter of from about 0.02 mm to about 10 cm and is positioned
from about 1.2 cm to about 14 cm from the axis of rotation, more
proximal than boronate affinity matrix chamber 28. Column wash
buffer reservoir 30 comprises from about 250 .mu.L to about 350
.mu.L of column wash buffer as described above. Fluid flow through
capillary 31 is connected to sacrificial valve 32. Sacrificial wax
valve 32 is further fluidly connected with wax recrystallization
chamber 32A that is from about 0.03 mm to about 2.2 mm deep and has
a volumetric capacity sufficient to sequester melted wax from a
released wax valve and prevent occlusion of the lumen of the
capillary 32 controlled by the valve.
[0202] Boronate affinity matrix chamber 28 is further fluidly
connected to capillary 37. Capillary 37 is from about 0.02 mm to
about 2 mm deep and has a cross-sectional diameter of from about
0.02 mm to about 2 mm and is connected to sample collection cuvette
array 12. Sample collection cuvette array 12 is from about 0.02 mm
to about 3 cm deep and has a cross-sectional diameter of from about
0.02 mm to about 10 cm and is positioned from about 1.2 cm to about
14 cm from the axis of rotation. Sample collection cuvette array 12
is separated into a multiplicity of individual chambers, each
separated from one another by septa that extend from the distal
wall of the cuvettes to a position adjacent to the proximal wall of
the cuvettes, so that a fluid passage 50 is maintained between each
of the cuvettes. The fluid passage 50 is formed by the back
(proximal wall) of the sample collection cuvette array 12 and the
row of septa separating each of the sections of the sample
collection cuvettes 12. Capillary 33 is fluidly connected to sample
collection cuvette array 12 at a position adjacent to the proximal
wall of the array and directed to the cuvette most proximal to the
boronate affinity matrix chamber 28. In alternative embodiments,
collection cuvette array 12 can be constructed without such septa,
and this structure is then just a single collection chamber.
[0203] Capillary 22 is fluidly connected to secondary metering
structure 19. Capillary 22 is from about 0.02 mm to about 2 mm deep
and has a cross-sectional diameter of from about 0.02 mm to about 2
mm and is connected to secondary metering structure 19 at a
position between the metering section and the overflow section.
Capillary 22 is further fluidly connected with total hemoglobin
read chamber 23. Total hemoglobin read chamber 23 is from about
0.02 mm to about 3 cm deep, is positioned from about 1.2 cm to
about 14 cm from the center of rotation, and has a volumetric
capacity of from about 5 .mu.L to about 100 .mu.L. Total hemoglobin
read chamber 23 is positioned radially more distal from the center
of rotation than secondary metering structure 19, and comprises a
read window translucent to light having a wavelength of from about
400 nm to about 950 nm. In addition, there is no capillary or
sacrificial valving controlling fluid flow in capillary 23.
[0204] The platform also comprises control sample read cuvettes 13
and 14, advantageously positioned in proximity to total hemoglobin
read chamber 23. Control sample read cuvettes 13 and 14 are each
from about 0.02 mm to about 3 cm deep, positioned from about 1.2 cm
to about 14 cm from the center of rotation, and have a volumetric
capacity of from about 5 .mu.L to about 100 .mu.L. Control sample
read cuvettes 13 and 14 comprise a read window translucent to light
having a wavelength of from about 400 nm to about 950 nm. Control
sample read cuvettes 13 and 14 are not fluidly connected to any
other structure on the platform.
[0205] Air displacement channels 33 and capillary junction(s) 34,
that permit venting of air displaced by fluid movement on the
platform, are fluidly connected to the components of the platform
to permit unimpeded fluid flow.
[0206] As illustrated in FIGS. 12A through 12Q, in the use of this
platform a volume of blood from about 15 .mu.L to about 150 .mu.L
is applied to entry port 1. Blood enters lysis subvolume 2 and
lateral passageway 3 under the influence of gravity and capillary
forces in the absence of rotation of the platform, as shown in FIG.
12A. Upon rotation of the platform at a first rotational speed
f.sub.1 of from about 50 rpm to about 1000 rpm, blood completely
fills lysis subvolume 2 and also flows through passageway 3 and
into blood glucose metering chamber 4 and overflow chamber 5, shown
in FIG. 12B. Blood is retained in blood glucose chamber 4 either
due to capillary pressure or by a sacrificial valve 6, most
preferably a wax valve. Similarly, blood is retained in blood lysis
subvolume 2 by a valve, most preferably a sacrificial valve 7.
Excess blood flows at rotational speed f.sub.1 through overflow
channel 8 and into overflow chamber 9, shown in FIGS. 12C and 12D.
Typical values for the first rotational speed are an acceleration
of about 20 to about 60 rpm/sec to a final radial velocity of about
600 rpm.
[0207] After blood is metered and excess blood delivered to
overflow chamber 9, the rotational speed of the disc is reduced to
a rotational speed f.sub.1a of from about 0 rpm to about 500 rpm,
typically to about 60 rpm, to perform a blanking measurement on
sample collection cuvette array 12, total hemoglobin read chamber
23 and blood glucose assay chamber 11. Measurements of blanking
cuvettes 13 and 14 are also advantageously performed.
[0208] The disc is then accelerated to a second rotational speed
f.sub.2 of about 200 rpm to about 2000 rpm, greater than f.sub.1,
and typically in the range of from about 800 rpm to about 1000 rpm.
At this speed, capillary valve 6 is overcome or sacrificial valve 6
is released, and from about 1 .mu.L to about 50 .mu.L of blood from
blood glucose metering chamber 4 flows through capillary 15 and
into blood glucose assay chamber 11, shown in FIG. 12E. Upon
entering assay chamber 11, blood fluid components are forced into
absorbent matrix 10 through depression 11A. The blood fluid is
incubated in matrix 10 for a time sufficient for the reagents 10A
to produce a colored product 10B in an amount proportional to the
amount of glucose in the blood fluid sample. The disc is slowed,
typically to a rotational speed f.sub.4 of from about 0 rpm to
about 500 rpm, typically about 100 rpm, for glucose data
acquisition using reflectance spectrometry; data acquisition as the
disc is spinning down also enables to instrument to set t=0 for the
assay, based on a decrease in reflectance when the matrix 10 is wet
by the blood fluid components and hence the matrix's scattering
decreases. Development of colored product 10B is shown in FIG.
12G.
[0209] The disc is then accelerated to rotational speed f.sub.3 of
about 500 rpm to about 3000 rpm, typically about 1000 rpm, with
release of sacrificial valve 7A and fluid flow of from about 1
.mu.L to about 50 .mu.L, typically about 5 .mu.L of blood from
metered subvolume 2 through capillary 7 and into blood lysis
chamber 16 containing from about 25 .mu.L to about 90 .mu.L,
typically about 45 .mu.L of blood lysis buffer. This is shown in
FIG. 12H. The mixture of blood and blood lysis buffer in blood
lysis chamber 16 is mixed by agitation, wherein the platform is
accelerated repeatedly from about +2000 rpm/sec to -2000 rpm/sec
(wherein + and - indicate rotation in different directions),
typically from about 250-500 rpm/sec, over a time period of about
30 seconds to about 5 min, typically 1-2 min, as shown in FIGS. 12I
and 12J.
[0210] The disc then is accelerated to a rotational speed f.sub.5
from about 200 rpm to about 2000 rpm and typically about 750 rpm,
and sacrificial valve 18 is released. Lysed blood from blood lysis
chamber 16 flows through capillary 17 and into secondary metering
structure 19, as shown in FIG. 12K. The lysed blood solution
sequentially fills throwaway section 20, which is used as a trap
for cell debris, metering section 21 and excess lysed blood then
fills overflow section 24. Filling of metering chamber 21 is
immediately followed by fluid flow through capillary 22 and filling
of total hemoglobin read chamber 23. The disc is spun at rotational
speed f.sub.5 for a time sufficient to substantially completely
drain blood lysis chamber 16. The configuration of the blood fluids
on the disc after this spin is shown in FIG. 12L.
[0211] The disc is then accelerated to a rotational speed f.sub.?
from about 200 rpm to about 3000 rpm and typically about 750 rpm,
and sacrificial valve 26 is released. A metered volume of about 1
.mu.L to about 50 .mu.L, typically about 6 .mu.L of lysed blood
from metering section 21 flows through capillary 27 and into
boronate affinity matrix chamber 28 (shown in FIG. 12M). The lysed
blood solution is allowed to incubate in the chamber for a time
from about 30 seconds to about 5 min, typically about 1 min,
sufficient for glycated hemoglobin to bind to the matrix. This
aspect of the disc is illustrated in FIG. 12N.
[0212] The disc is then accelerated to a rotational speed f.sub.?
from about 500 rpm to about 3000 rpm and typically about 1000 rpm,
and sacrificial valve 32 is released. A volume of about 250 .mu.L
to about 350 .mu.L, typically about 290 .mu.L of column wash buffer
as described above flows from wash buffer reservoir 30 though
capillary 31 and into boronate affinity matrix chamber 28 (shown in
FIG. 12O). The wash buffer displaces the non-glycated hemoglobin
and other components of the lysed blood fluid from the affinity
column matrix and into sample collection cuvette array 12. FIGS.
12P through 12Q show sequential filling of the individual cuvettes
in sample collection cuvette array 12. The rotation speed of the
disc is reduced, to from about 0 rpm to about 500 rpm and typically
to about 60 rpm for sample collection cuvette array 12 and total
hemoglobin read chamber 23 to be interrogated
spectrophotometrically. The glycated fraction of the blood sample
is determine algorithmically by subtracting the non-glycated
hemoglobin fraction in sample collection cuvette array 12 from the
total hemoglobin detected in total hemoglobin read chamber 23.
2. Resistive Heater and Temperature Sensing Components
[0213] Temperature control elements are provided to control the
temperature of the platform. The invention provides heating
elements, specifically resistive heating elements, and elements for
detecting temperature at specific positions on the platform.
Heating devices are preferably arrayed to control the temperature
of the platform over a particular and defined area, and are
provided having a steep temperature gradient with distance on the
platform from the heater.
[0214] Certain resistors, including commercially-available
resistive inks (available from Dupont) exhibit a positive
temperature coefficient (PTC), i.e., an increase in resistance with
increasing temperature. Applying a fixed voltage across a PTC
resistor screen-printed on a plastic substrate results in rapid
heating, followed by self-regulation at an elevated temperature
defined by the circuit design heat sink and ambient temperature. In
such screen-printed resistors, connection to a power source is made
by first printing parallel silver conductors followed by printing
the PTC ink between the conductors.
[0215] A resistive heating element comprises a conductive ink
connected with electrical contacts for activation of the heater,
and resistive inks applied between the conductive ink and in
electrical contact therewith, wherein application of a voltage
(direct or alternating current) between the conductive inks results
in current flow through the resistive inks and production of heat.
There are two important types of resistive inks used in the
resistive heating elements of this invention The first is a
standard polymer thick film ink, such as Dupont 7082 or Dupont 7102
ink. These inks produce a surface temperature that is not
self-limiting, and the temperature resulting from the use of these
inks is dependent primarily on the magnitude of the applied
voltage. In contrast, the positive temperature coefficient (PTC)
inks show increase resistivity with increasing voltage, so that
surface temperature is self-limiting because the amount of
heat-producing current goes down as the applied voltage goes up.
PTC inks are characterized as having a particular temperature where
this self-limiting property is first exhibited; at voltages that
produce temperatures less than the critical temperature, the amount
of heat is dependent on the magnitude of the applied voltage.
[0216] Resistive inks useful according to the invention include
Dupont 7082, 7102, 7271, 7278 and 7285, and other equivalent
commercially available polymer thick film ink and PTC inks.
[0217] Conductive inks useful according to the invention include
Dupont 5028, 5025, Acheson 423SS, 426SS and SS24890, and other
equivalent commercially available conductive inks.
[0218] Additional components of the dielectric layer that serves to
insulate the electrical circuit. Dielectric layers advantageously
comprise dielectric inks such as Dupont 5018A. Insulation can also
be achieved using pressure sensitive transfer adhesive such as
7952MP (3M Co.), or a pressure sensitive transfer adhesive
deposited onto a polyester carrier layer such as 7953MP (3M Co.) or
thermoplastic bonding films such as 3M 406, 560 or 615.
[0219] Resistive heaters of the invention are advantageously used
to incubate fluids at a stable temperature and for melting
sacrificial valves as described below, and also for thermal
cyclic.
[0220] Resistive and conductive inks are preferably screen-printed
using methods and techniques well known in the art. See Gilleo,
1995, Polymer Thick Film (Van Nostrand Reinhold). Inks are
typically screen printed to a thickness of about 10 microns;
however, repetitive screen printing of resistive inks can be used
to deposit thicker layers having reduced resistances. Both
conductive and resistive inks are heat cured, typically at between
110.degree. C. and 120.degree. C. for about 10 minutes. The outline
of this printing process is shown in FIG. 30. Importantly, each of
the layers must be correctly registered with one another for
resistive heating to be provided. Heaters can be screen printed to
any required size; a minimum area for a screen-printed heater has
been determined to be about 0.25 mm.sup.2 (0.5 mm.times.0.5
mm).
[0221] The ability to tailor the resistance (and hence the
temperature profile) of the resistive heaters using choice of ink
formulation and reprinting of heater circuits provides control of
the final electrical and thermal properties of the resistive
heating elements of the invention. The resistance can also be
controlled through connection of series and parallel configurations
of resistive elements.
3. Sacrificial Valves
[0222] The ability to specifically generate heat at a particular
location on a Microsystems platform of the invention also enables
the use of sacrificial valves that can be released or dissolved
using heat. For the purposes of this invention, the term
sacrificial valve is intended to encompass materials comprising
waxes, plastics, and other material that can form a solid or
semi-solid fluid-tight obstruction in a microchannel, capillary,
chamber, reservoir or other microfluidics component of the
platforms of the invention, and that can be melted or deformed to
remove the obstruction with the application of heat. Sacrificial
valves are preferably made of a fungible material that can be
removed from the fluid flow path. In preferred embodiments, said
sacrificial valves are wax valves and are removed from the fluid
flow path by heating, using any of a variety of heating means
including infrared illumination and most preferably by activation
of resistive heating elements on or embedded in the platform
surface as described herein. For the purposes of this invention,
the term wax is intended to encompass any solid, semi-solid or
viscous liquid hydrocarbon, or a plastic. Examples include
mondisperse hydrocarbons such as eicosane, tetracosane and
octasone, and polydisperse hydrocarbons such as paraffin. In the
use of wax sacrificial valves, application of a temperature higher
than the melting temperature of the wax melts the valve and removes
the occlusion from the microchannel, capillary or other fluidic
component of the microsystems platforms of the invention.
Particularly when the sacrificial valve is melted on a rotating
Microsystems platform of the invention, the melted wax to flow
through the microchannel, capillary or other fluidic component of
the Microsystems platforms of the invention and away from the
original site of the valve.
[0223] One drawback, however, is the possibility that the wax will
recrystallize as it flows away from the original valve site, and
concomitantly, away from the localized heat source.
Recrystallization results in re-occlusion of the microchannel,
capillary or other fluidic component of the Microsystems platforms
of the invention, potentially and most likely at a site other than
the site of a localized heat source, and therefore likely to foul
fluid movement on the disc. One solution for this problem is the
inclusion in the sacrificial wax valves of the invention of a wax
recrystallization chamber positioned downstream from the position
of the wax valve. Preferably, the wax recrystallization chamber is
fluidly connected with the microchannel, capillary or other fluidic
component of the Microsystems platforms of the invention that was
occluded by the wax sacrificial valve. Typically, the wax
recrystallization chamber is a widening of the microchannel,
capillary or other fluidic component of the Microsystems platforms
of the invention so that recrystallized wax can harden on the walls
of the microchannel, capillary or other fluidic component of the
Microsystems platforms of the invention with enough distance
between said walls that the recrystallized wax does not re-occlude
the microchannel, capillary or other fluidic component of the
Microsystems platforms of the invention. Preferably, the heating
element, most preferably the resistive heating element of the
invention, extends past the site of the wax valve and overlaps at
least a portion of the wax recrystallization chamber, thereby
retarding the propensity of the wax valve to recrystallize.
[0224] It is also recognized that this propensity of wax valves to
recrystallize can be exploited to create a wax valve at a
particular location in a microchannel, capillary or other fluidic
component of the microsystems platforms of the invention. In this
embodiment, a particular location can be kept below a threshold
temperature by failing to apply heat at that location, and a wax
valve material can be mobilized from a storage area on a platform
by heating and them allowed to flow under centripetal acceleration
to a particularly cold site where a wax valve is desired. An
advantage of wax valves in this regard is that the proper
positioning an activation of resistive heater elements enables
flexibility in choosing when and whether a particular microchannel,
capillary or other fluidic component of the Microsystems platforms
of the invention is to be occluded by a wax sacrificial valve.
[0225] In particularly preferred embodiments, the sacrificial
valves of the invention comprise a cross-linked polymer that
displays thermal recover, most preferably a cross-linked,
prestressed, semicrystalline polymer; an example of a commercially
available embodiment of such a polymer is heat recoverable tubing
(#FP301H, 3M Co., Minneapolis, Minn.). Using these materials, at a
temperature less than the melting temperature (T.sub.m), the
polymer occludes a microchannel, capillary or other fluidic
component of the Microsystems platforms of the invention. At a
temperature greater than T.sub.m,, however, the polymer reverts to
its pre-stressed dimensions by shrinking. Such shrinking is
accompanied by release of the occlusion from the microchannel,
capillary or other fluidic component of the microsystems platforms
of the invention. Such embodiments are particular preferred because
the polymer remains in situ and does not recrystallize or otherwise
re-occlude the microchannel, capillary or other fluidic component
of the microsystems platforms of the invention. Also, such
embodiments do not require the more extensive manipulation in
preparing the platforms of the invention that wax valves
require.
[0226] In another embodiment, the sacrificial valves of the
invention comprise a thin polymeric layer or barrier dividing two
liquid-containing microchannel, capillary or other fluidic
component of the Microsystems platforms of the invention, that can
burst when sufficient temperature and/or pressure is applied.
[0227] Another embodiment of the sacrificial valves of the
invention are provided wherein a screen-printed resistive heater
element is itself a valve. In this embodiment, the resistive heater
element is screen-printed on a substrate such as polyester that
divides two liquid-containing microchannel, capillary or other
fluidic component of the Microsystems platforms of the invention.
In these embodiments, localized application of heat using a
resistive heating element is used to melt the substrate dividing
the liquid-containing microchannel, capillary or other fluidic
component of the microsystems platforms of the invention.
Preferably, in this embodiment the two liquid-containing
microchannel, capillary or other fluidic component of the
microsystems platforms of the invention are positioned in adjacent
layers through the vertical thickness of the platform.
[0228] As described above, the screen-printed resistive heater
elements of this invention provide localized application of heat to
a microsystems platform. The degree of localization achieved using
these resistive heating elements is sufficient to provide for the
placement of two adjacent sacrificial valves separated by a
distance of 0.15 cm.
4. Detectors and Sensors
[0229] Detection systems for use on the microsystem platforms of
the invention include spectroscopic, electrochemical, physical,
light scattering, radioactive, and mass spectroscopic detectors.
Spectroscopic methods using these detectors encompass electronic
spectroscopy (ultraviolet and visible light absorbance,
fluorescence, luminescence, and refractive index), vibrational
spectroscopy (IR and Raman), and x-ray spectroscopies (x-ray
fluorescence and conventional x-ray analysis using micromachined
field emitters, such as those developed by the NASA Jet Propulsion
Lab, Pasadena, Calif.).
[0230] General classes of detection and representative examples of
each for use with the microsystem platforms of the invention are
described below. In addition, the detection implementation systems
utilizing the detectors of the invention can be external to the
platform, adjacent to it or integral to the disk platform.
Spectroscopic Methods
1. Fluorescence
[0231] Fluorescence detector systems developed for macroscopic uses
are known in the prior art and are adapted for use with the
microsystem platforms of this invention. For example, an excitation
source such as a laser is focused on an optically-transparent
section of the disk. Light from any analytically-useful portion of
the electromagnetic spectrum can be coupled with a disk material
that is specifically transparent to light of a particular
wavelength, permitting spectral properties of the light to be
determined by the product or reagent occupying the reservoir
interrogated by illumination with light. Alternatively, the
selection of light at a particular wavelength can be paired with a
material having geometries and refractive index properties
resulting in total internal reflection of the illuminating light.
This enables either detection of material on the surface of the
disk through evanescent light propagation, or multiple reflections
through the sample itself, which increases the path length
considerably.
[0232] Alternative configurations appropriate for evanescent wave
systems are provided as understood in the art (see Glass et al.,
1987, Appl. Optics 26: 2181-2187). Fluorescence is coupled back
into a waveguide on the disk, thereby increasing the efficiency of
detection. In these embodiments, the optical component preceding
the detector can include a dispersive element to permit spectral
resolution. Fluorescence excitation can also be increased through
multiple reflections from surfaces in the device whenever noise
does not scale with path length in the same way as with signal.
[0233] In another type of fluorescence detection configuration,
light of both the fluorescence excitation wavelength and the
emitted light wavelength are guided through one face of the device.
An angle of 90 degrees is used to separate the excitation and
collection optical trains. It is also possible to use other angles,
including 0 degrees, whereby the excitation and emitted light
travels colinearly. As long as the source light can be
distinguished from the fluorescence signal, any optical geometry
can be used. Optical windows suitable for spectroscopic measurement
and transparent to the wavelengths used are included at appropriate
positions (i.e., in "read" reservoir embodiments of detecting
chambers) on the disk. The use of this type of fluorescence in
macroscopic systems has been disclosed by Haab et al. (1995, Anal.
Chem. 67: 3253-3260).
2. Absorbance Detection
[0234] Absorbance measurements can be used to detect any analyte
that changes the intensity of transmitted light by specifically
absorbing energy (direct absorbance) or by changing the absorbance
of another component in the system (indirect absorbance). Optical
path geometry is designed to ensure that the absorbance detector is
focused on a light path receiving the maximum amount of transmitted
light from the illuminated sample. Both the light source and the
detector can be positioned external to the disk, adjacent to the
disk and moved in synchrony with it, or integral to the disk
itself. The sample chamber on the disk can constitute a cuvette
that is illuminated and transmitted light detected in a single pass
or in multiple passes, particularly when used with a stroboscopic
light signal that illuminates the detection chamber t a frequency
equal to the frequency of rotation or multiples thereof.
Alternatively, the sample chamber can be a planar waveguide,
wherein the analyte interacts on the face of the waveguide and
light absorbance is the result of attenuated total internal
reflection (i.e., the analyte reduces the intensity source light if
the analyte is sequestered at the surface of the sample chamber,
using, for example, specific binding to a compound embedded or
attached to the chamber surface; see Dessy, 1989, Anal. Chem. 61:
2191).
[0235] Indirect absorbance can be used with the same optical
design. For indirect absorbance measurements, the analyte does not
absorb the source light; instead, a drop in absorbance of a
secondary material is measured as the analyte displaces it in the
sample chamber. Increased transmittance therefore corresponds to
analyte concentration.
3. Light Scattering
[0236] Turbidity can also be measured on the disk. Optics are
configured as with absorbance measurements. In this analysis, the
intensity of the transmitted light is related to the concentration
of the light-scattered particles in a sample. An example of an
application of this type of detection method is a particle
agglutination assay. Larger particles sediment in a rotating disk
more rapidly than smaller particles, and the turbidity of a
solution in the sample chamber before and after spinning the disk
can be related to the size of the particles in the chamber. If
small particles are induced to aggregate only in the presence of an
analyte, then turbidity measurements can be used to specifically
detect the presence of an analyte in the sample chamber. For
example, small particles can be coated with an antibody to an
analyte, resulting in aggregation of the particles in the presence
of the analyte as antibody from more than one particle bind to the
analyte. When the disk is spun after this interaction occurs,
sample chambers containing analyte will be less turbid that sample
chambers not containing analyte. This system can be calibrated with
standard amounts of analyte to provide a gauge of analyte
concentration related to the turbidity of the sample under a set of
standardized conditions.
[0237] Other types of light scattering detection methods are
provided for use with the Microsystems platforms and devices of the
invention. Monochromatic light from a light source, advantageously
a laser light source, is directed across the cross-sectional area
of a flow channel on the disk. Light scattered by particles in a
sample, such as cells, is collected at several angles over the
illuminated portion of the channel (see Rosenzweig et al., 1994,
Anal. Chem. 66: 1771-1776). Data reduction is optimally programmed
directly into the device based on standards such as
appropriately-sized beads to relate the signal into interpretable
results. Using a calibrated set of such beads, fine discrimination
between particles of different sizes can be obtained. Another
application for this system is flow cytometry, cell counting, cell
sorting and cellular biological analysis and testing, including
chemotherapeutic sensitivity and toxicology.
Analytic Methods
[0238] It will be understood that the interpretation of the optical
detection data from performance of analytical assays as provided by
the invention may require transformation of "raw" data into
information useful for the operator. For example, "reflectance" is
determined as the difference between the "signal" and the "dark
signal" (i.e., the signal detected in the absence of transmitted
light), normalized by the difference between the "reference" and
the "dark reference," wherein each of the reference values is
determined for a "blank" cuvette not containing a sample.
Similarly, the detection methods used by the invention also include
methods for eliminating artifactual signal produced by interfering
absorbing or light-scattering components, such as scratches or
particulate matter. Advantageously, normalized readings are
collected at a wavelength greater than 650 nm, more preferably
660.+-.10 nm as a reference. This reading is then subtracted from
the sample reading to as a correction thereof.
[0239] Absorbance from certain components of biological fluid
sample, such as hemoglobin in blood, are advantageously removed by
using a light source with a wavelength at the absorbance peak of
the interfering material; for hemoglobin, this is about 425 nm. In
preferred embodiments, light of this wavelength is provided by a
blue light-emitting diode (LED) having a wavelength of about 430
nm. The light emission profile of the LED was found to overlap
sufficiently with the hemoglobin absorbance profile to effectively
quench the hemoglobin absorbance signal in the glucose assays of
the invention. The glucose signal was then further treated
analytically by subtracting a factor times the (blue-red) signal
from the (orange-red) signal, wherein the (orange) signal was the
wavelength specific for the glucose assay product. The factor used
is dependent on the optics, the reading chamber structure, spectral
properties of the light sources and filters, and the spectral
absorbance characteristics of hemoglobin and the colored product of
the glucose oxidase reaction. The factor can be determined
empirically using solutions of known glucose and hemoglobin
concentrations, both singly and in combination.
[0240] It will be understood in the art that similar combinations
of light sources and interrogated wavelengths can be advantageously
used to reduce or eliminate the contribution of other interfering
species in optical detection methods used according to the
invention.
5. Chemistries
[0241] As described above with regard to the microfluidic
components of the Microsystems platforms of the invention, the
present invention provides platforms for performing chemical,
biochemical, enzymatic, immunological and other assays on fluid
samples, most preferably wherein the fluid sample is a biological
fluid sample.
[0242] Two exemplary types of assay formats are explicitly set
forth herein; one of ordinary skill will recognize that the
disclosure is generally applicable to a variety of assay systems as
set forth, for example, in CLINICAL GUIDE TO LABORATORY TESTS,
Tietz, ed., W. B. Sanders Co: Philadelphia, 1995. A representative
and non-limiting sample of assays advantageously performed using
the microsystems platforms of the invention are set forth in Table
I below.
1TABLE I Assay for: Analyte/Detection Type Components on solid
phase: Acid Phosphatase enzyme/colorimetric Alpha-naphthol
phosphate, Fast Red TR dye.sup.1 Alanine enzyme/colorimetric
Alanine, alpha-ketoglutarate, 2,4- Aminotransferase
dinitrophenylhadrazine dye Albumin protein/colorimetric Bromcresol
green dye Alkaline Phosphatase enzyme/colorimetric p-nitrophenyl
phosphate Amylase enzyme/colorimetric
4,6-ethylidine(G7)-p-nitrophenyl(G1)-alpha,D- maltoheptaside,
alpha-glucosidase Apolipoprotein A-1
lipoprotein/immunoturbidimetric Anti-ApoA1 antibody, polyethylene
glycol.sup.2 Direct Bilirubin organic compound/colorimetric
Diazotized sulfanilic acid Total Bilirubin organic
compound/colorimetric Caffeine, benzoate, acetate, diazotized
sulfanilic acid Calcium mineral/colorimetric Cresolphthalein
complexone Cholesterol lipid/colorimetric Cholesterol esterase,
cholesterol oxidase, 4- aminoantipyrine, p-hydroxybenzene sulfonate
Fructosamine glycated serum Nitroblue tetrazolium
protein/colorimetric Gamma-glutamyl enzyme/colorimetric
L-gamma-glutamyl-3-carboxy-4-nitroanilide, transferase
glycylglycine Iron mineral/colorimetric Ferrozine, reducing agent
Microprotein (urine protein/colorimetric Pyrogallol red-molybdate
complex or CSF) Urea organic compound/colorimetric
Diacetylmonoxime, heat Specific Protein immunologically reactive
Antibody reactive with unique species, etc, site; species,
subspecies or proteins/immunoturbidimetry precipitation enhancers
(e.g., anti-alpha-1- variant antitrypsin IgG, polyethylene
glycol).sup.3 Antibodies against immunoreaction to bacteria or
Immunoreactive component from infective infectious agents virus
infection/heterogeneous agent, enzyme linked to antibody against
the enzyme immunoassay primary immunoglobulin species of reaction
to the agent, enzyme substrate linked to color generation (e.g.,
Epstein-Barr early antigen, anti-human IgG conjugated to
horseradish peroxidase, 3-3'-5-5'-tetramethylbenzidine) Drugs
immunologically reactive Competition between drug and drug-enzyme
therapeutic drugs or drugs of reagent for anti-drug antibody
binding sites abuse/homogeneous enzyme where antibody binding
inhibits enzyme immunoassay activity, enzyme substrate linked to
color generation (e.g., phenobarbital-glucose-6- phosphatase,
anti-phenobarbital IgG, NAD).sup.4 .sup.1Saw, D., et. al, Clin.
Biochem., 1990; 23: 505. .sup.2Rifai, N., Warnick, G. R., eds.,
Methods for Clinical Laboratory Measurement of Lipid and
Lipoprotein Risk Factors, Washington, D. C.: AACC Press, 1990
.sup.3Hills, L. P., and Tiffany, T. O., Comparison of turbidimetric
and light-scattering measurements of immunoglobulins by use of a
centrifugal analyzer with absorbance and fluorescence/light
scattering optics. Clin. Chem. 1980; 26: 1466. .sup.4Oellerich, M.:
Enzyme immunoassays in clinical chemistry: Present status and
trnds. J. Clin. Chem. Clin. Biochem. 1980;18: 197.
[0243] The requirements for performing such assays on the solid
phase (such as the matrices disclosed herein) include: the ability
to link, by one or a series of chemical reaction steps, the
presence of an analyte of interest in a fluid sample, most
preferably a biological fluid sample, to the quantitative
generation of a product detectable by optical methods as disclosed
herein; stabilization of the necessary reagents (chemicals or
biochemicals) onto the solid phase so that the reagents retain
their potencies or activities.
[0244] A second general scheme for performing assays on the
Microsystems platforms of the invention involve miniaturized
versions of affinity chromatography column separations, wherein the
analyte specifically binds to a material in a chamber or on a
surface, most preferably a derivatized surface, of the platform, or
is bound to a material such as a bead, chromatography resin, or
membrane on the surface of the platform, so that the remainder of
the fluid sample can be washed from the affinity matrix and the
analyte separated thereby. In certain preferred embodiments, the
analyte is detected indirectly, wherein the biological fluid sample
is interrogated after passage of the sample over the chromatograohy
matrix. Such detection methods can be subtractive, wherein the
interrogated optical property of the biological fluid sample after
passage over the chromatography matrix is compared with the same
property of a portion of the sample that has not been passed over
the matrix; or directly, wherein the analyte is dissociated from
the chromatography matrix (either non-specifically, using for
example a salt or dielectric gradient, or specifically, using a
binding competitor that displaces the analyte from the
chromatography matrix).
[0245] Examples of analytes advantageously separated from
biological fluid samples and the column affinity material(s) used
therefore are set forth in Table II.
2TABLE II Column Affinity Material Bound Species Diatomateous earth
DNA Protein A-agarose IgG Heparin-agarose coagulation proteins,
Protein C, growth factors, lipoproteins, steroid receptors Blue
agarose albumin, coagulation factors, interferon, enzymes requiring
cofactors with adenyl group Streptavidin-agarose biotinylated
molecules Con A-agarose glycoproteins, polysaccharides with
terminal mannose or glucose Lentil lectin-agarose glycoproteins,
polysaccharides with branched mannose with fucose linked
.alpha.(1,6) to N-acetyl-glucosamine Wheat gern lectin-agarose
glycoproteins, polysaccharides with chitobiose core of N-linked
oligosaccharides Peanut lectin-agarose glycoproteins,
polysaccharides with terminal .beta.-galactose Arginine-agarose
serine proteases Calmodulin-agarose ATPases, phosphodiesterases,
neurotransmitters, protein kinases Gelatin-agarose Fibronectin
Glutathione-agarose S-transferases, glutathione-dependent proteins
Lysine-agarose plasminogen, plasminogen activator, ribosomal RNA
DNA (denatured)-agarose DNA polymerase, RNA polymerase, T4
polynucleotide kinase, exonuclease, deoxyribonucleases DNA
(native)-cellulose Glucocorticoid receptor, DNA polymerase, DNA
binding proteins 2'5' ADP-agarose NADP-dependent dehydrogenases 5'
AMP-agarose NAD-dependent dehydrogenases, ATP-dependent kinases
7-Methyl-GTP-agarose eukaryotic mRNA, cap-binding protein
Poly(U)-agarose mRNA, reverse transcriptase, interferon, plant
nucleic acids C8-silica Proteins
[0246] It will be appreciated that preparative embodiments of the
affinity chromatographic column separations are within the scope of
the invention.
[0247] The following Examples are intended to further illustrate
certain preferred embodiments of the invention and are not limiting
in nature.
EXAMPLE 1
Blood Glucose Assay Fluidics Structure
[0248] A microsystems platform provided by the invention and
specifically designed for performing blood glucose assay is
illustrated in FIG. 1. Disk embodiments of the platforms of the
invention were fashioned from machined acrylic. The overall disc
dimensions include an outer radius of about 6 cm and an inner
radius of about 0.75 cm, wherein the disk was mounted on the
spindle of a rotary device. The thickness of the fluidics disc was
3.2 mm, which was mounted on a conventional compact disc (CD)
having a thickness of 1.2 mm. All surfaces coming into contact with
blood on the platform are advantageously treated with heparin, EDTA
or other anticoagulants to facilitate fluid flow thereupon.
[0249] The components of the blood glucose assay were prepared as
follows. Blood sample entry port chamber 101 having a depth in the
platform surface of about 0.32 cm and lateral dimensions of about 1
cm was constructed on the platform, and designed to accommodate a
volume of 100 .mu.L. This entry port was fluidly connected with a
metering capillary 102 having a square cross-sectional diameter of
about 0.1 cm deep.times.0.5 cm wideand proximal ends rounded with
respect to entry port 101; the length of this metering capillary
array was sufficient to contain a total volume of about 16 .mu.L.
The entry port was also fluidly connected with an overflow
capillary 103 having a cross-sectional diameter of about 0.05
cm.times.0.075 cm and proximal ends rounded with respect to entry
port 101. The overflow capillary was fluidly connected with a
two-layered overflow chamber 105 having a first depth in the
platform of about 0.025 cm and a second depth in the platform of
about 0.25 cm, greater than the depth of the overflow capillary
103. Metering capillary 102 was fluidly connected to metered blood
fluid chamber 104 having a depth in the platform surface of 1 mm
and greater than the depth of the metering capillary 102. Each of
the overflow and fluid chambers was also connected with air ports
or air channels, such as 114, that have dimensions of 0.025 cm deep
and permitted venting of air displaced by fluid movement on the
platform. A capillary junction 115 that was 0.051 cm deep was
present in the air channel to prevent fluid flow into the air
channel.
[0250] Entry port 101 was positioned on the platform about 2 cm
from the center of rotation. Metering chamber 102 extended about 1
cm from entry port 101. The extent of the length of overflow
capillary 103 was 300% greater than the extent of the length of
metering capillary 102. The position of blood fluid chamber 104 was
about 4.6 cm from the center of rotation, and the position of
overflow chamber 105 was about 4 cm from the axis of rotation.
[0251] Blood fluid chamber 104 acted as a capillary barrier that
prevented fluid flow from metering chamber 102 at a first, non-zero
rotational speed f.sub.1 of about 3000 rpm that was sufficient to
permit fluid flow comprising overflow from the entry port 101
through overflow capillary 103 and into overflow chamber 105. This
capillary boundary was constructed to be overcome at a second
rotational speed f.sub.2 of about 600 rpm (so that
f.sub.2>f.sub.1). Blood fluid chamber 104 was fluidly connected
to capillary 110 that was about 0.025 cm deep and had a
cross-sectional diameter of about 0.025 cm and was connected to
capillary or sacrificial valve 111. Sacrificial valve 111 was
further fluidly connected with capillary 112 that was about 0.25 mm
deep and had a cross-sectional diameter of about 0.05 cm, and
capillary 112 was fluidly connected to assay chamber 107.
Sacrificial valve chamber 111 was positioned to sequester melted
wax produced by release of the sacrificial valve.
[0252] Assay chamber 107 comprised a depression in the surface of
the platform having a depth of about 0.1 cm, and further comprised
a circular or rectangular concave depression 113 connected to
capillary 112. Assay chamber 107 also comprised a pad or matrix 106
comprising a positively-charged nylon matrix having a pore size of
about 0.8 .mu.m. The pore size of matrix 106 was chosen to inhibit
or prevent blood cell entry into the matrix. The matrix was
positioned in assay chamber 107 to be in fluidic contact with
depression 113, covering depression 113 and having a surface area
greater than the surface area of depression 113. The matrix was
impregnated with immobilized reagents 108 which produce a
detectable product proportional to the concentration of glucose in
a blood sample. The detectable product was a colored product 109,
i.e., a product absorbing light at a detectable, visible
wavelength.
[0253] As illustrated in FIGS. 2A through 2E, in the use of this
platform an imprecise volume (about 30 .mu.L of fluid) of blood was
applied to the entry port 101. The fluid wicked into air channel
114 and was stopped by capillary junction 115. Fluid also wicked
into metering capillary 102 and overflow capillary 103. Fluid
flowed through the metering capillary 102 and overflow capillary
103 at no rotational speed until the fluid reached capillary
junctions at the junction between metering chamber 102 and blood
fluid chamber 104 and overflow capillary 103 and overflow chamber
105. Metering capillary 102 was constructed to define a precise
volume of about 15 .mu.L of blood between entry port 101 and the
capillary junction at fluid chamber 104, which was designed to be
at least the amount of the fluid placed by the user in entry port
101.
[0254] After sample loading by a user and filling of metering
chamber 102 and overflow capillary 103 at zero rotational speed,
the platform was spun at a first rotational speed f.sub.1 of about
300 rpm, which was sufficient to motivate fluid flow through the
overflow capillary 103 in this microfluidics array, wherein entry
port 101 had a depth of about 0.3 cm, metering chamber 102 had
dimensions of about 0.1 cm deep.times.0.4 cm wide in cross-section
and about 0.5 cm in length from the center of rotation, and
overflow capillary 103 had dimensions of about 0.05 cm.times.0.075
cm in cross-section and about 2.7 cm in length from the center of
rotation.
[0255] Due to the greater distance from the center of rotation of
the end of overflow capillary 103 than the end of metering chamber
102, at rotational speed f.sub.1 fluid flowed through overflow
capillary 103 into overflow chamber 105. The platform was spun
until all excess fluid was evacuated from entry port 101 and into
overflow chamber 105, except the fluid contained in metering
capillary 102.
[0256] At a second rotational speed f.sub.2 of about 1000 rpm, the
precise amount of fluid (16 .mu.L) contained in metering capillary
102 was delivered into fluid chamber 104. Fluid movement into fluid
chamber 104 was accompanied by filling of capillary 110.
[0257] In embodiments comprising a sacrificial valve 111 in-line
with capillary 110 at a position between capillary 110 and 112
shown in FIG. 2A, release of the sacrificial valve resulted in
fluid flow through capillary 112 and into assay chamber 107. In
said embodiments, fluid flow was achieved at rotational speed
f.sub.2 with removal of the sacrificial valve. In embodiments of
the platforms of the invention comprising capillary valve 111 at a
position between capillary 110 and 112 shown in FIG. 2B, capillary
110 filled along with filling of blood fluid chamber 104 until
blood reached capillary junction 111 at the junction between
capillary 110 and capillary 112; in such embodiments, the capillary
junction had a depth of about 0.05 cm. At a third rotational speed
f.sub.3 of about 500 rpm, the fluid contained in blood fluid
chamber 104 was delivered into assay chamber 107 (FIG. 2B). Blood
flowing into assay chamber 107 was preferentially directed to
depression 113 in the assay chamber; the dimensions of depression
113 are conveniently chosen to be able to contain substantially all
of the blood fluid of the sample metered through metering chamber
102 into assay chamber 107 (FIG. 2C). Displaced air flows through
air channel 114, and was vented to the surface of the disc.
[0258] As blood flowed into depression 113, the fluid component of
the blood is driven by pressure and hydrophilic forces into matrix
106, comprising a positively-charged nylon matrix having a pore
size of about 0.8 .mu.m; this pore size was chosen to prevent the
cellular components of the blood from entering the matrix (FIG.
2D). The cellular blood components were retained in depression 113
and the fluid component was efficiently distributed into matrix
106. As the fluid component of the blood entered matrix 106, dried
reagents 108 were solubilized and the reaction of the blood
component catalyzed by said reagents proceeded. This reaction(s)
went to completion within about 1 min. Reaction of the blood
component(s) with reagents 108 produce colored product 109 which
was then detected (FIG. 2E), as described below in Example 4.
EXAMPLE 2
Glycated Hemoglobin Assay--Fluidics Structure
[0259] A Microsystems platform provided by the invention and
specifically designed for performing a glycated hemoglobin assay is
illustrated in FIG. 11.
[0260] Construction of the disk embodiments of the platforms of the
invention are as described in Example 1. The blood application and
metering components and their dimensions and relationships to one
another are identical to those described above, comprising sample
entry port chamber 801, metering capillary 802, overflow capillary
803, metered blood fluid chamber 804 and overflow chamber 805. As
in Example 1, each of the overflow and fluid chambers is also
connected with air ports or air channels, such as 814, and
capillary junction(s) 815, that permit venting of air displaced by
fluid movement on the platform.
[0261] Blood fluid chamber 804 was fluidly connected to capillary
810 that was about 0.25 mm deep and had a cross-sectional diameter
of about 0.25 mm and was connected to sacrificial valve 811.
Sacrificial valve 811 was further fluidly connected with capillary
812 that was about 0.25 mm deep and had a cross-sectional diameter
of about 0.25 mm. Capillary 812 was further fluidly connected to
mixing chamber 815 that was about 0.25 cmcm deep, had a
cross-sectional diameter of about 1 cm, and was positioned about
2.5 cm from the center of rotation. Lysis buffer was loaded
directly onto mixing chamber 815 in this embodiment and did not use
capillary 818 or lysis buffer chamber 816 as shown in the Figure.
45 .mu.L of lysis buffer was applied to the mixing chamber as a
solution of 0.1% Triton X100 in 50 mM Tris pH 9.5.
[0262] Mixing chamber 815 was fluidly connected to capillary 817
that was about 0.25 mm deep and had a cross-sectional diameter of
about 0.25 mm, and was connected to secondary metering structure
819. Secondary metering structure 819 was about 0.1 cm deep and was
positioned about 3.5 cm from the center of rotation. Secondary
metering structure 819 was constructed to comprise two sections. A
metering section was arranged proximal to the entry position of
capillary 817 and was separated from an overflow section by a
septum that extended from the distal wall of the structure to a
position just short of the proximal wall of the structure. This
arrangement produced a fluid connection between the first metering
section having a volumetric capacity of about 6.4.mu.L and the
overflow section having an excess volumetric capacity of 90 .mu.L.
The volumetric capacity of the overflow section was sufficient to
accommodate the largest blood fluid volume applied to the disk.
[0263] Capillary 821 was in fluid connection with secondary
metering structure 819 at the distal wall of the metering section.
Capillary 821 was about 0.15 cm deep and had a cross-sectional
diameter of about 0.05 cm and was connected to boronate affinity
matrix chamber 822. Boronate affinity matrix chamber 822 was about
0.15 cm deep, had a cross-sectional diameter of about 0.3 cm and
was positioned about 4.8 cm from the axis of rotation. Boronate
affinity matrix chamber 822 was filled with boronate-functionalized
agarose beads having a mean diameter of about 60 .mu.m; the beads
were maintained in the chamber 822 using a porous frit 827. Fluid
flow through capillary 821 was connected to capillary or
sacrificial valve 823. Boronate affinity matrix chamber 822 was
further comprised of a translucent window that permitted reflective
spectrophotometry of the contents of the chamber.
[0264] Boronate affinity matrix chamber 822 was further fluidly
connected to capillary 828. Capillary 828 was about 0.25 mm deep
and had a cross-sectional diameter of about 0.25 mmand was
connected to column wash buffer reservoir 829. Column wash buffer
reservoir 829 was about 0.25 cm deep and had a cross-sectional
diameter of about 2 cm and was positioned about 3.6 cm from the
axis of rotation, more proximal to the axis of rotation than
boronate affinity matrix chamber 822. Column wash buffer reservoir
829 comprises 290 .mu.L of column preparation buffer that was a
solution of magnesium chloride, taurine, D,L-methionine, sodium
hydroxide, antibiotics and stabilizers constituted according to the
manufacturer's instructions (Isolab Inc. #SG-6220). Fluid flow
through capillary 828 was connected to capillary or sacrificial
valve 836.
[0265] Boronate affinity matrix chamber 822 was further fluidly
connected to capillary 832. Capillary 832 was about 0.5 m deep and
had a cross-sectional diameter of about 0.5 mm and was connected to
non-glycated hemoglobin read chamber 834. Chamber 834 was about
0.25 cm deep and had a cross-sectional diameter of about 2 cm and
was positioned about 5 cm from the axis of rotation and was further
comprised of a translucent window that permitted reflective
spectrophotometry of the contents of the chamber at 430 nm.
[0266] As illustrated in FIG. 1, in the use of this platform about
a 6.4 .mu.L volume of blood was applied to blood fluid chamber 804,
either directly or using the metering components of the platform
described above. Blood flowing through capillary 810 and lysis
buffer contained in mixing chamber 815 were mixed in the mixing
chamber. A 45 .mu.L volume of lysis buffer was mixed with the blood
sample. Fluid flow within mixing chamber 815 was turbulent, in
contrast to fluid flow through capillaries 810 or 818, which was
primarily laminar, so that mixing occurred predominantly in mixing
chamber 815. Fluid flow proceeded through channel 817 and into
secondary metering structure 819. The mixture of lysis buffer and
blood, comprising a lysed blood sample 841, flowed at a rotational
speed f.sub.? of about 750 rpm into secondary metering structure
819 with release of a sacrificial valve 853. Lysed blood sample 841
entered and filled the metering section of secondary metering
structure 819. Any additional lysed blood sample then emptied into
the overflow chamber of secondary metering structure 819 and filled
the total hemoglobin read chamber. Most preferably, a sufficient
volume of lysis buffer and blood sample was applied to the disc to
fill at least the metering sections of secondary metering structure
819 and the total hemoglobin read chamber.
[0267] After the lysed blood sample 841 was completely transferred
to secondary metering structure 819, capillary or sacrificial valve
823 was released, allowing the metered lysed blood sample from the
metering section of secondary metering structure 819 through
capillary 821 and into boronate affinity matrix 822. Capillary or
sacrificial valve 836 was then released, allowing about 290 .mu.L
of column wash buffer 843 to flow at rotational speed f.sub.?
through capillary 830 and into boronate affinity matrix 822.
Continued or discontinuous rotation motivates column preparation
buffer through boronate affinity matrix 822 and into non-glycated
hemoglobin read chamber 834. The control sample read cuvettes 8-
and 8.about. were then then illuminated by light at a wavelength of
430 nm and the blank reading, i.e. reflectance from cuvettes
containing only buffer, was determined. The non-glycated hemoglobin
read window was then illuminated by light at a wavelength of 430 nm
and the concentration of non-glycated hemoglobin in the sample that
has eluted from the column was determined by reflectance
spectroscopy. The total hemoglobin read window was also illuminated
by light at a wavelength of 430 nm and the concentration of total
hemoglobin in the lysed sample was determined by reflectance
spectroscopy.
[0268] The amount of non-glycated hemoglobin in the sample is
determined by dividing the amount of hemoglobin obtained by
illuminating the eluted fraction by the amount of hemoglobin
obtained in the total fraction.
EXAMPLE 3
Combination Glucose Concentration--Glycated Hemoglobin Assay
Platform
[0269] A microsystems platform provided by the invention and
specifically designed for performing both a determination of blood
glucose concentration and a glycated hemoglobin assay is
illustrated in FIGS. 12A through 12Q and 13A through 13E.
[0270] Construction of the disk embodiments of the platforms of the
invention were as described above. FIG. 13B shows a detailed
description of the microfluidics components of the platform, which
are described in additional detail below. FIG. 13C shows the
geometry of a screen printed electrical lead layer deposited on a
mylar substrate. FIG. 13D shows the positions of screen printed
heaters activated by the electrical leads of the lead layer and
screen printed on mylar. FIG. 13E shows a overlay of these
components in the assembled disc.
[0271] Referring to the microfluidics components of the platform
shown in FIG. 13B, an entry port 1 is positioned on the top surface
of the disc and is open for the user to apply an unmetered sample.
Entry port 1 is about 0.32 cm deep, has a cross-sectional diameter
of about 1 cm, is positioned about 2 cm from the center of
rotation, and is fluidly connected to capillary channel 1A that is
about `1 mm deep and has a cross-sectional diameter of about 1 mm.
Capillary channel 1A is fluidly connected to metering component 2,
which comprises four sections. The first section is
rectangularly-shaped and extends in a direction proximal to the
axis of rotation away from its fluid connection with capillary
channel 1A. About half way up this rectangular section is a lateral
chamber 3, which empties into a blood glucose metering chamber 4
and an overflow chamber 5. The first section of the metering
component is about 0.1 cm deep, has a cross-sectional diameter of
about 0.4 cm, is positioned about 2.2 cm from the center of
rotation, and has a volumetric capacity of about 5 microliters for
Hb assay. The lateral chamber 3 is 1 mm deep, has a cross-sectional
diameter of 1 mm, and is positioned 2.3 cm from the center of
rotation. Blood glucose metering chamber 4 is 1 mm deep, has a
cross-sectional diameter of 4.5 mm, is positioned 2.6 cm from the
center of rotation and has a volumetric capacity of 16 .mu.L.
Overflow chamber 5 is 1 mm deep, has a cross-sectional diameter of
2 mm, is positioned 2.6 cm from the center of rotation and has a
volumetric capacity of 7 .mu.L.
[0272] Overflow chamber 5 is fluidly connected to overflow channel
8 that is 0.75 mm deep, has a cross-sectional diameter of 0.75 mm
and extends 3.8 cm from overflow chamber 5. Overflow channel 5 is
fluidly connected to short sample detection cuvette 9 that is two
depths: 0.25 mm and 2.2 mm(inner) deep, has a cross-sectional
diameter of 2.5 mm, is positioned 4-5.8 cm from the center of
rotation and has a volumetric capacity of 50 .mu.L.
[0273] Blood glucose metering chamber 4 is fluidly connected to
capillary 15 that is about 0.25 mm deep, has a cross-sectional
diameter of about 0.25 mm and extends about 0.1 cm from blood
glucose metering chamber 4. Capillary 15 is connected to
sacrificial wax valve 6, which is further fluidly connected with
wax recrystallization chamber 6A that is about 0.5 mm deep and has
a volumetric capacity sufficient to sequester melted wax from a
released wax valve and prevent occlusion of the lumen of the
capillary 15 controlled by the valve. Capillary 15 is further
fluidly connected with glucose assay chamber 11 that is about 0.1
cm deep, has a cross-sectional diameter of about 0.5 mm, is
positioned about 5 cm from the center of rotation and has a
volumetric capacity of about 10 .mu.L. Glucose assay chamber 11
comprises a depression 11A in the surface of the platform having a
depth of about 0.1 cm, most preferably comprising a circular or
rectangular depression connected to capillary 15 so that blood
flows into the chamber through the bottom of depression 11A.
Depression 11A is constructed to have a volumetric capacity of from
half to twice the assay volume. Blood glucose assay chamber 11 also
comprises a pad or matrix 10 of a positively-charged nylon matrix
having a pore size of about 0.8 .mu.m. The upper limit on pore size
of matrix 10 is chosen to inhibit or prevent blood cell entry into
the matrix. The matrix is positioned in blood glucose assay chamber
11 to be in fluidic contact with depression 11A, more preferably
covering depression 11A, and most preferably having a surface area
greater than the surface area of depression 11A. The matrix was
further impregnated with immobilized reagents 11B which produce a
detectable product proportional to the amount or concentration of
glucose in a blood sample. Most preferably, the detectable product
is a colored product 11C, i.e., a product absorbing light at a
detectable, most preferably a visible, wavelength.
[0274] Lysis metering chamber 2 is fluidly connected to capillary 7
controlled by sacrificial valve 7A. Sacrificial wax valve 7A is
further fluidly connected with wax recrystallization chamber 7B
that is 1 mm deep and has a volumetric capacity sufficient to
sequester melted wax from a released wax valve and prevent
occlusion of the lumen of the capillary 7 controlled by the valve.
Capillary 7 is 0.25 mm deep, has a cross-sectional diameter of 0.25
mm, extends 0.1 cm from lysis metering chamber 2 and is fluidly
connected to blood lysis chamber 16. Blood lysis is contained
within the mixing chamber Blood lysis chamber 16 is 2.3 mm deep,
has a cross-sectional diameter of 1 cm, is positioned 3 cm from the
center of rotation, and contains 45 .mu.L of blood lysis solution
(0.1% Triton-X100 in 50 mM Tris, pH 9.5).
[0275] Blood lysis chamber 16 is fluidly connected at a distal
aspect to capillary 17 controlled by sacrificial valve 18.
Capillary 17 is about 0.25 mm deep and has a cross-sectional
diameter of about 0.25 mm Sacrificial wax valve 18 is further
fluidly connected with wax recrystallization chamber 18A that is
about 0.5 mm deep and has a volumetric capacity sufficient to
sequester melted wax from a released wax valve and prevent
occlusion of the lumen of the capillary 17 controlled by the valve.
Capillary 17 is fluidly connected to secondary metering structure
19. Secondary metering structure 19 is 1.5 mm deep, and is
positioned 3.8 cm from the center of rotation. Secondary metering
structure 19 is constructed to comprise three sections. A first
section 20 comprises a throwaway section that is used to discard
the first sample and taking the second sample to provide a better
sample for analysis, having a volumetric capacity of about 6.4
.mu.L. Throw away section 20 is arranged proximal to the entry
position of capillary 17 and is separated from a metering section
21 by a septum that extends from the distal wall of the structure
to a position just short of the proximal wall of the structure.
This arrangement produces a fluid connection between throwaway
section 20 and the metering section 21. Metering section has a
volumetric capacity of about 6.4 .mu.L and is fluidly connected to
an overflow section 24 having an excess volumetric capacity of
about 90 .mu.L. The volumetric capacity of the overflow section is
sufficient to accommodate the largest blood fluid volume applied to
the disk.
[0276] Capillary 25 is in fluid connection with secondary metering
structure 21 at the distal wall of the metering section. Capillary
25 is about 0.1 5 cm deep and has a cross-sectional diameter of
about 0.5 mm and is connected to boronate affinity matrix chamber
28. Capillary 25 is fluidly connected to sacrificial wax valve 26
that is further fluidly connected with wax recrystallization
chamber 26A Wax recrystallization chamber 26A is about 0.3 cm deep
and has a volumetric capacity sufficient to sequester melted wax
from a released wax valve and prevent occlusion of the lumen of the
capillary 25 controlled by the valve.
[0277] Boronate affinity matrix chamber 28 is about 0.15 cm deep,
has a cross-sectional diameter of about 0.3 cm and is positioned
about 4.8 cm from the axis of rotation. Boronate affinity matrix
chamber 28 is filled with boronate-functionalized agarose beads
(Isolab, $SG-6220) having a mean diameter of about 60 .mu.m; the
beads are maintained in the chamber 28 using a porous frits 29.
Boronate affinity matrix chamber 28 is further fluidly connected to
capillary 31. Capillary 31 is about 0.25 mm deep and has a
cross-sectional diameter of about 0.25 mm and is connected to
column wash buffer reservoir 30. Column wash buffer reservoir 30 is
about 0.25 cm deep and has a cross-sectional diameter of about 2 cm
and is positioned about 3.6 cm from the axis of rotation, more
proximal than boronate affinity matrix chamber 28. Column wash
buffer reservoir 30 comprises about 290 .mu.L of column wash buffer
comprising a solution of asparagine, magnesium chloride, taurine
and D,L-methionine (Isolab, #SG-6220). Fluid flow through capillary
31 is connected to sacrificial valve 32. Sacrificial wax valve 32
is further fluidly connected with wax recrystallization chamber 32A
that is about 0.5 mm deep and has a volumetric capacity sufficient
to sequester melted wax from a released wax valve and prevent
occlusion of the lumen of the capillary 32 controlled by the
valve.
[0278] Boronate affinity matrix chamber 28 is further fluidly
connected to capillary 37. Capillary 37 is about 0.5 mm deep and
has a cross-sectional diameter of about 0.5 mm and is connected to
sample collection cuvette array 12. Sample collection cuvette array
12 is about 0.25 cm deep and has a cross-sectional diameter of
about 2.1 cm and is positioned about 5 cm from the axis of
rotation. Sample collection cuvette array 12 is separated into a
multiplicity of individual chambers, each separated from one
another by septa that extend from the distal wall of the cuvettes
to a position adjacent to the proximal wall of the cuvettes, so
that a fluid passage 50 is maintained between each of the cuvettes.
The fluid passage 50 is formed by the back (proximal wall) of the
sample collection cuvette array 12 and the row of septa separating
each of the sections of the sample collection cuvettes 12.
Capillary 33 is fluidly connected to sample collection cuvette
array 12 at a position adjacent to the proximal wall of the array
and directed to the cuvette most proximal to the boronate affinity
matrix chamber 28.
[0279] Capillary 22 is fluidly connected to secondary metering
structure 19. Capillary 22 is about 0.25 mm deep and has a
cross-sectional diameter of about 0.25 mm and is connected to
secondary metering structure 19 at a position between the metering
section and the overflow section. Capillary 22 is further fluidly
connected with total hemoglobin read chamber 23. Total hemoglobin
read chamber 23 is about 0.25 cm deep, is positioned about 5 cm
from the center of rotation, and has a volumetric capacity of about
50 .mu.L. Total hemoglobin read chamber 23 is positioned radially
more distal from the center of rotation than secondary metering
structure 19, and comprises a read window translucent to light
having a wavelength of 300-1000 nm. In addition, there is no
capillary or sacrificial valving controlling fluid flow in
capillary 23.
[0280] The platform also comprises control sample read cuvettes 13
and 14, advantageously positioned in proximity to total hemoglobin
read chamber 23. Control sample read cuvettes 13 and 14 are each
about 0.25 cm deep, positioned about 5 cm from the center of
rotation, and have a volumetric capacity of about 50 .mu.L. Control
sample read cuvettes 13 and 14 comprise a read window translucent
to light having a wavelength of 410 nm. Control sample read
cuvettes 13 and 14 are not fluidly connected to any other structure
on the platform.
[0281] Air displacement channels 33 and capillary junction(s) 34,
that permit venting of air displaced by fluid movement on the
platform, are fluidly connected to the components of the platform
to permit unimpeded fluid flow.
[0282] As illustrated in FIGS. 12 A through 12Q, in the use of this
platform a volume of blood about 30 .mu.L is applied to entry port
1. Blood enters lysis subvolume 2 and lateral passageway 3 under
the influence of gravity and capillary forces in the absence of
rotation of the platform, as shown in FIG. 12A. Upon rotation of
the platform at a first rotational speed f.sub.1 of 400-600 rpm,
blood completely fills lysis subvolume 2 and also flows through
passageway 3 and into blood glucose metering chamber 4 and overflow
chamber 5, shown in FIG. 12B. Blood is retained in blood glucose
chamber 4 either due to capillary pressure or by a sacrificial
valve 6, most preferably a wax valve. Similarly, blood is retained
in blood lysis subvolume 2 by a valve, most preferably a
sacrificial valve 7. Excess blood flows at rotational speed f.sub.1
through overflow channel 8 and into overflow chamber 9, shown in
FIGS. 12C and 12D. Typical values for the first rotational speed
are an acceleration of about 20 to 60 rpm/sec to a final radial
velocity of about 600 rpm.
[0283] After blood is metered and excess blood delivered to
overflow chamber 9, the rotational speed of the disc is reduced to
a rotational speed f.sub.1a of 600 to 100 rpm, typically 60 rpm, to
perform a blanking measurement on sample collection cuvette array
12, total hemoglobin read chamber 23 and blood glucose assay
chamber 11. Measurements of blanking cuvettes 13 and 14 are also
advantageously performed.
[0284] The disc is then accelerated to a second rotational speed
f.sub.2 greater than f.sub.1, and typically in the range of 800 rpm
to about 1000 rpm. At this speed, capillary valve 6 is overcome or
sacrificial valve 6 is released, and about 16 .mu.L of blood from
blood glucose metering chamber 4 flows through capillary 15 and
into blood glucose assay chamber 11, shown in FIG. 12E. Upon
entering assay chamber 11, blood fluid components are forced into
absorbent matrix 10 through depression 11A. The blood fluid is
incubated in matrix 10 for a time sufficient for the reagents 10A
to produce a colored product 10B in an amount proportional to the
amount of glucose in the blood fluid sample. The disc is slowed,
typically to a rotational speed f.sub.4 of about 100 rpm, for
glucose data acquisition using reflectance spectrometry; data
acquisition as the disc is spinning down also enables to instrument
to set t=0 for the assay, based on a decrease in reflectance when
the matrix 10 is wet by the blood fluid components and hence the
matrix's scattering decreases. Development of colored product 10B
is shown in FIG. 12G.
[0285] The disc is then accelerated to rotational speed f.sub.3 of
about 1000 rpm, with release of sacrificial valve 7A and fluid flow
of about 5-6.4 .mu.L of blood from metered subvolume 2 through
capillary 7 and into blood lysis chamber 16 containing about 40
.mu.L of blood lysis buffer. This is shown in FIG. 12H. The mixture
of blood and blood lysis buffer in blood lysis chamber 16 is mixed
by agitation, wherein the platform is accelerated repeatedly +250
rpm/sec to -250 rpm/sec (wherein "+" and "-" indicate acceleration
in different directions), typically 250-500 rpm/sec, over a time
period of about 2 min, typically 1-3 min, as shown in FIGS. 12I and
12J.
[0286] The disc then is accelerated to a rotational speed f.sub.5
of 1000 rpm and sacrificial valve 18 is released. Lysed blood from
blood lysis chamber 16 flows through capillary 17 and into
secondary metering structure 19, as shown in FIG. 12K. The lysed
blood solution sequentially fills throwaway section 20, which is
used as a trap for cell debris, metering section 21 and excess
lysed blood then fills overflow section 24. Filling of metering
chamber 21 is immediately followed by fluid flow through capillary
22 and filling of total hemoglobin read chamber 23. The disc is
spun at rotational speed f.sub.5 for a time sufficient to
substantially completely drain blood lysis chamber 16. The
configuration of the blood fluids on the disc after this spin is
shown in FIG. 12L.
[0287] The disc is then decelerated to a rotational speed of 750
rpm, and sacrificial valve 26 is released. A metered volume of
about 6.4 .mu.L of lysed blood from metering section 21 flows
through capillary 27 and into boronate affinity matrix chamber 28
(shown in FIG. 12M). The lysed blood solution is allowed to
incubate in the chamber for a time of about 1 min, sufficient for
glycated hemoglobin to bind to the matrix. This aspect of the disc
is illustrated in FIG. 12N.
[0288] The disc is then accelerated to a rotational speed of 1000
rpm, and sacrificial valve 32 is released. A volume of about 290
.mu.L of column wash buffer (Isolab #SG-6220) flows from wash
buffer reservoir 30 though capillary 31 and into boronate affinity
matrix chamber 28 (shown in FIG. 12O). The wash buffer displaces
the non-glycated hemoglobin and other components of the lysed blood
fluid from the affinity column matrix and into sample collection
cuvette array 12. FIGS. 12P through 12Q show sequential filling of
the individual cuvettes in sample collection cuvette array 12. The
rotation speed of the disc is reduced, to 60 rpm for sample
collection cuvette array 12 and total hemoglobin read chamber 23 to
be interrogated spectrophotometrically. The glycated fraction of
the blood sample is determine algorithmically by subtracting the
non-glycated hemoglobin fraction in sample collection cuvette array
12 from the total hemoglobin detected in total hemoglobin read
chamber 23.
EXAMPLE 4
Blood Glucose Assay--Chemistries
[0289] Microsystems platforms as provided by the invention were
used to perform blood glucose and glycated hemoglobin assays as
described herein.
Blood Glucose Assay
[0290] A series of blood glucose assays were performed to determine
the precision of repeated glucose assays using the Microsystems
platforms of the invention.
[0291] The assays were performed on a Microsystems platform
according to Examine 3 using a round reagent cuvette (113). 60-70
.mu.L of human whole blood was applied to the disk, and 15 .mu.L
metered into the metering capillary. A glucose reagent pad obtained
from LifeScan was used for performing the glucose determination.
Optical absorbance readings were obtained at 430 nm, 590 nm and 660
nm on the unreacted reagent pad as a control. Blood was released
into the reaction chamber and optical data gathered at one-second
intervals for about 1 min. Absorbance at 590 nm was specific for
the amount of glucose in the sample; absorbance at 430 nm was
specific for hemoglobin in the sample; and absorbance at 660 nm was
used to detect non-specific background absorbance, as described
above.
[0292] A total of seven assays were performed for each of two
samples and the amount of glucose (in mg/dL) determined as
follows:
3 Sample 1 Sample 2 Glucose mg/dL 136.0 113.2 % CV 4.4% 4.5%
[0293] These assays were repeated as above using a Microsystems
platform according to Example 1 comprising a rectangular reagent
cuvette (113). A Biodyne-B membrane (Pall) was impregnated with
glucose oxidase reagents as described above and used for performing
the glucose determination. In these embodiments, chamber 113 had
dimensions of 4 mm.times.5 mm.
[0294] The glucose determination was performed exactly as described
in the first series of experiments. Standard blood glucose assays
were run on these samples in parallel to compare the results
obtained with the Microsystems platforms of the invention with
conventional assays. These results were as follows:
4 mg/dL Reference Method mg/dL Invention 46.8 61.9 85.9 76.8 107.0
115.8 149.0 144.2
[0295] These results established that the microsystems platforms of
the invention were capable of determining blood glucose
concentrations.
Correction for Interfering Substances
[0296] In addition, assays were performed on a Microsystems
platform to Example 3 using a round reagent cuvette (113) and the
glucose results obtained were corrected for hemoglobin
interferences as discussed herein. In these experiments, the
spectrophotometric results obtained were used to calculate blood
glucose with and without correction for the hemoglobin absorbance
signal at 590 nm. The correction factor was determined empirically
using samples comprised of phosphate buffered saline to which known
qualities of glucose and/or hemoglobin were added. The resulting
samples were assayed on the Microsystems platform according to
Example 3 using a round reagent cuvette (113) to give the following
data:
5 Glucose Hemoglobin Concentration Concentration Reflectance
Reflectance Sample (mg/dL) (mg/dL) (590 nm) (430 nm) A 0 0 0.0152
-0.0204 B 0 1.25 0.2116 0.3041 C 75 5 1.2767 0.3452 D 78 0 0.9576
0.1311
[0297] For glucose chromagen alone, Sample D, the Reflectance(430
nm) is 16.1% of the Reflectance(590 nm)
[(0.131-(-0.0204)/(0.9576-0.152)]. For hemoglobin alone, Sample B,
the Reflectance(590 nm) is 60.5% of the Reflectance (430)
[(0.2116-0.0152)/(0.3041-(-0.0204))]. For the combination of
glucose and hemoglobin, Sample C, the Reflectance(430 nm) is the
sum of the Reflectance (430 nm) due to the hemoglobin plus 16.1% of
the Reflectance(590 nm) due to the glucose chromagen; similarly,
the Reflectance(590 nm) is the sum of the Reflectance(590 nm) due
to the glucose chromagen plus 60.5% of the Reflectance(430 nm) due
to the hemoglobin. Using standard algebraic methods, the factor to
be used was calculated to be:
[0298] Reflectance(590 nm) due to glucose chromagen=Reflectance(590
nm)-F.times.Reflectance(430 nm) where F=0.274
[0299] The results shown in FIGS. 14A and 14B illustrate the
difference in the accuracy of the data obtained with and without
correcting for the hemoglobin absorbance signal.
Glycated Hemoglobin Assay
[0300] A series of glycated hemoglobin assays were performed to
determine the precision of repeated binding of glycated hemoglobin
by measuring the amount of hemoglobin put on a phenyl boronate
column and the amount of (non-glycated) hemoglobin that could be
eluted from the column.
[0301] These experiments were performed using a microsystems
platform of the invention as disclosed in Example 3; the phenyl
boronate column material was from a kit obtained from IsoLab
Glyc-Affin GHb. 60-70 .mu.L of human whole blood was applied to the
disk, and 5 .mu.L metered into and mixed with 45 .mu.L of lysis
buffer in the mixing chamber of the platform. 5 .mu.L of the lysed
blood was metered onto the phenyl boronate column and non-glycated
hemoglobin eluted with 290 .mu.L of elution buffer. The reflective
absorbance of the lysed blood and the eluted non-glycated
hemoglobin was determined spectrophotometrically at 430 nm and used
to calculate the amount of non-glycated hemoglobin in the
sample.
[0302] Seven separate assays were performed on each of 2 difference
whole blood samples. These results are as follows:
6 Sample 1 Sample 2 % NGHb, reference method 93 90 % NGHb,
invention 93 92 % CV 1.2% 1.7%
DNA Binding Assays
[0303] These experiments were performed to show that the
microsystems platforms of the invention could efficiently isolate
plasmid DNA from a sample. In these experiments, diatomeceous earth
was used to specifically bind plasmid DNA, and the results were
compared with a standard plasmid DNA extraction kit (Qiagen).
[0304] In these experiments, the Qiagen assay was performed until
the bacterial culture had been disrupted with chaotropic salts. At
this point, the solution was split in half, and one half assayed
using the Qiagen reagents and columns, and the other half 300 .mu.L
assayed using a microsystem platform adapted from the one described
in Example 2. In this embodiment, the diatomeceous earth column was
loaded onto the platform and the sample applied thereto by
centrifugation at about 800 rpm. The column was washed twice with
100 .mu.L of Qiagen buffer PE at about 850 rpm, and the plasmid DNA
was then eluted twice, with 50 .mu.L of Qiagen Buffer EB, once at
600 rpm and again at at 900 rpm. The eluted plasmid DNA from each
preparation was precipitated by the addition of ice cold ethanol to
70% and the DNA pellet collected by centrifugation and resuspended
in 50 .mu.L of 10 mM Tris-HCl, pH 8. 15 .mu.L were analyzed by
conventional agarose gel electrophoresis, stained with ethidium
bromide and osverved under ultraviolet light illumination.
[0305] The results of these assays are shown in FIG. 15, and
illustrate the use of the Microsystems platforms of the invention
for isolating plasmid DNA from a biological fluid sample.
[0306] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims.
* * * * *