U.S. patent application number 10/359395 was filed with the patent office on 2003-09-04 for apparatus and methods for correcting for variable velocity in microfluidic systems.
This patent application is currently assigned to Caliper Technologies Corp.. Invention is credited to Chow, Andrea W., Cohen, Claudia B., Kopf-Sill, Anne R., Parce, John Wallace, Sundberg, Steven A..
Application Number | 20030165960 10/359395 |
Document ID | / |
Family ID | 26726787 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165960 |
Kind Code |
A1 |
Kopf-Sill, Anne R. ; et
al. |
September 4, 2003 |
Apparatus and methods for correcting for variable velocity in
microfluidic systems
Abstract
Electrokinetic devices having a computer for correcting for
electrokinetic effects are provided. Methods of correcting for
electrokinetic effects by establishing the velocity of reactants
and products in a reaction in electrokinetic microfluidic devices
are also provided. These microfluidic devices can have substrates
with channels, depressions, and/or wells for moving, mixing and
monitoring precise amounts of analyte fluids.
Inventors: |
Kopf-Sill, Anne R.; (Portola
Valley, CA) ; Chow, Andrea W.; (Los Altos, CA)
; Cohen, Claudia B.; (Palo Alto, CA) ; Sundberg,
Steven A.; (San Francisco, CA) ; Parce, John
Wallace; (Palo Alto, CA) |
Correspondence
Address: |
QUINE INTELLECTUAL PROPERTY LAW GROUP, P.C.
P O BOX 458
ALAMEDA
CA
94501
US
|
Assignee: |
Caliper Technologies Corp.
1275 California Avenue
Palo Alto
CA
94304
|
Family ID: |
26726787 |
Appl. No.: |
10/359395 |
Filed: |
February 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10359395 |
Feb 5, 2003 |
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10102149 |
Mar 19, 2002 |
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10102149 |
Mar 19, 2002 |
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09093542 |
Jun 8, 1998 |
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6524790 |
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60049013 |
Jun 9, 1997 |
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60076468 |
Mar 2, 1998 |
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Current U.S.
Class: |
435/6.19 ;
435/7.1; 702/19 |
Current CPC
Class: |
Y10S 436/805 20130101;
B01J 2219/00995 20130101; B01L 3/5027 20130101; B01L 2200/14
20130101; G01N 27/447 20130101; G01N 27/44791 20130101; B01L
2300/0627 20130101; G01N 27/44726 20130101; B01J 2219/0097
20130101; B01L 9/527 20130101; B01J 2219/0095 20130101; B01L
2200/143 20130101; Y10S 435/81 20130101; B01L 2300/0816 20130101;
B01L 3/563 20130101; B01L 2400/0415 20130101; Y10S 436/809
20130101; B01L 3/502746 20130101; B01L 3/50273 20130101; B01L
2300/0867 20130101; G01N 33/557 20130101; B01J 19/0093
20130101 |
Class at
Publication: |
435/6 ; 435/7.1;
702/19 |
International
Class: |
C12Q 001/68; G01N
033/53; G06F 019/00; G01N 033/48; G01N 033/50 |
Claims
What is claimed is:
1. A method for determining the rate or extent of a reaction or
assay in a microfluidic system, comprising: converting a first
reaction or assay component having a first velocity (U.sub.1) into
a product having a second velocity (U.sub.p) in a microfluidic
channel; determining at least one velocity selected from the group
consisting of U.sub.1 and U.sub.p; determining the concentration of
the reaction or assay product in a portion of the microfluidic
channel, whereby determining the at least one velocity and the
concentration of the reaction or assay product provides for
determination of the rate or extent of the reaction or assay.
2. The method of claim 1, wherein the first reaction or assay
component is converted into the product by exposing the product to
heat, light, acid, or base.
3. The method of claim 1, wherein the first reaction or assay
component is converted into the product by contacting the first
reaction or assay component with a second reaction or assay
component.
4. A method for determining the rate or extent of a reaction or
assay in a microfluidic system, comprising: contacting a first
reaction or assay component having a first velocity (U.sub.1) to a
second reaction or assay component having a second velocity
(U.sub.2) in a microfluidic channel, thereby permitting formation
of a reaction or assay product with a third velocity (U.sub.p);
determining at least one velocity selected from the group
consisting of U.sub.1, U.sub.2, and U.sub.p; determining the
concentration of the reaction or assay product in a portion of the
microfluidic channel, whereby determining the at least one velocity
and the concentration of the reaction or assay product provides for
determination of the rate or extent of the reaction or assay.
5. A method for determining the rate or extent of a reaction or
assay in an electrokinetic microfluidic system, comprising:
providing an electrokinetic microfluidic device having a
microfluidic channel; applying an electric field along the length
of the microfluidic channel; contacting a first reaction or assay
component having a first charge mass ratio (CM.sub.1) and a first
velocity (U.sub.1) to a second reaction or assay component having a
second charge mass ratio (CM.sub.2) and a second velocity (U.sub.2)
in the microchannel, thereby permitting formation of a reaction or
assay product with a third charge mass ratio (CM.sub.p) and a third
velocity (U.sub.p); determining at least one velocity selected from
the group consisting of U.sub.1, U.sub.2, and U.sub.p; determining
the concentration of the reaction or assay product in a portion of
the microfluidic channel, whereby determining the at least one
velocity and the concentration of the reaction or assay product
provides for a determination of the rate or extent of the
reaction.
6. The method of claim 5, wherein U.sub.1 is proportional to
CM.sub.1, U.sub.2 is proportional to CM.sub.2, and U.sub.p is
proportional to CM.sub.P.
7. The method of claim 1, 4 or 5, the first reactant or product
further comprising a detectable label.
8. The method of claim 4 or 5, wherein the velocity of U.sub.1 or
U.sub.2 is zero.
9. The method of claim 4 or 5, further comprising measuring the
velocity of the first reaction component or the second reaction
component and determining U.sub.p.
10. The method of claim 4 or 5, further comprising measuring
U.sub.p.
11. The method of claim 1, 4 or 5, further comprising determining
the reaction rate constant (k) for the formation of the
product.
12. The method of claim 4 or 5, wherein the second velocity U.sub.2
and the third velocity U.sub.p are different.
13. A method of determining concentration of a reaction or assay
product (C.sub.p) in a microfluidic device, the method comprising
the steps of: (i) converting a labeled first reactant or assay
component having a velocity (U.sub.r) and a label (L.sub.r), the
labeled first reactant or assay component producing a signal
(S.sub.as) in a signal detection system, to a reaction or assay
product comprising a label L.sub.p, having a velocity (U.sub.p),
wherein (U.sub.r) does not equal (U.sub.p) and wherein L.sub.p
comprises component elements of L.sub.r; and, (ii) detecting a
resulting change in S.sub.as, wherein the change in S.sub.as is an
indicator of C.sub.p.
14. The method of claim 13, wherein the resulting change in
S.sub.as is an indicator of U.sub.r.
15. The method of claim 13, wherein L.sub.r comprises a
fluorophore.
16. The method of claim 13, wherein the first reactant or assay
component is contacted by the second reactant or assay component in
microfluidic channel in a first microfluidic channel region and
wherein S.sub.as is detected by monitoring an output from a label
detection device which is mounted to view a second microfluidic
channel region in fluid communication with the first microfluidic
channel region.
17. The method of claim 1, 4, 5 or 16, further comprising the step
of injecting one or more fluorescent dyes or other flow markers
into the microfluidic channel to generate a flow profile versus
time mask file.
18. The method of claim 1, 4, 5 or 16, further comprising the step
of injecting one or more labeled size markers into the microfluidic
channel to generate a fluorescence intensity versus time mask
file.
19. The method of claim 1, 4, 5 or 16, further deconvolution of a
complex signal with a time mask file.
20. The method of claim 1, 4, 5 or 16, further comprising baseline
subtraction by injecting a series of blanks into the microfluidic
channel in a control experiment to measure a time dependent
baseline.
21. The method of claim 1, 4, 5 or 16, further comprising injecting
at least one flow marker into the microfluidic channel, sampling
signal from the flow marker and generating a flow profile versus
time mask file.
22. The method of claim 13, further comprising baseline subtraction
of reactant signal (S.sub.r) produced by the labeled first reactant
from S.sub.as to provide a normalized signal (S.sub.n) produced by
the product.
23. The method of claim 13, wherein the step of converting the
labeled first reactant or assay component to a reaction or assay
product is performed by contacting the labeled first reactant or
assay component with a second reactant or assay component to form a
reaction or assay product comprising a label L.sub.p having a
velocity (U.sub.p), wherein (U.sub.r) does not equal (U.sub.p) and
wherein L.sub.p comprises component elements of L.sub.r.
24. The method of claim 13 wherein L.sub.p is formed from the
components of L.sub.r by treating the first reactant with a label
modifier selected from light, heat, electrical charge, a
polymerization agent, and a catalyst.
25. The method of claim 13, wherein L.sub.p and L.sub.r comprise
the same label moiety.
26. The method of claim 13, wherein L.sub.p and L.sub.r comprise
different label moieties.
27. The method of claim 4, 5 or 13, wherein the assay or reaction
is in a continuous flow format.
28. The method of claim 4, 5 or 13, wherein flux is conserved in
the assay or reaction.
29. The method of claim 4, 5 or 13, wherein the reaction or assay
is a non-fluorogenic reaction or assay.
30. The method of claim 4, 5 or 13, wherein the first reactant or
assay component is contacted to the second reactant or assay
component in a microfluidic channel.
31. The method of claim 4, 5 or 13, wherein the first reactant
flows down a first channel and the second reactant is periodically
injected into the first channel to contact the first reactant.
32. The method of claim 4, 5 or 13, wherein the first reactant or
assay component flows down a first microfluidic channel and the
second reactant or assay component is periodically injected into
the first microfluidic channel, whereby the first reactant or assay
component contacts the second reactant or assay component in the
first microfluidic channel.
33. The method of claim 4, 5 or 13, wherein the second reactant or
assay component is injected into a microfluidic channel comprising
the first reactant for a duration of from 0.001 to 10 min.
34. The method of claim 4, 5 or 13, wherein the second reactant or
assay component is reacted with the first reactant or assay
component in a non-fluorogenic continuous flow mode.
35. The method of claim 4, 5 or 13, wherein the first reactant or
assay component comprises a moiety derived from an antibody, an
antigen, a ligand, a receptor, an enzyme, an enzyme substrate, an
amino acid, a peptide, a protein, a nucleoside, a nucleotide, a
nucleic acid, a fluorophore, a chromophore, biotin, avidin, an
organic molecule, a monomer, a polymer, a drug, a polysaccharide, a
lipid, a liposome, a micelle, a toxin, a biopolymer, a
therapeutically active compound, a I molecule from a biological
source, a blood constituent, or a cell.
36. The method of claim 4, 5 or 13, wherein the first assay
component is a component of a biological assay.
37. The method of claim 4, 5 or 13, wherein the first assay
component is a component of a non-biological assay.
38. The method of claim 4, 5 or 13, wherein the first assay
component is a component of a chemical synthetic reaction.
39. The method of claim 4, 5 or 13, further comprising contacting
the first or second reactant or assay component with at least one
additional reactant.
40. The method of claim 4, 5 or 13, further comprising the
formation of at least one additional reactant or product.
41. The method of claim 4, 5 or 13, further comprising determining
the velocity of an additional reactant, assay component, or
product.
42. The method of claim 4, 5 or 13, further comprising the step of
injecting a series of blanks into a channel comprising the first
reactant to determine a time-dependent baseline.
43. The method of claim 4, 5 or 13, wherein the first or second
reactant or assay component is dissolved in an aqueous buffer.
44. The method of claim 4, 5 or 13, wherein the first or second
reactant or assay component is dissolved in an aqueous buffer
having a pH between 3 and 11.
45. The method of claim 4, 5 or 13, further comprising determining
the flux for the first reaction component, the second reaction
component and the product.
46. The method of claim 4, 5 or 13, wherein the first reaction
component and the second reaction component are mixed at a first pH
which facilitates reaction of the first and second reaction
component, wherein unreacted first component, unreacted second
component or product are subsequently electrokinetically
transported at a second pH which inhibits reaction of the first and
second components.
47. The method of claim 4, 5 or 13, wherein the first reaction
component is an enzyme.
48. The method of claim 4, 5 or 13, wherein the first reactant or
assay component is an enzyme, the second reactant or assay
component is a substrate and the product is formed by conversion of
the substrate by the enzyme into the product.
49. The method of claim 4, 5 or 13, wherein the first reactant or
assay component and the second reactant or assay component
hybridize to form the product, which product has a velocity faster
than either the first component or the second component.
50. The method of claim 4, 5 or 13, wherein the first reactant or
assay component and the second reactant or assay component
hybridize to form the product, which product has a velocity slower
than either the first component or the second component.
51. The method of claim 4, 5 or 13, further comprising measuring
the concentration of the product spectrophotometrically, or
optically.
52. The method of claim 4, 5 or 13, wherein the first reactant or
assay component and the second reactant or assay component comprise
a ligand and a ligand binder, wherein the first component
hybridizes to the second component.
53. The method of claim 4, 5 or 13, wherein the first reactant or
assay component and the second reactant or assay component comprise
a ligand and a ligand binder wherein the first reactant or assay
component hybridizes to the second reactant or assay component, and
the ligand and ligand binder are selected from the group consisting
of: a first nucleic acid and a second nucleic acid; an antibody and
an antibody ligand; a receptor and a receptor ligand; biotin and
avidin; a protein and a complementary protein; and, a carbohydrate
and a carbohydrate binding moiety.
54. The method of claim 4, 5 or 13, the first reactant or assay
component further comprising a biotin moiety, the second reactant
or assay component further comprising a streptavidin moiety and the
product further comprising the biotin moiety hybridized to the
streptavidin moiety.
55. The method of claim 13, wherein the step of converting the
labeled first reactant or assay component to a reaction or assay
product is performed by contacting the labeled first reactant or
assay component with a second reactant or assay component to form a
reaction or assay product comprising a label L.sub.p having a
velocity (U.sub.p), wherein (U.sub.r) does not equal (U.sub.p) and
wherein L.sub.p comprises component elements of L.sub.r, wherein
the first reactant or assay component and the second reactant or
assay component are contacted in a microfluidic channel.
56. The method of claim 4, 5 or 55, further comprising measuring
the concentration of the product in a microfluidic channel, and,
optionally, measuring the concentration of the first reaction or
assay component in a portion of the microfluidic channel and,
optionally, measuring the concentration of the second reaction or
assay component in a portion of the microfluidic channel.
57. The method of claim 4, 5 or 55, further comprising measuring a
length of time for travel of the first reaction component or the
second reaction component along a selected length of the
microfluidic channel.
58. The method of claim 4, 5 or 55, further comprising measuring a
length of time for travel of the product along a selected length of
the microfluidic channel.
59. The method of claim 4, 5 or 55, wherein the first component,
the second component, and the product are soluble in an aqueous
solvent, wherein the microchannel comprises said aqueous
solvent.
60. The method of claim 4, 5 or 55, further comprising providing an
electrokinetic microfluidic device having the microfluidic channel;
and, applying an electric field along the length of the
microchannel.
61. The method of claim 4, 5 or 55, wherein the first and second
component have a K.sub.a of between about 10.sup.5 and
10.sup.15.
62. A method of detecting a product formed by contacting a first
and second component of a reaction comprising: contacting the first
and second reactant in a microfluidic channel, wherein the first
reactant comprises a detectable label, thereby producing a product
comprising 5 the detectable label, which product has a different
electrokinetic mobility than the first or second reactant; flowing
the product and any first or second reactant remaining in the
channel subsequent to said contacting step past a detector, wherein
the label on the first reactant and the label on the product
comprise the same detectable moiety; and, determining at least one
of: concentration of the product, rate of product formation, or
amount of product produced.
63. The method of claim 62, wherein the detectable label is a
fluorophore.
64. The method of claim 62, wherein the detectable label is a
fluorophore and the reaction is non-fluorogenic.
65. The method of claim 62, the method further comprising measuring
or calculating the velocity of the first reactant, the second
reactant, or the product.
66. The method of claim 62, wherein flux of the detectable label is
conserved.
67. The method of claim 62, wherein the first or second reactant is
periodically injected into the channel.
68. The method of claim 62, wherein the first reactant, the second
reactant and the product are flowed continuously in the
channel.
69. The method of claim 62 further comprising detecting phase shift
of reactant and product waves.
70. A method of dispensing representative mixtures in a
microfluidic system, comprising: (i) introducing a first mixture
into a first microfluidic channel, the mixture comprising at least
first and second materials; (iii) transporting the first and second
materials through the first channel, wherein the first and second
mixtures travel at different velocities in the channel; (iv) gating
an aliquot of first and second materials into the second channel
for a selected period of time, the relative amount of first and
second materials in the aliquot being proportional to the flux of
first and second materials in the first mixture in the first
channel, thereby dispensing a representative mixture of the first
and second components.
71. The method of claim 70, wherein flux is conserved in the
system.
72. The method of claim 70, wherein the first and second compounds
have different fluxes during electrokinetic movement.
73. The method of claim 70, wherein the first or second material is
labeled, the method comprising measuring signal from the aliquot of
first or second labeled material, wherein the amount of labeled
material is determined by measuring the signal.
74. The method of claim 70, comprising providing a microfluidic
device comprising a body structure having at the first channel and
at least a second channel disposed therein, the first and second
channels communicating at a first intersection.
75. The method of claim 74, wherein the first and second channels
communicate at a crossing intersection.
76. The method of claim 70, wherein the first and second materials
are moved electrokinetically in the first channel.
77. The method of claim 70, further comprising measuring the amount
of first or second material in the aliquot.
78. The method of claim 70, wherein the first material is a
reactant and the second material is a product of a reaction of the
reactant.
79. The method of claim 70, wherein a separation of the first and
second materials occurs in the second channel within the
aliquot.
80. The method of claim 70, wherein the aliquot is injected into
the second channel by a voltage change.
81. The method of claim 70, wherein the aliquot is injected into
the second channel by a current change.
82. The method of claim 70, the method further comprising detecting
the first or second material using a total amount detector which
measures label across the entire aliquot.
83. The method of claim 70, the method further comprising detecting
the first or second material with a label detector comprising a
wide photomultiplier tube slit and a photomultiplier tube.
84. The method of claim 70, the method further comprising detecting
the first or second material by total photobleaching, a long window
fluorescent detector or an electrochemical detector which samples
the entire aliquot.
85. A method of correcting data in a microfluidic system for
effects of stacking of charged molecules in a microfluidic channel
comprising: injecting at least one labeled blank into the
microfluidic channel; monitoring control signal from the labeled
blank in the channel to determine the signal of the blank over
time; and, subtracting the control signal of the blank over time
from experimental data from an analyte in the microfluidic
channel.
86. The method of claims 85, further comprising injecting at least
one flow marker into the microfluidic channel, sampling signal from
the flow marker and generating a flow profile versus time mask
file.
87. A method of correcting data in a microfluidic system for
effects of stacking of charged molecules in a microfluidic channel
comprising: injecting a series of labeled control molecules in
discreet high-salt buffer control plugs into the microfluidic
channel to characterize timing of the control plugs as they pass
the detection point; creating a control label intensity versus time
data mask file; and, correlating the label intensity versus time
mask file to experimental data from an analyte in the microfluidic
channel to determine which times from the experimental data
correlate with a sample plug.
88. The method of claims 85 or 87 wherein the label is
fluorescent.
89. A method of regulating a flowing reaction in a microfluidic
channel comprising: mixing a plurality of reaction components in a
first buffer, thereby providing a mixture of reaction components;
electrokinetically transporting the mixture of reaction components
in a microfluidic channel, thereby permitting the mixture of
reaction components to react; applying a reaction inhibitor to at
least a portion of the reaction mixture, thereby inhibiting further
reaction of the reaction components in the portion.
90. The method of claim 89, wherein the inhibitor is selected from:
an aliquot of high pH buffer; an aliquot of low pH buffer; an
aliquot of buffer comprising an ion chelator; an aliquot of high
temperature buffer, an aliquot of low temperature buffer, heat, and
light.
91. The method of claim 89, wherein the inhibitor is applied to
selected regions of the flowing mixture of reaction components,
wherein the selected regions bracket regions which are not selected
in which the inhibitor is not applied.
92. The method of claim 89, wherein the inhibitor is added in a
time-gated aliquot.
93. The method of claim 89, wherein the mixture of reaction
components are electrokinetically flowed for a selected period of
time.
94. The method of claim 89, wherein the mixture of reaction
components are electrokinetically flowed for a selected distance in
the microfluidic channel.
95. A microfluidic apparatus for determining a rate of formation of
a moving analyte on an electrokinetic microfluidic substrate
comprising: a microfluidic substrate holder for receiving a
microfluidic substrate during operation of the apparatus, which
substrate holder has a microfluidic substrate viewing region; an
analyte detector mounted proximal to the substrate viewing region
to detect the moving analyte in a portion of the substrate viewing
region; and, a computer operably linked to the analyte detector,
which computer determines the rate of formation of the analyte,
correcting for the effects of the motion of the analyte.
96. A microfluidic apparatus for determining a rate of formation of
a moving analyte on an electrokinetic microfluidic substrate
comprising: a microfluidic substrate holder for receiving a
microfluidic substrate during operation of the apparatus, which
substrate holder has a microfluidic substrate viewing region;
analyte movement means for imparting velocity to the analyte in a
channel of the microfluidic substrate during operation of the
apparatus; detection means for detecting the moving analyte in the
substrate viewing region; and, correction means for correcting the
observed rate of formation of the moving analyte for the effects of
the velocity of the analyte, which means are operably linked to the
means for detecting the moving analyte.
97. The microfluidic apparatus of claim 96, wherein the correction
means comprise a computer operably linked to the detection means,
which computer determines the rate of formation of the analyte, and
which computer corrects for the effects of the motion of the
analyte.
98. The microfluidic apparatus of claim 95, 96, wherein the
apparatus is in use and further comprises a microfluidic substrate
mounted in the microfluidic substrate holder.
99. A microfluidic apparatus, comprising: a microfluidic substrate
comprising a body having a top portion, a bottom portion and an
interior portion; the interior portion comprising at least two
intersecting channels, wherein at least one of the two intersecting
channels has at least one cross sectional dimension between about
0.1 .mu.m and 500 .mu.m; a detection zone for detecting the analyte
in at least one of the two intersecting channels, when the analyte
is in motion; and, a data analyzer which determines a rate of
formation of the analyte in motion, wherein the analyzer comprises
a processor which calculates the flux or velocity of the analyte in
the detection zone.
100. The microfluidic apparatus of claim 99, wherein the apparatus
is formed by etching at least two intersecting groves in a top
surface of the bottom portion, the top portion being fused to the
top surface of the bottom portion, thereby forming the interior
portion.
101. The microfluidic apparatus of claim 99, the data analyzer
comprising a computer with software for determining the rate of
formation of moving analytes on a microfluidic device in which flux
is conserved.
102. The microfluidic apparatus of claim 99, the top portion of the
device further comprising a plurality of wells in fluid
communication, and an electrokinetic fluid direction system
comprising a plurality-of electrodes adapted to fit into the
plurality of wells.
103. The microfluidic apparatus of claim 95, 96 or 99, wherein the
apparatus comprises an optical or fluorescent detection system for
viewing the analyte.
104. The apparatus of claim 95, 96 or 99 comprising an
electrokinetic fluid direction system.
105. The apparatus of claim 104, comprising an electrode disposed
within a well formed in the top portion of the body.
106. The apparatus of claim 95, 96 or 99 further comprising a
microscope.
107. An apparatus for determining the concentration of a product in
a non-fluorogenic format, comprising: conversion means for
converting a labeled first reactant or assay component to a second
labeled component; signal detection means for detecting signal
amplitude from the labeled first reactant or assay component and
second labeled component; concentration calculation means for
calculating the concentration of the product by measuring a change
in signal amplitude which results from converting the first
reactant or assay component into the second labeled component.
108. The apparatus of claim 107, wherein the signal detection means
comprises an optical detector for detecting a light signal.
109. The apparatus of claim 107, wherein the signal detection means
comprises an optical detector for detecting a fluorescent
signal.
110. The apparatus of claim 107, wherein the concentration
calculation means comprises a digital computer.
111. The apparatus of claim 107, wherein the conversion means
comprises a microfluidic substrate having at least two intersecting
channels fabricated therein.
112. An apparatus for determining the concentration of a product in
a non-fluorogenic format, comprising: a microfluidic substrate
holder for receiving a microfluidic substrate, the holder
comprising a substrate viewing region; a signal detector mounted
proximal to the substrate viewing region; a signal output processor
which converts variations in signal amplitude from the signal
detector into concentration measurements for at least one of a
plurality of moving analytes comprising one or more labels which
have the same signal output, which plurality of analytes are
detected by the signal detector.
113. The apparatus of claim 112, wherein the signal detector
detects one or more label selected from the group of a fluorescent
label, a calorimetric label, and, a radioactive label.
114. The apparatus of claim 112, comprising: a microfluidic
substrate having a plurality of microchannels fabricated therein,
the substrate mounted in the substrate holder, wherein the
apparatus in use comprises a first analyte comprising a first
label, and a second analyte comprising the same first label,
wherein the mobility of the first and second analyte are
different.
115. The apparatus of claim 107 or 112, further comprising an
electrokinetic fluid control means.
116. The apparatus of claim 107 or 112, comprising a microfluidic
substrate with a reaction channel fabricated therein, wherein,
during use of the apparatus, first and second reactants are
contacted in the reaction channel, wherein the reaction channel is
in fluid communication with a first reagent introduction channel,
wherein the second reactant is introduced into the reaction channel
from the first reagent introduction channel by time gated
injection.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
60/049,013 filed Jun. 9, 1997 entitled "APPARATUS AND METHODS FOR
CORRECTING FOR ELECTROKINETIC EFFECTS IN MICROFLUIDIC SYSTEMS" by
Kopf-Sill and Parce (Attorney docket no. 017646-00360) and U.S.
Ser. No. 60/076,468 filed Mar. 2, 1998 "HIGH THROUGHPUT SCREENING
APPLICATIONS OF MICROFLUIDIC SYSTEMS" by Cohen et al. (Attorney
docket number 100/04000); the present application claims priority
to each of these applications and incorporates each of the
applications herein in their entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention provides microfluidic apparatus,
methods and integrated systems for the separation and analysis of
reaction components, fluid velocities, component velocities and
reaction rates. Exemplary software is provided.
BACKGROUND OF THE INVENTION
[0003] There exists a need for assay methods and associated
equipment and devices that are capable of performing repeated,
accurate assays that operate at very small volumes. U.S. Ser. No.
08/761,575 entitled "High Throughput Screening Assay Systems in
Microscale Fluidic Devices" by Parce et al. (see also, U.S. Ser.
No. 08/881,696) provides pioneering technology related to
microscale fluidic devices, including electrokinetic devices. The
devices are generally suitable for assays relating to the
interaction of biological and chemical species, including enzymes
and substrates, ligands and ligand binders, receptors and ligands,
antibodies and antibody ligands, as well as many other assays.
[0004] In the electrokinetic devices provided by Parce et al., an
appropriate fluid is placed in a microchannel etched into a
substrate having functional groups present at the surface. The
groups ionize when the surface is contacted with an aqueous
solution. For example, where the surface of the channel includes
hydroxyl functional groups at the surface, protons can leave the
surface of the channel and enter the fluid. Under such conditions,
the surface possesses a net negative charge, whereas the fluid will
possess an excess of protons, or positive charge, particularly
localized near the interface between the channel surface and the
fluid. By applying an electric field along the length of the
channel, cations will flow toward the negative electrode. Movement
of the positively charged species in the fluid pulls the solvent
with them.
[0005] Improved methods and devices for monitoring reactions
between chemical or biological species would be desirable.
Electrokinetic microfluidic devices and assays using such devices
are particularly desirable, due to the general adaptability of
electrokinetic movement of small volumes of fluids to high
throughput assay systems. The present invention fulfills these and
a variety of other needs.
SUMMARY OF THE INVENTION
[0006] It has now been discovered that accurate determination of
the reaction rate of a reaction conducted in a microscale fluidic
device is facilitated by consideration of the velocity of the
components in the reaction. In a microscale system in which the
flux of reactants and reaction products is conserved, the velocity
of at least one reactant or product is determined and the
concentration of a reaction product is measured or calculated,
facilitating determination of the reaction rate.
[0007] The concentration of products and reactants is typically
measured at a selected position on the microscale fluidic device,
e.g., spectrophotometrically, radioscopically, electrochemically,
or optically. Velocity rates are optionally determined by measuring
the speed of a component in a portion of the microscale fluidic
device over time, or are determined by consideration of the
parameters influencing velocity, e.g., the charge and mass of the
component in an electric field. As described herein, methods of
determining velocities are also provided in a constant flux state
by indirect measurements, e.g., the velocity of a reactant or
product can be determined by measuring a different reactant or
product. Thus, any or all reactants or product velocities can be
observed or determined. Velocity markers are also optionally used
to approximate velocity. In one series of embodiments,
electrokinetic devices and fluid injection schemes are described
which self-correct for velocity effects on fluids.
[0008] A variety of reactants and products are assessed by these
methods, including ligand and ligand binders such as an antibody
and an antibody ligand, a receptor and a receptor ligand, biotin
and avidin, proteins and complementary binding proteins,
carbohydrates and carbohydrate binding moieties, nucleic acids,
etc. Reactions which are monitored are fluorogenic or
non-fluorogenic. A variety of microscale apparatus are adaptable to
the methods such as microvalve and micropump arrangements, and
particularly electrokinetic devices and the like. Multiple
reactants and products are optionally assessed by serial or
simultaneous detection methods or a combination thereof.
[0009] In one preferred class of embodiments, the microscale
fluidic device provides for electrokinetic movement of reactants
and products along a microfluidic channel. An electrokinetic
microfluidic device is provided, having a microfluidic channel. An
electric field is applied along the length of the microchannel,
thereby causing charged species such as reactants, solvent
molecules and products to move along the length of the channel due
to electrophoretic flow, as well as by electroosmotic flow of the
solvent in the channel. A first reaction component having a first
charge mass ratio (CM.sub.1) and a first velocity (U.sub.1) is
contacted to a second reaction component having a second charge
mass ratio (CM.sub.2) and a second velocity (U.sub.2) in the
microchannel, thereby permitting formation of a reaction product
with a third charge mass ratio (CM.sub.p) and a third velocity
(U.sub.p). Additional reaction components and products are
optionally provided and assessed for velocities and concentrations.
In one embodiment, a reactant can have a velocity of zero, e.g.,
because it is fixed to a substrate of the detection apparatus.
However, the more typical case is for flowing reactants, where all
reactants and products are flowing in channels of the system.
Typically, the product has a velocity different from one or more
reactants in the system.
[0010] Apparatus for practicing the methods of the invention are
provided. For example, a microfluidic detection apparatus for
determining the rate of formation of a moving analyte on an
electrokinetic microfluidic substrate is provided. The apparatus
has a microfluidic substrate holder for receiving a microfluidic
substrate during operation of the apparatus, having a microfluidic
substrate viewing region. An analyte detector such as a phototube,
photodiode, a charge coupled device, a camera, a microscope, a
spectrophotometer, or the like is mounted proximal to the substrate
viewing region to detect the moving analyte in a portion of the
substrate viewing region. A computer operably linked to the analyte
detector is provided. The computer determines the rate of formation
of the analyte, correcting for the effects of the motion of the
analyte, e.g., by determining or collating the velocities of one or
more components and the concentrations of one or more components
and calculating the rate of formation of one or more components,
correcting for the velocity of the components. In preferred
embodiments, the apparatus also includes an electrokinetic fluid
direction system for moving fluids in the microfluidic substrate,
such as one or more electrodes which fit into wells of the
substrate, operably coupled to one or more electrical power
supply.
[0011] Electrokinetic microfluidic devices are also provided. The
devices have a substrate or body with a top portion, a bottom
portion and an interior portion. The interior portion has at least
two intersecting channels, with at least one of the two
intersecting channels having at least one cross sectional dimension
between about 0.1 .mu.m and 500 .mu.m. The device has an
electrokinetic fluid direction system for moving an analyte through
at least one of the two intersecting channels, a detection zone for
detecting the analyte within at least one of the two intersecting
channels, when the analyte is in motion, and a data detection
device for detecting the analyte in the detection zone. A data
analyzer which determines a rate of formation of the analyte in
motion, such as a computer, is operably connected to the
microfluidic device, e.g., with cables to the data detection
device, or by recording data on the data collection device and
transporting the recorded data (e.g., on a computer-readable
storage medium) to the computer. Typically, the computer has
appropriate software for determining reaction rates and other
related information.
[0012] In one embodiment, at least two intersecting channels are
etched in a top surface of the bottom portion, with the top portion
being fused to the top surface of the bottom portion, thereby
forming the interior portion disposed between the top portion and
the top surface of the bottom portion. When heat lamination of
glass or polymeric surfaces is performed, the glass or polymer
fuses, typically with no seam existing between the top and bottom
portion of the resulting microfluidic chip. In one preferred
embodiment, the top portion of the device has a plurality of wells
in fluid communication with the electrokinetic fluid direction
system comprising an electrode adapted to fit into at least two of
the plurality of wells. By applying an electric current with the
electrode, solvent and analyte molecules are moved through the
channels.
BRIEF DESCRIPTION OF THE DRAWING
[0013] FIG. 1 is a schematic depiction of the basic concept of
continuous flow non-fluorogenic binding assays on microchips
showing changes in electrophoretic mobility over time and distance,
including signal output.
[0014] FIG. 2 is a graph providing model predictions of a
non-fluorogenic binding assay with large association constant
K.sub.a.
[0015] FIG. 3 is a graph providing model predictions of the
fluorescence signal of a non-fluorogenic binding assay at three
values of the association constant K.sub.a.
[0016] FIGS. 4A-4C provide a schematic of an integrated apparatus
of the invention and flowchart operations of software for data
manipulation.
[0017] FIG. 5 is a schematic of an exemplar fluorescent assay
apparatus of the invention.
[0018] FIG. 6 is a schematic representation of a fluorescent assay
of the invention.
[0019] FIG. 7 is a schematic of the channel and reagent well layout
of Caliper LabChip.TM. designated "7A."
[0020] FIG. 8 is a mobility shift signal measured (solid curve) for
the binding reaction of B-T.sub.10-FL and streptavidin under a
continuous flow mode for injection times of 2.5 s, 5 s, 10 s, 15 s.
The concentrations for B-T.sub.10-F and streptavidin were 3.1 .mu.m
and 78 nM, respectively. The dashed curve depicts model
predictions.
[0021] FIG. 9 is a mobility shift signal measured (solid curve) for
the competitive binding reaction of B-T.sub.10-Fl and biotin with
streptavidin for a 12-s injection time in a continuous flow mode.
The concentrations for B-T.sub.10-F and streptavidin were 3.1 .mu.M
and 78 nM, respectively. The concentrations of biotin were 0, 0.78,
1.6, 2.3, and 3.1 .mu.M. The dashed curve depicts model
predictions.
[0022] FIG. 10 is a plot of the fluorescence level versus the
reciprocal of the total concentration of biotin-containing
species.
[0023] FIG. 11 shows experimental data and model calculations of a
non-fluorogenic PKA enzyme assay in a continuous flow mode.
[0024] FIG. 12 shows an example of the progression of a phosphatase
reaction on the exemplar fluorogenic substrate dFMUP.
[0025] FIG. 13 is a fluorescence trace of the titration of
substrate in a microchip phosphatase assay.
[0026] FIG. 14 is a fluorescence trace of the titration of
substrate in a microchip phosphatase assay as a function of
inhibitor concentration.
[0027] FIG. 15 is a Lineweaver Burke plot used to determine Km and
Ki for the phosphatase assay.
[0028] FIG. 16 is the third hour of an eight-hour experiment for a
continuous flow phosphatase assay on a microchip with enzyme
inhibition.
[0029] FIG. 17 is the summary of the eight-hour phosphatase
inhibition experiment showing continuous inhibition for the
duration of the study.
[0030] FIG. 18 is a schematic of the exemplar protease reaction on
a microchip.
[0031] FIG. 19 is the raw data from an exemplar protease reaction
on a microchip as a function of increasing FRET substrate
concentration.
[0032] FIG. 20 is a Lineweaver Burke plot for determination of Km
for the protease assay.
[0033] FIG. 21 is the third hour of a twelve-hour inhibition
experiment for a continuous flow protease assay on a microchip.
[0034] FIG. 22 is the raw data for the first 1000 seconds of each
of the first nine hours of a protease reaction for the continuous
flow inhibition assay.
[0035] FIG. 23 is a summary of the inhibition observed for the
first nine hours of the protease assay on a microchip.
[0036] FIG. 24 is a schematic of an exemplar kinase reaction on a
microchip.
[0037] FIG. 25 is a schematic of microfluidic devices used in
performing the non-fluorogenic kinase assays described herein (the
"28A" and "28B" LABCHIPS.TM.).
[0038] FIG. 26 is the fluorescence data and a Lineweaver Burke plot
for the Km determination for PKA in a microchip.
[0039] FIG. 27 is a fluorescence trace for the PKA assay
demonstrating the mobility shift observed when enzyme is pulsed
into a continuous stream of fluorescent substrate for various
periods of time.
[0040] FIG. 28 is a fluorescence trace for the protease assay
demonstrating the concentration dependent mobility shift observed
when inhibitor is pulsed into a continuous stream of substrate and
enzyme for two concentrations of inhibitor.
[0041] FIG. 29 is an extended time phosphatase assay (hour 3 of an
8 hour data run) with no reagent replacement.
DEFINITIONS
[0042] Flux ("J") is equal to the velocity of analyte molecules
(generally referred to herein as "U") times the concentration of
the analyte molecules (generally referred to as "C") in a selected
microfluidic system. Flux is "conserved" in a microfluidic system,
such as a microchannel, when U times C is constant for a selected
set of analyte molecules, such as reactants, products or both. For
example, in a three component system, having a first reaction
component with a mass concentration C.sub.1 and a velocity U.sub.1,
a second reaction component with velocity U.sub.2 and concentration
C.sub.2, and a product, with velocity U.sub.p and concentration
C.sub.p, flux is constant when
U.sub.1wC.sub.1w+U.sub.2wC.sub.2w+U.sub.pwC.sub.pw=U.sub.1zC.sub.1z+U.sub-
.2zC.sub.2z+U.sub.pzC.sub.pz where w is one point in the channel
and z is a second point in the channel. An alternative notation is
[U.sub.1C.sub.1+U.sub.2C.sub.2+U.sub.pC.sub.p].sub.w=[U.sub.1C.sub.1+U.su-
b.2C.sub.2+U.sub.pC.sub.p].sub.z. A more general notation that
allows for multiple product (P) or reactant (R) species is: 1 h = 1
m C R h U h = i = 1 n C P i U i
[0043] where C is mass concentration (not molar concentration), m
is the number of species before the reaction, and n is the number
of species after the reaction. Thus, the sum of the mass
concentration times the velocity of each of the species before a
reaction is equal to the sum of the mass concentration times the
velocity of each of the species after a reaction. In the cases when
the reaction yields no net change in the total number of molecules,
the molar flux as well as the mass flux are conserved.
[0044] "Velocity" typically refers to the distance a selected
component travels (l) divided by the time (t) required for the
travel. In many embodiments, the velocity under consideration is
essentially constant, e.g., for the travel of reaction components
along the length of a microchannel under a constant rate of current
in an electrokinetic system. Although products of reactions
typically change velocity as they are made from, or by, reactants,
the velocity change is often considered to be instantaneous because
the product reaches its terminal velocity in the system in a very
short period of time. Thus, the velocity of a product is
essentially constant immediately following formation of the
product. Where the velocity changes significantly over time, due,
e.g., to change of applied current in an electrokinetic system, or
where a change from substrate to product results in a slow
acceleration (or deceleration) in the system, an "instantaneous
velocity" equal to the change in distance for a selected time
(.DELTA.l/.DELTA.t) can be determined by graphing distance against
time and taking the tangent of the resulting function at a
particular point in time.
[0045] A "microfluidic" channel is a channel (groove, depression,
tube, etc.) which is adapted to handle small volumes of fluid. In a
typical embodiment, the channel is a tube having at least one
subsection with a cross-sectional dimension of between about 0.1
.mu.m and 500 .mu.m; ordinarily, the channel is closed over a
significant portion of its length, having top, bottom and side
surfaces.
[0046] As used herein, "electrokinetic material transport systems"
or "electrokinetic devices" include systems which transport and
direct materials within an interconnected channel and/or chamber
containing structure, through the application of electrical fields
to the materials, thereby causing material movement through and
among the channel and/or chambers, i.e., cations will move toward
the negative electrode, while anions will move toward the positive
electrode. Such electrokinetic material transport and direction
systems include those systems that rely upon the electrophoretic
mobility of charged species within the electric field applied to
the structure. Such systems are more particularly referred to as
electrophoretic material transport systems. Other electrokinetic
material direction and transport systems rely upon the
electroosmotic flow of fluid and material within a channel or
chamber structure which results from the application of an electric
field across such structures. In brief, when a fluid is placed into
a channel which has a surface bearing charged functional groups,
e.g., hydroxyl groups in etched glass channels or glass
microcapillaries, those groups can ionize. In the case of hydroxyl
functional groups, this ionization, e.g., at neutral pH, results in
the release of protons from the surface and into the fluid,
creating a concentration of protons at near the fluid/surface
interface, or a positively charged sheath surrounding the bulk
fluid in the channel. Application of a voltage gradient across the
length of the channel, will cause the proton sheath to move in the
direction of the voltage drop, i.e., toward the negative electrode.
The steady state velocity of this fluid movement is generally given
by the equation: 2 v = E 4
[0047] where v is the solvent velocity, .epsilon. is the dielectric
constant of the fluid, .xi..multidot.is the zeta potential of the
surface, E is the electric field strength, and .eta. is the solvent
viscosity. The solvent velocity is, therefore, directly
proportional to the surface potential. Use of electrokinetic
transport to control material movement in interconnected channel
structures was described in WO 96/04547 to Ramsey, which is
incorporated by reference.
[0048] A "ligand" is a molecule which selectively binds or
"hybridizes" to a "ligand binding partner". Many examples of
ligands and ligand binding partners are known, including biotin and
avidin or steptavidin, substantially complementary strands of
nucleic acids, proteins and molecules bound by proteins (including
cell receptors and cognate receptor binding molecules, antibodies
and cognate antigens, etc.), proteins and "complementary proteins"
(proteins which are specifically bound by other proteins, such as a
cell receptor and a peptide which specifically binds the cell
receptor), carbohydrates and carbohydrate binding molecules,
engineered associating peptides and the like.
[0049] An "aqueous" solvent comprises primarily water, and
optionally further comprises other chemical species, depending on
the intended application, such as buffers, dyes, preservatives, or
the like.
[0050] A "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and unless otherwise limited, encompasses known analogues of
natural nucleotides that hybridize to nucleic acids in manner
similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence optionally includes
the complementary sequence thereof.
[0051] An "antibody" is a polypeptide substantially encoded by an
immunoglobulin gene or immunoglobulin genes, or fragments thereof
which specifically bind and recognize an analyte ("antigen" or
"antibody ligand").
[0052] A "label" is any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means. A "label moiety" is the detectable portion of the
composition, e.g., the fluorophore, radioactive element or the
like.
DETAILED DESCRIPTION
[0053] In some assays it is useful to determine the concentrations
of products and related reaction rates for reactions in
microfluidic devices. In standard laboratory devices where products
or reaction rates are determined, such as cuvettes, or systems
where reactants are delivered to reaction chambers, the analysis of
reaction rates is straightforward, since all components of the
reaction are maintained in one location. The reaction rate is
related to the concentration of reagents and the time between the
mixing of reagents and detection of the product. It has now been
discovered that this simple analysis is not applicable to
microfluidic systems in which reaction components and products have
differing velocities through the channels of the device. Methods of
determining the reaction kinetics in electrokinetic systems are
provided.
[0054] In the case of electrokinetic movement of chemicals, the
velocity of different chemical species in a single flowing system
is not necessarily identical. Velocity for a particular component
depends on the charge of the particular species, the size of the
species, the solvent, and the like. For example, in a standard
electrophoresis gel, analytes such as nucleic acids move through
the matrix of the gel at different rates, depending on the size of
the molecule and the charge of the molecule. Large molecules move
more slowly in the matrix of the gel. Highly charged molecules have
a greater attraction for an oppositely charged electrode than more
modestly charged molecules, making more highly charged molecules
travel toward an oppositely charged electrode with a higher
velocity. These basic properties are understood, and form the basis
for purification and analysis of biological and chemical molecules.
However, mixing of components in such standard electrophoretic
systems is not performed. No attempt is made during standard
electrophoresis to determine reaction rates for the mixing of
reactants. Accordingly, the special problems encountered during
electrokinetic mixing were not considered in the electrophoretic
art, and, of course, solutions to these unknown problems were not
proposed.
[0055] In the special case of electrokinetic movement of fluids in
a microfluidic device, different species are commonly mixed to form
one or more product. Any or all of the reactant species or reaction
products can have differing mobilities. Thus, for example, an
enzyme can be reacted with a substrate which is modified to form a
product. The substrate, modified substrate (i.e., product) and
enzyme will often all have different mobilities. Detection
equipment downstream from a reaction site in the microfluidic
device will perceive the concentration of reactants and products
based, in part, on the differing velocities of the components. For
example, if an enzyme and a substrate are mixed at the start of a
microchannel down which the components travel, the appearance of
any product of the reaction downstream to the reaction site will
depend on standard considerations such as the actual rate of the
reaction (i.e., the number of product molecules made per unit time
in the reaction), and the concentration of the reactants (until
non-rate limiting amounts of reactants are provided, the more
reactants provided, the faster the reaction will proceed--a simple
result of chemical equilibrium). However, the perceived
concentration of product downstream of the reaction site also
depends on the velocity of the product. For example, if the
velocity of the product is substantially slower than the velocity
of the substrate in the system, then the product concentration will
be substantially higher than the decrease in the substrate
concentration that produced it. This is in contrast to the standard
non-flowing system in which product concentration would be equal to
the substrate that produced it. Thus, the reaction rates determined
without consideration of velocities of the system components were
discovered not to match results for reactions obtained by standard
techniques, where the velocity of the components is zero (or at
least not changing). Accordingly, the present invention relates to
the discovery of a problem not previously known to exist, and to
non-obvious solutions to this new problem.
[0056] Although the analysis of reaction rates in an electrokinetic
system requires corrections for velocity changes, the value of
determining reaction rates for many different concentrations in
very short periods of time and in very small volumes of fluids
makes the effort worthwhile. Accordingly, the present invention
makes possible, for the first time, the accurate and simple
analysis of accurate reaction kinetics in an electrokinetic system.
The ability to assess reaction kinetics "on the fly" Le., with the
reaction occurring while the components have velocity relative to
the observer, greatly speeds the rate at which such reactions can
be assessed. This, in turn facilitates accurate high-throughout
determination of reaction kinetics, and of a variety of other
flowing interactions with applicability to drug screening, nucleic
acid sequencing, enzyme kinetics, and the like.
[0057] Uses for Correcting for Electrokinetic Effects
[0058] It will be appreciated that the ability to quickly and
accurately monitor and determine reaction kinetics has broad
applicability to many different combinatorial approaches in biology
and chemistry, for medical diagnostics, basic research, quality
control, and the like. For example, the ability to correct for
electrokinetic effects in microfluidic electrokinetic systems
enhances the versatility of such systems. Any and all uses
contemplated for electrokinetic systems can benefit from the
present methods of correcting for electrokinetic effects.
[0059] The present methods and compositions are useful in measuring
the rate of essentially any chemical or biological reaction,
including particularly those which occur in an aqueous or other
flowable solution. The methods are particularly desirable where
repetitive screening of reactants is needed. This has general
applicability to assessing the purity and activity of industrial
and laboratory reagents (See, e.g., Kirk-Othmer Encyclopedia of
Chemical Technology third and fourth editions, Martin Grayson,
Executive Editor, Wiley-Interscience, John Wiley and Sons, NY, and
in the references cited therein ("Kirk-Othmer") for a basic
discussion of industrial chemical processes). Combinatorial
screening of large libraries of compounds for biological activities
provides the basis for finding new therapeutics. Thus, the ability
to monitor the effect of compounds on biologically relevant
reaction rates is of great importance and is of immediate
commercial value to a variety of pharmaceutical, agricultural and
chemical industries.
[0060] Similarly, the ability to rapidly and accurately screen
large patient populations for evidence of infection, genetic
disease, or the like, is typically performed by monitoring the
interaction of chemical or biological components. For example,
binding of HIV antigens to antibodies in a patient's blood is
commonly used to detect whether a patient has been exposed to HIV.
In a system in which the binding constant between the antibody and
the relevant antigen can easily be monitored, it is possible to
reduce the incidence of false-positives. Thus, the present
invention provides for increased sensitivity in biological assays,
as well as increased throughput.
[0061] In addition to monitoring antibody-antigen and other
protein-protein interactions, it is possible to monitor the
affinity of nucleic acid-nucleic acid interactions. This is
particularly useful for empirically determining percent similarity
for complementary related nucleic acids, and for detecting nucleic
acids in various biological samples (including PCR samples; See,
PCR Protocols A Guide to Methods and Applications (Innis et al.
eds) Academic Press Inc. San Diego, Calif. (1990) (Innis)). As an
alternative to standard solid state Southern or northern analysis
(See, Sambrook, Ausubel, or Berger, supra.) the assay provides
increased automation, a clear indication of the efficiency of
nucleic acid hybridization (providing an increase in signal to
noise ratios) and the like.
[0062] Monitoring reaction rates between enzymes and substrates has
applicability as a general laboratory tool for basic research,
where the reaction rate is unknown, and as a quality control tool
for the assessment of the quality of reagents such as enzymes or
substrates. And in diagnostic assays. Enzymes and other chemical
and biological catalysts are in common use as components of foods,
food supplements, detergents, therapeutics, and, e.g., as
laboratory tools for recombinant nucleic acid manipulation (e.g.,
restriction enzymes, see, Berger and Kimmel, Guide to Molecular
Cloning Techniques, Methods in Enzymology volume 152 Academic
Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)
Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3, Cold
Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook);
and Current Protocols in Molecular Biology, F. M. Ausubel et al.,
eds., Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement)
(Ausubel) for a discussion of some enzymes commonly used in
molecular biology). Defective enzymes also serve as the direct
cause for the etiology of many inherited diseases, including, e.g.,
ADA and phenylketonuria. The ability to screen enzymes rapidly from
patients suffering enzyme defects is of considerable medical
diagnostic value.
[0063] Methods of Correcting for Electrophoretic Effects
[0064] The present invention provides methods of accurately
determining the rate of a chemical reaction. The reaction can be
between two or more components that chemically join (by forming a
covalent or non-covalent association) to form a new component or
complex, or between a component such as an enzyme, catalyst or
electromagnetic radiation that converts a first reactant or other
component into a product, or due to spontaneous degradation of a
component. In the methods, a first component and a second component
are contacted, often by mixing, typically in a channel in an
electrokinetic device. The components react to form a product.
[0065] Flux (J), with units of molecules/(cross sectional
area.times.time) or mass/cross sectional area.times.time, is equal
to the velocity of the molecules under consideration (U) times the
concentration of molecules (C); thus, J=U.times.C. Flux is
conserved in the microchannel. In other words, the number of
analyte molecules (enzymes, substrates and products, or ligands and
ligand partners) times the velocity of the components in a
microchannel is constant along the channel.
[0066] The components and the solvent all travel along the length
of the channel at different velocities to a position downstream of
the mixing point where they are detected, typically by detecting a
label (a variety of labels are described supra).
[0067] The velocity of one or more reaction components (U.sub.r1,
U.sub.r2, U.sub.r3 . . . ) or products (U.sub.p1, U.sub.p2,
U.sub.p3 . . . ) in the channel are determined. As explained in the
examples below, in a system in which flux is conserved, if the
velocity of one component is known, the velocities of the other
components can be determined, given concentration information,
charge mass ratios (ordinarily, the charge mass ratio (CM) is
proportional to velocity in a flowing system, i.e., U.sub.r1 is
proportional to CM.sub.r1, U.sub.r2 is proportional to CM.sub.r2,
U.sub.3 is proportional to CM.sub.r3, U.sub.p1 is proportional to
CM.sub.p1 . . . .), or the like. In some unusual instances,
velocity (U) and charge mass ratios (CM) are not directly
proportional due to unusual molecular shapes which either shield
charge on portions of the molecules, or which cause molecular drag
during electrophoretic motion.
[0068] In one convenient embodiment, the velocities of the
reactants are known, either from direct measurement, or from
previous measurements in a similar system, or by comparison to
known velocity markers. Velocity markers are components which are
run in the system which are detectable and known to have a
particular velocity relative to an analyte. Measurement of the
marker is used to estimate the velocity of the analyte (reactant,
product or the like). The product velocity may be similarly known,
or directly measured, e.g., by measuring the velocity of a
detectable product over a section of the microchannel. Similarly,
the velocities of the reactants can be measured over a section of
the microchannel.
[0069] The concentration of the reaction product is determined in)
a portion of the microchannel. This determination can be done by
measuring the number of molecules with the detector as described
above, typically in a given section of an electrokinetic channel.
Alternatively, the concentration can be determined indirectly, by
measuring velocities and concentrations of other components in the
system. Where flux is conserved, the sum of the concentration of
reactants and products times the respective velocity of reactants
and products is constant. Accordingly, the concentration of
particular components can be measured, or determined from
measurements for other components in the system, e.g., using simple
algebra. For example, in a simple system having reactant 1 (R1)
reactant 2 (R2) and a product (P) where J is constant, and
J=(U.sub.R1)[R1]+(U.sub.r2)[R2]+(U.sub.p)C.sub.p, one of skill can
easily determine C.sub.P where J is constant and
J=[U.sub.1C.sub.1+U.sub.2C.sub.-
2+U.sub.PC.sub.P].sub.w=[U.sub.1C.sub.1+U.sub.2C.sub.2+U.sub.PC.sub.P].sub-
.z. By algebraic manipulation,
C.sub.Pz=(U.sub.1/U.sub.p)(C.sub.1w-C.sub.1-
z)+(U.sub.2/U.sub.p)(C.sub.2w-C.sub.2z)+C.sub.Pw. Similar algebraic
considerations can be used to yield the velocities or
concentrations of other components where sufficient information is
available. Linear algebra techniques are conveniently used to solve
for the concentrations or velocities of components where there are
multiple unknowns related in multiple flux relationships.
[0070] Given the velocity of a product (U.sub.p) and the
concentration of a product (C.sub.p), it is possible to correctly
determine the rate of a reaction. In particular, it is possible to
determine the rate at which a product is formed, by conversion of
one or more of the reactants into a product.
[0071] In the system in which one of the reactants aids in
converting the other reactant into the product (e.g., where R1 is
an enzyme or catalyst and R2 is a substrate), the following flux
relationship can be used in determining a reaction rate: Flux
(3)=[R1].times.T.sub.LR2.times.k.times.-
U.sub.R2=[R2].sub.converted.times.U.sub.p=U.sub.p.times.C.sub.P,
where k is the turnover number for the enzyme reaction. Rearranging
and writing transit time (T.sub.LR2) of substrate as L/U.sub.R2
results in:
[R1].times.L/U.sub.R2.times.k.times.U.sub.R2=U.sub.p.times.C.sub.P.
Thus, [R1]/U.sub.p.times.L.times.k=C.sub.p. Substituting transit
time for product (.sub.TLP) for L/U.sub.p gives the result that
product concentration is proportional to the transit time of the
product, not the substrate as might have been extrapolated from the
stationary or non-mobility changing case above:
[R1].times.T.sub.LP.times.k=C.sub.P. Thus,
k=C.sub.P/([R1]T.sub.LP). In one embodiment, where the product
concentration before a reaction is zero and the enzyme
concentration, R1 remains essentially constant, then, rearranging,
C.sub.P=([R2].sub.total-- [R2].sub.unreacted)U.sub.2/U.sub.p.
[0072] Consideration of the case in which two or more components
are joined to form a product is similar. When two reactants join,
they typically result in a product with a different velocity than
either of the two individual reactants (R1 and R2). With the flux
being conserved, the concentration of detected species changes as a
result of a change in velocity. The product optionally results in a
different detectable label than either of the reactants, or can
have the same label. Where R1 and R2 molecules are converted to P,
taking the principle of the conservation of flux into account:
[R1].times.U.sub.R1=C.sub.P.times.U.sub.P
[0073] Recognition of this relationship allows quantification of
the amount of R2 present in the system by detecting downstream
fluorescence (all R2 is bound to R1). The relationship between the
concentrations of R1 bound to R2 (i.e., forming P) and unbound R1
is proportional to their mobilities:
C.sub.P=[R1].times.U.sub.R1/U.sub.P.
[0074] At intermediate amounts of R2, where a portion of R1 is
bound to R2, the concentration is proportional to the fraction
(Y.sub.R1) of R1 that is bound to R2:
C.sub.P=Y.sub.R1([R1]U.sub.R1/U.sub.p).
[0075] Without the knowledge that concentration changes as velocity
changes, as taught herein, the assay is necessarily more
complicated. For example, one could sample the mixture into a
separation column which separated reacted and unreacted molecules,
and detected florescence. The amount of material coming off of the
column per unit time could be detected (see also, the Examples
below). However, using conservation of flux, much simpler
arrangements are possible. For instance, an electrokinetic system
with one channel and two electrodes driving fluid flow in an
electrokinetic device is used to monitor formation of reaction
products.
[0076] It will be appreciated that products and reactants need not
be fluorogenic (producing or quenching a fluorescent signal), but
only need to be "velocitigeneic," i.e., a reaction need only
produce a detectable change in velocity of a product compared to a
substrate. This ability to sort signals based on the velocity of
products as compared to reactants provides for the detection of
multiple reactions and multiple products in a single electrokinetic
device. Additional assays utilizing non-fluorogenic assays are
described below.
[0077] A mass balance on the substrate of an enzyme reaction
yields:
[S].sub.total=a[S].sub.converted+(1-a)[S].sub.remaining,
[0078] where "a" is the fraction of substrate (S) that is converted
to product. By definition, [S].sub.converted=C.sub.P.
[0079] From the conservation of flux:
C.sub.P=[S].times.U.sub.s/U.sub.P. Therefore,
[S]=a[S]U.sub.s/U.sub.p+(1-a)[S]. After measuring the signal before
the reaction (l.h.s.) and after the reaction (.tau..h.s.), it is
possible to solve for "a" if the velocity of substrate and product,
U.sub.s and U.sub.P, are known.
[0080] In many enzyme reactions, enzyme kinetics are studied in a
range in which a very small portion of substrate is converted into
product; in these cases, the substrate concentration can be treated
as a constant. This makes the signal change due to formation of the
product relatively small. To optimize the signal to noise ratio for
observation of the product, it is possible to optimize
electrokinetic flow so that the product velocity is slow (or close
to zero) when the substrate velocity is relatively high, or to make
product velocity fast while substrate mobility is slow.
[0081] When reactions are performed on microsubstrates with
electrokinetic movement of solutions, the analysis of reaction
rates and product formation is done from a starting point of
conservation of flux. This is in contradistinction from prior art
systems in which the velocities of reactants and products do not
differ, permitting analysis from a simple standpoint of
concentration balance. The present invention, therefore, provides
for correct determination of reaction rates, a wider range of
detectable reagents (e.g., velocitigenic, rather than flourescent),
and simpler electrokinetic movement and detection apparatus.
[0082] Non-Fluorogenic Assays
[0083] The detection of results for many biochemical assays in
conventional cuvette experiments, as well as in microfluidic
devices has primarily been based on fluorogenic or chromogenic
reactions in which the quantum efficiency of a labeling fluorescent
moiety or the amount of colored label (chromophore) changes as a
result of the reaction. However, for certain classes of assays the
reactions are non-fluorogenic (i.e., there is no change in the
quantum efficiency of the labeling fluorescent species upon
reaction by the enzyme). As noted above, a reaction need only be
velocitigenic for accurate rate determination; the formation of a
new detectable element is not necessary in the practice of the
invention.
[0084] It will be appreciated that the concepts described for
non-fluorogenic assays are equally applicable for non-fluorescent
systems, in which the label is other than a fluorophore, i.e., a
calorimetric label, a radioactive label, an electrochemical label,
or the like; for example, a non-chromogenic assay is an assay in
which the color or intensity of a label does not change upon
reaction; a non-radiogenic assay is an assay in which the
radioactive component of the label is not modified by the reaction.
Again, the relevant criterion is that a product have a different
velocity than a reactant. For simplicity, fluorogenic assays and
non-fluorogenic assays are discussed in more detail; it will be
appreciated upon review of this disclosure that similar
considerations apply for radio labels, chromophore labels, pH
labels, ionic labels, or other common labels known to one of
skill.
[0085] Detection of non-fluorogenic assays is possible in an
electroosmotically driven microfluidic device using periodic
injections of reaction mixture into a separation channel, in which
reactants and products are separated by electrophoresis due to
changes in the electrophoretic mobility resulting from the
reaction, as discussed above (see also, A. R. Kopf-Sill, T.
Nikiforov, L. Bousse, R. Nagel, & J. W. Parce, "Complexity and
performance of on-chip biochemical assays," in Proceedings of
Micro- and Nanofabricated Electro-Optical Mechanical Systems for
Biomedical and Environmental Applications, SPIE, Vol. 2978, San
Jose, Calif., February 1997, p. 172-179). The periodic injections
are typically on the order of from about 0.0001 to 10 minutes,
typically about 0.001 to 1 minute, often about 0.1 seconds to 10
second. See also, concurrently filed U.S. application Ser. No.
______ (attorney docket number 100/04200).
[0086] In an alternate non-fluorogenic continuous flow mode assays
of the invention, the injection/separation step is eliminated. The
binding reaction of fluorescently-labeled biotin to streptavidin
was chosen as a model system for non-fluorogenic continuous flow
mode.
[0087] The following discussion provides the basic concept of
continuous flow non-fluorogenic assay on microchips, the use of
conservation of flux to predict and interpret non-fluorogenic assay
data quantitatively, modeling and experimental information to
validate these concepts, applications of the format to biochemical
assays on microchips, and the applicability of non-fluorogenic
assays e.g., to high throughput drug screening.
[0088] The Continuous Flow Non-Fluorogenic Assay Format
[0089] In an electroosmotically driven microfluidic device, each
type of dissolved species in a buffer moves down a channel at a
velocity (U.sub.tot) equal to the vector sum of the electroosmotic
velocity of the buffer (U.sub.eo) and the electrophoretic velocity
of the molecule (U.sub.ep):
U.sub.tot=U.sub.eo+U.sub.ep=(.mu..sub.eo.+-..mu..sub.ep)E.
[0090] In this equation, .mu..sub.eo and .mu..sub.ep are the
electroosmotic mobility of the buffer and the electrophoretic
mobility of the dissolved species, respectively, and E is the
applied electric field. The electrophoretic mobility in turn
depends on the charge-to-mass ratio of the molecule. In most
biochemical reactions, the charge-to-mass ratio of the reactant
molecule changes as a result of the reaction, thus changing the
electrophoretic mobility of the molecules. This change in mobility,
and therefore velocity, is the basis for detection of
non-fluorogenic reactions in a continuous flow format.
[0091] Accordingly, methods of determining concentration of a
reaction or assay product (C.sub.p) in a channel of a microfluidic
device are provided. In the methods, a labeled first reactant or
assay component having a velocity (U.sub.r) and a label (L.sub.r),
such as a fluorophore, chromophore or other label (see, supra for a
discussion of labels) is flowed down a microfluidic channel and
past a signal detector (detectors are also described supra). The
labeled first reactant or assay component produces a signal
(S.sub.as) detectable by the detector. The labeled first reactant
or assay component is converted to a reaction or assay product
comprising a label L.sub.p, the product having a velocity
(U.sub.p). In the typical case, (U.sub.r) does not equal (U.sub.p),
resulting in a change in signal from L.sub.p, thereby providing an
indication of C.sub.p. Because the assay is non-fluorogenic,
L.sub.p comprises component elements of L.sub.r (i.e., the labels
are typically essentially the same for the product and reactant,
i.e., providing the same detectable output). Reactant or assay
component signal (S.sub.as of a labeled first reactant or component
prior to addition of a second reactive component, termed "S.sub.r")
can be subtracted from S.sub.as after the addition of additional
components which react with the first reactant or component to
provide a normalized signal (S.sub.n) produced by the product.
[0092] In non-fluorogenic assays, a molecule comprising L.sub.p is
converted from a molecule comprising L.sub.r by treating the
molecule with any physical component or force which results in a
modification of the molecule, including light, heat, electrical
charge, a polymerization agent, a catalyst, or a binding molecule.
L.sub.r and L.sub.P are optionally identical after the conversion,
with only distal portions of the molecule being affected.
Alternatively, L.sub.r can be modified so that a new label,
L.sub.p, is produced; however, the output of the label typically
does not change in a non-fluorogenic assay. Of course, where the
label does change, the concepts herein can also be applied, as the
velocity will typically also concomitantly change.
[0093] The basic concept of the continuous flow mode of a
non-fluorogenic assay can easily be illustrated with a schematic
drawing of a binding reaction as shown in FIG. 1. In FIG. 1, the
fluorescently-labeled reactant molecules are denoted by circles and
the unlabeled reactant are denoted by squares. The reaction product
molecules, denoted by the combined shape of a circle and a square,
are shown lighter toned as a result of a binding reaction which,
for the sake of simplifying this discussion, is fast and has a high
association constant (K.sub.a). (K.sub.a=[P]/[A][B] for a reaction
A+B.fwdarw.P, where the brackets denote concentrations.) The
labeled reactant (circles) flows continuously down the main channel
at a constant concentration, whereas the unlabeled reactant
(squares) is injected in a short pulse from a side channel into the
main channel. In this illustration, the labeled reactant is assumed
to move slow whereas the product moves fast (in the figure, motion
is from left to right).
[0094] As the squares are injected into the main channel, they bind
to the circles and convert them to fast moving molecules (for
purposes of simplification, the binding is considered to be
instantaneous). Downstream of the injection point, the faster
moving product catches up with the slower reactant, giving rise to
a higher local concentration of fluorescent species (i.e., the sum
of labeled reactants and labeled products) ahead of the injection
plug, and a lower concentration at the trailing end of the
injection plug due to the depletion in reactants. Quantitatively,
it is important to recognize that the product zone occupies a
larger volume in the channel than the depleted reactant zone due to
the higher product velocity. Consequently, the apparent
concentration of product in the channel is less than the
concentration of the reacted reactant, since the same number of
molecules are now spread out in a larger volume. Interestingly, in
the time domain as illustrated in the bottom of FIG. 1, the widths
of the peak and valley are the same because the spatially wider
product zone, which has been increased by a factor equal to the
ratio of product velocity (U.sub.p) to reactant velocity (U.sub.r),
moves past the detector faster by the same factor of
U.sub.p/U.sub.r. If the concentration of the reacted reactant
(C.sub.p) and the velocities U.sub.r and U.sub.p are known, the
concentration of the product (C.sub.p) can be calculated as:
C.sub.p=C.sub.r(U.sub.r/U.sub- .p). This equation makes use of the
concept of conservation of flux (flux is defined as the product of
velocity and concentration as discussed above).
[0095] When a label detector is placed downstream of the injection
point (e.g., a photomultiplier tube, photo diode, or the like),
depending on the distance between the injection and detection
points, the length of the injection plug, and the species
velocities, the plug of faster moving product can be partially or
totally separated from the slower moving depletion hole of the
reactant. In the case of partial separation, the detector signal
(S.sub.as) displayed in time will show a characteristic shape of a
peak followed by a plateau region and a valley. The ratio of the
magnitude of the peak to valley is (C.sub.p/C.sub.r), which, by
algebraic manipulation, is equal to (U.sub.r/U.sub.p). The plateau
region is lower in fluorescence than the background level. The
ratio of the magnitude of the plateau region to the valley is
1-(C.sub.p/C.sub.r) or 1-(U.sub.r/U.sub.p). In the case of total
separation, the signal shows a peak and a valley separated by the
baseline fluorescence level instead of the plateau region.
[0096] Mobility Shift Modeling
[0097] For the case of a fast binding assay with a high K.sub.a
(e.g., between about 10.sup.5 and 10.sup.15 or higher, typically
higher than about 10.sup.8M.sup.-1 for a 1 .mu.M concentration of
reactants) as described in the last section, the fluorescence
signal can easily be modeled in the time domain, e.g., using an
Excel.TM. spreadsheet. Input parameters include reactant
concentrations, electroosmotic mobility of the buffer,
electrophoretic mobilities of the labeled reactant and product,
distance between injection and detector locations, injection pulse
time, and applied field strength. See, Appendix 1.
[0098] Two cases of the model predictions are shown in FIG. 2. The
first case, denoted by the solid curve in FIG. 2, is for a long
injection time such that the signal peak and valley are only
partially separated and a plateau region is clearly seen. The
second case (dash curve) is for a short injection time such that
the peak and valley are fully separated by the baseline
fluorescence level. Note that in both cases, the magnitude of the
peak height is smaller than the magnitude of the valley depth due
to the principle of conservation of flux in flowing systems.
[0099] For the more general case of a reaction with variable
reaction rates and K.sub.a values, continuous flow non-fluorogenic
assays can be modeled in the spatial domain. In one convenient
embodiment, an Excel spreadsheet is again utilized. The basic
construct of the spatial domain model is to split the channel into
discrete sections spatially and in time. At an initial time, the
channel is filled with the labeled reactant. For each subsequent
time step, the second reactant is allowed to be injected into the
channel and then reacted with the labeled reactant to form products
at some prescribed reaction kinetics, which are required as input
parameters. In this model, an algorithm is included to ensure that
the concentration flux of each species moving down the channel is
conserved. The Macro program listing, in Visual Basic Applications
(VBA), for binding assays with variable K.sub.a values is included
in Appendix A. FIG. 3 illustrates model predictions of the
fluorescence signal at various values of K.sub.a when the
concentrations of the reactants were chosen to be 1 .mu.M.
[0100] Integrating Non-Fluorogenic Assays in High-Low Salt
Format
[0101] In one series of high throughput screening embodiments,
compounds of interest (e.g., potential drugs, or other analytes)
are dissolved in a high salt buffer and placed in a source of
materials, such as the wells of a microtiter dish, with a low salt
buffer used as the running buffer to pipette the compounds from the
wells into the planar LabChip.TM., e.g., through a capillary. A
variety of source-chip arrangements and interfaces are described in
08/835,101 and CIP application 09/054,962 by Knapp et al. See also,
U.S. Ser. No. 08/671,986. In brief, an electropipettor pipettor
having one or several separate channels is fluidly connected to an
assay portion of the microfluidic device (i.e., a microfluidic
substrate having the reaction and/or analysis and/or separation
channels, wells or the like). In one typical embodiment, the
electropipettor has a tip fluidly connected to a channel under
electroosmotic control. The tip optionally includes features to
assist in sample transfer, such as a recessed region to aid in
dissolving samples. Fluid can be forced into or out of the channel,
and thus the tip, depending on the application of current to the
channel. Generally, electropipettors utilize electrokinetic or
"electroosmotic" material transport as described herein, to
alternately sample a number of test compounds, or "subject
materials," and spacer compounds. The pipettor then typically
delivers individual, physically isolated, sample or test compound
volumes in subject material regions, in series, into the sample
channel for subsequent manipulation within the device. Individual
samples are typically separated by a spacer region of low ionic
strength spacer fluid. These low ionic strength spacer regions have
higher voltage drop over their length than do the higher ionic
strength subject material or test compound regions, thereby driving
the electrokinetic pumping, and preventing electrophoretic bias. On
either side of the test compound or subject material region, which
is typically in higher ionic strength solution, are fluid regions
referred to as first spacer regions (also referred to as high salt
regions or "guard bands"), that contact the interface of the
subject material regions. These first spacer regions typically
comprise a high ionic strength buffer solution to prevent migration
of the sample elements into the lower ionic strength fluid regions,
or second spacer region, which would result in electrophoretic
bias. The use of such first and second spacer regions is described
in greater detail in U.S. patent application Ser. No. 08/671,986.
These electropipettors are used to physically sample a source of
materials of interest, such as a microtiter dish, a membrane having
dried or wet samples disposed thereon (dry samples can be
resolublized, e.g. by expelling fluids from the electropipettor
followed by drawing the expelled fluid into the device; for other
arrangements see 09/054,962) or the like.
[0102] In the high-low salt format, the electric field within the
high salt region in the channel of a pipettor chip is relatively
small compared to that in the low salt region, due to the lower
electrical resistance of the high salt buffer. Consequently,
electrophoresis of compounds in the high salt plug is greatly
retarded, whereas the high salt plug itself is dragged along by
electroosmosis driven primarily by the conditions in the low salt
region.
[0103] At least two general approaches to integrate non-fluorogenic
assays into this high-low salt pipettor chip format for high
throughput drug screening--continuous flow mode and
injection/separation mode are provided. In the continuous flow
format, integration of the two opposing principles of preventing
and encouraging electrophoresis at will into one simple chip design
requires careful chip and experimental design. One method is to
inject a buffer into the latter part of the main reaction channel
to "spoil" the high-low salt format after the assay has had
adequate incubation time to generate product.
[0104] Incorporating non-fluorogenic assays into the high-low salt
format by injection followed by separation in another channel is
likely to be less dependent on the buffer systems, and thus is
general in its applicability to a wide range of biochemical assays.
However, a control mechanism is used to time the injection.
External control mechanisms to time the arrival of the high salt
plug to trigger injection include use of an electromagnetic means
such as an in-situ conductivity probe in the channel and/or optical
methods based upon the intrinsic properties of the buffer (e.g.,
refractive index changes in high/low salt buffers), or placing a
dye marker in the buffer in conjunction with using an optical
detector to time the flow. Another method is to use the pressure
developed at the interfaces of the high and low salt regions to
induce injection at a channel intersection. In this case, the
injection is automatic; no external control and feedback means is
required. See also, concurrently filed U.S. application Ser. No.
______ (attorney docket number 100/04200).
[0105] Continuous Flow Assay Formats Using Interference Patterns of
Analyte
[0106] Concentration Waves in Electrokinetic Microfluidic
Systems
[0107] Methods to enhance the detection of non-fluorogenic assays
on chips for small mobility shifts are available. One approach is
to inject the reaction mixture into a planar cyclic capillary
electrophoresis channel to separate products from reactants. In
this case, the separation time can be made very long by
continuously cycling the voltage around the cyclic structure.
Another method is to use the concept of interference of
concentration waves in channels to enhance to the magnitude of
peaks and valleys in the non-fluorogenic assay fluorescence signal
(see, below).
[0108] Use of Concentration Waves for Data Correction
[0109] In a microfluidic device in which an electric field is
applied along the length of the microchannel, charged species such
as analytes, solvent molecules, reactants and products move along
the microchannel by the electrokinetic forces of electroosmosis and
electrophoresis. The net mobility of each species is determined by
the vectorial sum of the electroosmotic and electrophoretic
mobilities, the latter of which is a function of the hydrodynamic
radius-to-charge ratio of each species. During a chemical or
biological reaction such as ligand-receptor binding,
antibody-antigen binding, etc., the reactants in general have
different electrophoretic mobilities than those of the products.
The differences in mobilities are useful for detection, e.g., of
non-fluorogenic assays described above, in which reaction detection
is not dependent on the production or quenching of fluorescence as
a consequence of the reaction. Instead, the mobility difference
during flow in the microchannel is used to separate the "reactant
hole" (i.e., decrease in reactant concentration) of the labeled
reactant from the "product peak" (i.e., increase in product
concentration) under continuous flow, thereby providing a signature
from which quantitative information on the reaction kinetics can be
extracted from calculation methods based on conservation of species
flux discussed supra. Non-fluorogenic assay formats are unique to
electrokinetic microfluidic systems; there is no analogy for
cuvette assays.
[0110] The invention provides methods for performing continuous
flow assays in electrokinetic microfluidic devices to facilitate
determination of reaction kinetics using the generation and
detection of reactant and product "concentration waves" in
microchannels. The reactant concentration wave is generated
temporally by modulating the concentration of one or more reactants
using electroosmotic pumping. The product/concentration wave is
generated as a result of the reaction. At the point of reaction in
the microchannel, the product wave is inherently 180.degree.
out-of-phase with the reactant wave. If the reaction is
non-fluorogenic, a detection device placed very close to the point
of reaction along the microchannel measures a constant signal (such
as due to fluorescence of a labeling moiety covalently bonded to a
reactant), since the sum of the signals from the labeled reactant
and converted product is constant. Further downstream of the
microchannel, however, the reactant and product waves separate
spatially due to differences in electrophoretic mobility, and the
reaction can be detected. The measured signal can be viewed as
"interference" of the reactant and product waves, analogous to the
phenomenon of interference of electromagnetic (such as optical)
waves. The "phase shift" in the reactant and product waves is a
function of the net mobility difference of the labeled reactant and
product, the average flow velocity in the microchannel, and the
distance from the point of reaction. At the point of reaction, the
phase shift is zero and the waves interfere destructively. As the
phase shift approaches 1800, the waves interfere constructively and
the signal is maximized.
[0111] In studying the kinetics of a reaction in a microfluidic
device, analyte concentration waves with a constant frequency and
varying concentrations can be used to elucidate the dependence of
kinetics on concentration (analogous to analyte titration). An
"interference pattern" as a function of spatial position can be
measured by placing the detector at different points along the
microchannel. Deconvolving the interference patterns using wave
equations, conservation of flux, and diffusion equations provides
quantitative information on species mobilities and reaction
kinetics.
[0112] In many cases when the mobility shift of the reactant and
product is not known, a reactant concentration wave with varying
frequencies can conveniently be used to study the reaction. For
instance, the frequency of the reactant concentration wave can be
increased linearly with time. A detector located at a fixed
distance from the point of reaction can measure an increase in the
signal intensity as the mobility-induced phase shift becomes a
significant fraction of the wavelength of the concentration wave.
Again, kinetics data can be obtained by deconvolving the signal
using wave, diffusion, and flux conservation equations.
[0113] In general, this continuous flow assay format using
interference patterns of analyte concentration waves can be applied
to a wide range of assays. This format can be especially sensitive
to small changes in the mobility shift of the converted product,
such as in the case of ligand-receptor assay, in which the mobility
of the protein-ligand complex is expected to differ little from
that of the labeled protein of interest since the binding ligands
are usually small molecules. The following is an example to
illustrate the practicality and usefulness of this format. The
reaction of interest is:
P+L.fwdarw.PL
[0114] where P is a labeled protein with molecular weight of 10 to
100 kDaltons, L is a ligand with molecular weight of 50 to 500, and
PL is the protein-ligand complex. If a concentration wave of an
unlabeled ligand is electroosmotically pumped into a microchannel
containing a constant concentration of the labeled protein, the
binding reaction generates a complementary concentration wave of
the labeled complex.
[0115] Assume for illustrative purposes that the electroosmotic
(EO) mobility of the buffer is 0.4 cm.sup.2/kV-s and the protein
has an electrophoretic (EP) mobility of -0.2 cm.sup.2/kV-s. If the
EP mobility shift due to binding is only 1%, then the EP mobility
of the complex is -0.202 cm.sup.2/kV-s. In a nominal electric field
of 250 V/cm along the microchannel, the velocities of the protein
and complex is 0.5 and 0.495 mm/s, respectively. For a nominal
channel distance of 20 mm between the point of reaction and
detector location, the time for the protein and complex to arrive
the detector is 40 and 40.4 s, respectively. The time difference is
therefore 0.4 s between the 2 labeled species. If this time
difference is a significant fraction of the wavelength to achieve
noticeable constructive interference, say 1/4 (or 90.degree. in
phase shift), then a ligand concentration wave of frequency 0.625
Hz (=1/(4.times.0.4 s)) is needed. This frequency is practical
compared to the response time of a typical electrical controller
and a data acquisition rate of 20 Hz. Furthermore, for 0.5 mm/s
velocity, this frequency is equivalent to ligand injection plugs of
800 .mu.m per cycle spatially. This dimension is also reasonable
when compared to a nominal detector window of .about.50 .mu.m, and
a Brownian diffusion length of .about.70 .mu.m under the given flow
conditions and the assumption of a protein diffusion constant of
6.times.10.sup.-7 cm.sup.2/s.
[0116] Constant Flux Microchip Injector in Quantitative
Analysis
[0117] Essentially any analysis in which a starting compound is
converted to a product with a different mobility can be analyzed in
a microfluidic device of the invention. As noted above, essentially
any velocitogenic assay can be analyzed. One exemplar class of
velocitogenic assays includes enzymatic reactions. Kinases are a
specific example of enzymes of this type. Kinases recognize
specific polypeptide sequences and phosphorylate them.
Phosphorylation changes the peptide charge, mass and structure, and
thus the mobilities of the non-phosphorylated and phosphorylated
species are different. As a consequence of this change in mobility,
substrate and product move at different rates in an applied
field.
[0118] Enzyme kinetics (i.e., the determination of kcat, Km, and
Ki) may be performed in a microchip capillary electrophoresis
experiment by determining the extent of conversion of substrate to
product. Traditionally, kinetic analyses in a cuvette experiment
are performed under conditions such that the reaction is not
substrate limited and the enzymatic turnover is simply a function
of the solution conditions and the inherent catalytic nature of the
enzyme. Velocity is irrelevant in this format. In the microfluidic
system, the reaction is homogeneous in that it occurs in the
flowing stream in the capillary. There is typically no surface
immobilization of reagents (as described supra, the special case
where the velocity of a reagent is zero leads to special
considerations). Reagents are typically pumped electrokinetically
into a reaction channel. The field imposed on the flowing stream
results in a separation of each species according to its mobility.
In the case where the substrate concentration is high relative to
the Km of the enzyme reaction, the amount of product produced does
not depend on the concentration of substrate. The reaction rate
depends only on the reaction conditions and the inherent enzyme
reactivity. The signal generated in any unit volume is a function
of the amount of enzyme in the reaction mixture, the reaction time,
and the electrokinetic mobility of each species. Unlike the
homogeneous cuvette experiment, the electrokinetic forces used in
the microchip format to move reagents along the microchannels bias
the species concentrations in a reaction. On a microchip, substrate
and enzyme flow together through a capillary network, mix, and the
substrate is converted to product as the reaction mixtures flows
along the length of the mixing channel. Typically in fluorescence
detection, the substrate and product species are both labeled with
a fluorescent tag. After mixing, the reagents are pumped
electrokinetically through a portion of the channel passing in
front of the detector. Samples of the reaction mixture are analyzed
quantitatively as substrate and product moieties are separated by
their different electrokinetic mobilities either in the continuous
flow mode as described above or by injection followed by separation
in another channel.
[0119] In the injection/separation mode, one way to make injections
in a microchip is in a cross or orthogonal injector. In this
design, reagents flow in a fluid path along the length of the
applied field. They mix and react as they flow along the channel.
At some distance down the reaction channel, a perpendicular cross
channel is encountered. Injections can be made from the reaction
channel into the separation channel by modulating the voltages
applied at the end of the capillary length. The injection volume is
the volume mostly defined by the intersection of the orthogonal
channels. The consequence of this type of injection is that the
amount of reactants and products is a direct reflection of the
concentrations in the reaction channel at the injection point.
These concentrations are a function of the solution composition,
the enzyme reactivity, the reaction time, and the electrokinetic
mobilities of the reactants and products. Therefore, in order to
determine the substrate and product concentrations, the relative
mobilities of each reactant and product are determined. Kinetics
constant determination requires electrokinetic correction using the
relative mobilities of substrate and product as discussed
herein.
[0120] Alternatively, an injector that compensates for the
different mobilities of substrate and product in the microchip
reaction mixture can be used. The gated injector is realized in the
microchip design where the separation channel is collinear with the
reaction channel. In this case, the electric field for
electrokinetic pumping is applied along the axis of the reaction
channel. The fluid mixture flows along this axis but it is directed
off the main reaction channel into a side channel most of the time,
with periodic injection into the collinear separation channel
passing in front of the detector. Buffer or background electrolyte
from another side channel flows through the separation channel
between the injected aliquots from the reaction channel. The
injections of reaction mixture into the separation channel are
pulsed by voltage or current control. The bias imposed by the
electric field pulsing aliquots of reaction mixture to the detector
influences the rate at which reagents enter the separation channel.
The result is that species of highest apparent mobility move
fastest into the separation channel while the low mobility species
travel slowly into the separation channel. This electrokinetic bias
in the injection causes species that are concentrated in the
reaction channel because they move relatively slowly to map out a
smaller volume of injection into the separation channel.
[0121] Conversely, faster species that are diluted in the reaction
channel map out a proportionately larger volume into the separation
channel due to the higher velocity. Because the same electrokinetic
forces that result in the concentrating and diluting of analyte
concentrations in the reaction channel also cause the bias in the
injection volumes for the gated injector, the collinear chip
injector can be used to compensate for the effects of changes in
mobility on the determination of the extent of reaction in
microchips.
[0122] In a simple example in which a substrate with concentration
Cs and velocity Us is partially converted by an enzyme to a product
with concentration Cp and velocity U.sub.p, conservation of flux
dictates that J=C.sub.pU.sub.p=(yC.sub.s)U.sub.s, where y is the
fraction of substrate conversion. When this reaction mixture is
injected through a gated injector into a separation channel, the
length of the sample bands for the unconverted substrate (L.sub.s)
and for the product (L.sub.p) are proportional to their respective
velocities, U.sub.s and U.sub.p. L.sub.s.varies.U.sub.s;
L.sub.p.varies.U.sub.p. The total amount for each species in the
injection volume is the concentration times the volume injected. If
A is the cross-sectional area of the separation channel, then the
total amount of unconverted substrate injected is (y
C.sub.s)L.sub.sA, which is proportional to (yC.sub.sU.sub.s). The
total amount of product injected is (C.sub.pL.sub.pA), which is
proportional to (C.sub.pU.sub.p). Consequently, the total amount
injected for each species is representative of the flux of the
species in the reacting channel. Thus, the result of using a gated
injection is that the extent of chemical conversion can be
determined accurately without further electrokinetic correction if
the total amount of each species can be measured. A "total amount"
detector can be accomplished by setting the detector window (such
as a photomultiplier tube or PMT slit) spatially wider than the
longest sample band length, resulting in peaks whose amplitude is
proportional to the amount (as well as the concentration) one would
measure in a non-flowing cuvette experiment. Other examples of
detectors that report the total amount of reagent are ones based on
total photobleaching and total charge upon complete electrochemical
conversion. On the other hand, for "concentration" detectors such
as a narrow PMT slit compared to the sample band lengths, the
extent of reaction still requires the relative mobility correction
as disclosed herein because the gated injector does not alter the
species concentrations in the aliquot.
[0123] Accordingly, in one aspect, the invention provides methods
for dispensing representative mixtures by gated injection. In the
methods, a first fluidic mixture is introduced into a first
microfluidic channel. The mixture has at least first and second
materials; e.g., assay components, reactants or the like, and
optionally comprises any number of additional reaction components.
The first and second materials are transported through the first
channel at different velocities, i.e., due to differences in
charge/mass ratios, differing electrophoretic mobility or the like.
An aliquot of the first and second materials is gated (i.e.,
injected for a selected period of time) into the second channel.
The injection can be performed electrokinetically, i.e., by
applying a voltage or current difference at the intersection
between the first and second channel. The precise arrangement of
the first and second channel is not critical. For example, the
first and second channels optionally communicate at a crossing
intersection or a T intersection. The relative amount of first and
second materials in the aliquot are proportional to the flux of
first and second materials in the first mixture, thereby dispensing
a representative mixture of the first and second components.
[0124] Flux is ordinarily conserved in these methods. The flux of
the first and second components can be the same or different during
electrokinetic movement. The first or second material can be
labeled, and a product resulting from combining the first or second
material is optionally produced. This product is optionally
labeled; in non-fluorogenic labeled, the method comprising
measuring signal from the aliquot of first or second labeled
material, wherein the amount of labeled material is determined by
measuring the signal.
[0125] Modifying Detection Window Size to Analyze Velocitogenic
Reactions
[0126] As set forth above, the size of the detection region
compared to the size of a sample plug has an effect on the data
which is acquired. For a gated injection of a reaction produced on
the fly an "amount" detector such as a wide PMT slit (wider than
the longest sample plug) results in peaks whose amplitude is
proportional to the concentration (or amount) one would measure in
a cuvette experiment. For concentration detectors (e.g., narrow PMT
detection) the concentration is corrected by the velocity to
correctly calculate the percentage of reactant converted.
[0127] As noted above, a gated injection produces a sample plug in
a channel. As the sample plug travels in the channel, the molecules
separate in the sample plug based upon their respective
electrokinetic mobilities. As the sample plug passes a detector,
all or only a portion of the plug can be detected. If the entire
plug is detected, then the total amount of any detected species in
the plug can be detected. If only a portion of the plug is
detected, then the concentration of molecules in the detected
portion can be determined, by taking velocity into account as noted
herein. If the entire sample plug is detected, a velocity
correction does not have to be applied to correctly determine the
amount of product in the plug. Thus, by using gated injection as
noted above, in conjunction with a detection window as wide or
wider than a sample plug passing the detector, amounts of products,
reactants and the like can be determined. Several methods can be
used to vary the detection window size, including varying the slit
width where the detector is a photomultiplier or other similar
physical adjustments to the detector, or by data sampling
frequently in time and adding all of the data for an entire sample
plug.
[0128] Signal Processing, Digital Deconvolution, and Assay
Component Inactivation
[0129] Complex time dependent label signals are observed for
reactions in flowing microfluidic systems. Some of this complexity
is due to stacking of charged molecules in the low conductivity
running buffer used to separate high conductivity sample plugs and
to drive electroosmotic flow. These complex signals can hinder
direct interpretation of data for continuous flow enzyme inhibition
or receptor binding pipettor chip experiments that rely on the use
of the high/low conductivity format for electrokinetic
injection.
[0130] Digital signal processing techniques provide a way of
simplifying the interpretation of data in these types of
experiments. Examples of data analysis routines that are
implemented to simplify data interpretation include baseline
subtraction and masking.
[0131] In baseline subtraction, a series of blanks are injected in
a control experiment to measure the time dependent baseline, which
is then subtracted from an actual experiment to obtain a difference
signal that is proportional to the degree of inhibition of enzyme
activity or receptor binding.
[0132] In the masking approach, a series of label (e.g.,
fluorescent dye) injections are made in a control experiment to
characterize the timing of sample plugs as they pass a detection
point. For example, the dyes can be injected (e.g.,
electrokinetically or by pressure injection) into a channel of a
microfluidic apparatus and flowed in the channel through or past
the detection point. The resulting label intensity versus time data
is then normalized and subjected to a round off function to yield a
mask file which has values of 1 corresponding to points in time at
which sample plugs are positioned in front of the detector and
values of 0 for all other times. Multiplication of the mask file
with the data from an actual screening experiment then identifies
the time windows of interest.
[0133] In both of these approaches, the synchronization of data
acquisition and sample injection is optimally the same for control
experiments and screening experiments and light source intensities,
optics (or other appropriate detector) alignment and injection
cycle are optimally stable over the time course of the experiments.
In a preferred embodiment, the labels are fluorescent, although the
same approach is used with any label described herein, in
conjunction with an appropriate detector.
[0134] In addition to digital deconvolution techniques, assays are
optionally performed in a format which obviates some of the
difficulties observed for interpreting assays e.g., utilizing
fluidic regions comprising high conductivity and low conductivity
buffers (bracketing components in high or low salt buffers tends to
keep components together during electroosmotic flow; see, U.S. Ser.
No. 08/761,575 entitled "High Throughput Screening Assay Systems in
Microscale Fluidic Devices" by Parce et al. (see also U.S. Ser. No.
08/881,696)). In particular, assay components are optionally
deactivated in regions of fluid flowing past a detector. For
example, the interpretation of data for continuous flow enzyme
inhibition or receptor binding studies are optionally simplified by
using a running buffer having a pH sufficiently high (or low) to
deactivate an assay component in the running buffer, so that signal
is only generated in a sample plug (a region or fluid comprising a
high concentration of sample, typically bracketed by regions of
high or low salt buffer). Thus, buffers with pH in the range of
about 1-5 or about 8-14 are useful for inactivating components; for
ease of handling, buffers are typically in the range of about pH
3-11.
[0135] Alternatively, other inhibitors of the particular assay
component are optionally added to running buffer, e.g., to inhibit
enzyme activity or block receptor binding outside of the sample
plug. For example, ion chelators such as EDTA or EGTA are commonly
added to reactions to inhibit enzymatic reactions (e.g., where the
enzyme requires a Mg.sup.++ or Ca.sup.++ ion). Similarly, aliquots
of high or low temperature buffers, can be added to inhibit
reactions comprising temperature sensitive components. Similarly,
heat, cold or light can be applied to the flowing reaction, e.g.,
by contacting the microfluidic element comprising the microchannel
in which the reaction is run with heat, cold or light. In this
regard, reactants can be inactivated simply by running the
reactants through a region of high electrical resistance (e.g., a
narrowed portion of a microfluidic channel). Buffer traversing this
region of high electrical resistance heats up (a phenomenon
referred to as "joule heating"). Accordingly, by selecting current
and channel width, it is possible to inactivate selected portions
of flowing reaction components by joule heating. Thermocycling in
microscale devices utilizing joule heating is described in
co-pending application U.S. Ser. No. 60/056058, attorney docket
number 017646-003800 entitled "ELECTRICAL CURRENT FOR CONTROLLING
FLUID TEMPERATURES IN MICROCHANNELS" filed Sep. 2, 1997 by Calvin
Chow, Anne R. Kopf-Sill and J. Wallace Parce and in 08/977,528,
filed Nov. 25, 1997. see also, 08/835,101 and CIP application
09/054,962 by Knapp et al.
[0136] The reaction can proceed for either a selected time in the
channel prior to addition of the inhibitor, or for a selected
distance down the channel. The inhibitor can be added to the entire
reaction mixture, or any portion thereof; where the inhibitor is in
flowable form, the inhibitor can be added by time or volume gating
of the flowable inhibitor.
[0137] In addition to inactivating components in selected regions
of flow, inhibitors of reaction such as temperature, pH, ion
chelator or the like are optionally used to deactivate or stop a
reaction, e.g., where the reaction is only to be run for a set
period of time.
[0138] Microfluidic Detection Apparatus
[0139] The microfluidic apparatus of the invention often, though
not necessarily, comprise a substrate in which reactants are mixed
and analyzed. A wide variety of suitable substrates for use in the
devices of the invention are described in U.S. Ser. No. 08/761,575,
entitled "High Throughput Screening Assay Systems in Microscale
Fluidic Devices" by Parce et al. A microfluidic substrate holder is
typically incorporated into the devices of the invention for
holding and/or moving the substrate during an assay. The substrate
holder typically includes a substrate viewing region for analysis
of reactions carried out on the substrate. An analyte detector
mounted proximal to the substrate viewing region to detect
formation of products and/or passage of reactants along a portion
of the substrate is provided. A computer, operably linked to the
analyte detector, monitors reaction rates by taking velocities and
concentrations of reactants and products into account. An
electrokinetic component typically provides for movement of the
fluids on the substrate. Microfluidic devices and systems are also
described in Attorney Docket Number 17646-000100, filed Aug. 2,
1996, U.S. Ser. No. 08/691,632.
[0140] One of skill will immediately recognize that any, or all, of
these components are optionally manufactured in separable modular
units, and assembled to form an apparatus of the invention. See
also, U.S. Ser. No. 08/691,632, supra. In particular, a wide
variety of substrates having different channels, wells and the like
are typically manufactured to fit interchangeably into the
substrate holder, so that a single apparatus can accommodate, or
include, many different substrates adapted to control a particular
reaction. Similarly, computers, analyte detectors and substrate
holders are optionally manufactured in a single unit, or in
separate modules which are assembled to form an apparatus for
manipulating and monitoring a substrate. In particular, a computer
does not have to be physically associated with the rest of the
apparatus to be "operably linked" to the apparatus. A computer is
operably linked when data is delivered from other components of the
apparatus to the computer. One of skill will recognize that
operable linkage can easily be achieved using either electrically
conductive cable coupled directly to the computer (e.g., a
parallel, serial or modem cables), or using data recorders which
store data to computer readable media (typically magnetic or
optical storage media such as computer disks and diskettes, CDs,
magnetic tapes, but also optionally including physical media such
as punch cards, vinyl media or the like).
[0141] Substrates and Electrokinetic Modulators
[0142] Suitable substrate materials are generally selected based
upon their compatibility with the conditions present in the
particular operation to be performed by the device. Such conditions
can include extremes of pH, temperature, salt concentration, and
application of electrical fields. Additionally, substrate materials
are also selected for their inertness to critical components of an
analysis or synthesis to be carried out by the device.
[0143] Examples of useful substrate materials include, e.g., glass,
quartz and silicon as well as polymeric substrates, e.g. plastics,
particularly polyacrylates. In the case of conductive or
semi-conductive substrates, it is occasionally desirable to include
an insulating layer on the substrate. This is particularly
important where the device incorporates electrical elements, e.g.,
electrical fluid direction systems, sensors and the like. In the
case of polymeric substrates, the substrate materials may be rigid,
semi-rigid, or non-rigid, opaque, semi-opaque or transparent,
depending upon the use for which they are intended. For example,
devices which include an optical, spectrographic, photographic or
visual detection element, will generally be fabricated, at least in
part, from transparent materials to allow, or at least, facilitate
that detection. Alternatively, transparent windows of, e.g., glass
or quartz, are optionally incorporated into the device for these
types of detection elements. Additionally, the polymeric materials
optionally have linear or branched backbones, and may be
crosslinked or non-crosslinked. Examples of particularly preferred
polymeric materials include, e.g., polydimethylsiloxanes (PDMS),
polyurethane, polyvinylchloride (PVC) polystyrene, polysulfone,
polycarbonate and the like.
[0144] In certain embodiments, the substrate includes microchannels
for flowing reactants and products. At least one of these channels
typically has a very small cross sectional dimension, e.g., in the
range of from about 0.1 .mu.m to about 500 .mu.m. Preferably the
cross-sectional dimensions of the channels is in the range of from
about 0.1 to about 200 .mu.m and more preferably in the range of
from about 0.1 to about 100 .mu.m. In particularly preferred
aspects, each of the channels has at least one cross-sectional
dimension in the range of from about 0.1 .mu.m to about 100 .mu.m.
Although generally shown as straight channels for convenience of
illustration, it will be appreciated that in order to maximize the
use of space on a substrate, serpentine, saw tooth or other channel
geometries, are used to incorporate longer channels on less
substrate area. Substrates are of essentially any size, with area
typical dimensions of about 1 cm.sup.2 to 10 cm.sup.2.
[0145] Manufacturing of these microscale elements into the surface
of the substrates is generally be carried out by any number of
microfabrication techniques that are well known in the art. For
example, lithographic techniques are employed in fabricating, e.g.,
glass, quartz or silicon substrates, using methods well known in
the semiconductor manufacturing industries such as
photolithographic etching, plasma etching or wet chemical etching.
See, Sorab K. Ghandi, VLSI Principles: Silicon and Gallium
Arsenide, NY, Wiley (see, esp. Chapter 10). Alternatively,
micromachining methods such as laser drilling, air abrasion,
micromilling and the like may be employed. Similarly, for polymeric
substrates, well known manufacturing techniques are used. These
techniques include injection molding or stamp molding methods where
large numbers of substrates may be produced using, e.g., rolling
stamps to produce large sheets of microscale substrates or polymer
microcasting techniques where the substrate is polymerized within a
micromachined mold. Polymeric substrates are further described in
Provisional Patent Application Serial No. 60/015,498, filed Apr.
16, 1996 (Attorney Docket No. 017646-002600), and Attorney Docket
Number 17646-002610, filed Apr. 14, 1997.
[0146] In addition to micromachining methods, printing methods are
also used to fabricate chambers channels and other microfluidic
elements on a solid substrate. Such methods are taught in detail in
U.S. Ser. No. 08/987,803 by Colin Kennedy, Attorney Docket Number
017646-004400, filed Dec. 10, 1997 entitled "Fabrication of
Microfluidic Circuits by Printing Techniques." In brief, printing
methods such as ink-jet printing, laser printing or other printing
methods are used to print the outlines of a microfluidic element on
a substrate, and a cover layer is fixed over the printed outline to
provide a closed microfluidic element.
[0147] The substrates will typically include an additional planar
element which overlays the channeled portion of the substrate
enclosing and fluidly sealing the various channels. Attaching the
planar cover element may be achieved by a variety of means,
including, e.g., thermal bonding, adhesives or, in the case of
certain substrates, e.g., glass, or semi-rigid and non-rigid
polymeric substrates, a natural adhesion between the two
components. A preferred embodiment is heat lamination, which
results in permanent bonding of, e.g., glass substrates. In fact,
during heat lamination, the pieces fuse to form a single piece;
there is no joint between the pieces, even when viewed by electron
microscopy. The planar cover element can additionally be provided
with access ports and/or reservoirs for introducing the various
fluid elements needed for a particular screen, and for introducing
electrodes for electrokinetic movement.
[0148] The introduction of large numbers of individual, discrete
volumes of test compounds into the substrate is carried out by any
of a number of methods. For example, micropipettors are used to
introduce the test compounds to the substrate. In one embodiment,
an automated pipettor is used. For example, a Zymate XP (Zymark
Corporation; Hopkinton, Mass.) automated robot using Microlab 2200
(Hamilton; Reno, Nev.) pipeting station can be used to transfer
parallel samples to regularly spaced wells in a manner similar to
transfer of samples to microtiter plates.
[0149] In preferred aspects, an electropipettor is used. An example
of such an electropipettor is described in, e.g., U.S. patent
application Ser. No. 08/671,986, filed Jun. 28, 1996 (Attorney
Docket No. 017646-000500). Generally, this electropipettor utilizes
electrokinetic or "electroosmotic" fluid direction as described
herein, to alternately sample a number of test compounds, or
"subject materials," and spacer compounds. The pipettor then
delivers individual, physically isolated sample or test compound
volumes in subject material regions, in series, into the sample
channel for subsequent manipulation within the device. Individual
samples are typically separated by a spacer region of low ionic
strength spacer fluid. These low ionic strength spacer regions have
higher voltage drop over their length than do the higher ionic
strength subject material or test compound regions, thereby driving
the electrokinetic pumping. On either side of the test compound or
subject material region, which is typically in higher ionic
strength solution, are fluid regions referred to as first spacer
regions (also referred to as high salt regions on "guard bands"),
that contact the interface of the subject material regions. These
first spacer regions typically comprise a high ionic strength
solution to prevent migration of the sample elements into the lower
ionic strength fluid regions, or second spacer region, which would
result in electrophoretic bias. The use of such first and second
spacer regions is described in greater detail in U.S. patent
application Ser. No. 08/671,986, filed Jun. 28, 1996, (Attorney
Docket No. 017646-000500).
[0150] Alternatively, the channels are individually fluidly
connected to a plurality of separate reservoirs via separate
channels. The separate reservoirs each contain a separate analyte,
reagent, reaction component or the like, with additional reservoirs
being provided, e.g., for appropriate spacer compounds. The test
compounds and/or spacer compounds are transported from the various
reservoirs into the sample channels using appropriate fluid
direction schemes. In either case, it generally is desirable to
separate the discrete sample volumes, or test compounds, with
appropriate spacer regions.
[0151] In operation, a fluid first component of a biological
system, e.g., a receptor or enzyme, is placed in a first reservoir
on the substrate. This first component is flowed through a channel
past a detection window and toward a waste reservoir. A second
component of the biochemical system, e.g., a ligand or substrate,
is concurrently flowed into the channel, whereupon the first and
second components mix and are able to interact. Deposition of these
elements within the device are carried out in a number of ways. For
example, the enzyme and substrate, or receptor and ligand solutions
introduced into the device through open or sealable access ports in
the cover. Alternatively, these components are added to their
respective reservoirs during manufacture of the device. In the case
of such pre-added components, it is desirable to provide these
components in a stabilized form to allow for prolonged shelf-life
of the device. For example, the enzyme/substrate or receptor/ligand
components are provided within the device in lyophilized form.
Prior to use, these components are easily reconstituted by
introducing a buffer solution into the reservoirs. Alternatively,
the components are lyophilized with appropriate buffering salts,
whereby simple water addition is all that is required for
reconstitution.
[0152] Flowing and direction of fluids within the microscale
fluidic devices may be carried out by a variety of methods. For
example, the devices may include integrated microfluidic
structures, such as micropumps and microvalves, or external
elements, e.g., pumps and switching valves, for the pumping and
direction of the various fluids through the device. Examples of
microfluidic structures are described in, e.g., U.S. Pat. Nos.
5,271,724, 5,277,556, 5,171,132, and 5,375,979. See also, Published
U.K. Patent Application No. 2 248 891 and Published European Patent
Application No. 568 902.
[0153] Although microfabricated fluid pumping and valving systems
may be readily employed in the devices of the invention, the cost
and complexity associated with their manufacture and operation can
generally prohibit their use in mass-produced disposable devices as
are envisioned by the present invention. Furthermore, the velocity
of components in such systems is driven by overall fluid flow,
making consideration of velocity less relevant in these systems
(there is no electrophoretic component of velocity in a pure
pressure-driven system). For that reason, the devices of the
invention will typically include an electroosmotic fluid direction
system. Such fluid direction systems combine the elegance of a
fluid direction system devoid of moving parts, with an ease of
manufacturing, fluid control and disposability. Examples of
particularly preferred electroosmotic fluid direction systems
include, e.g., those described in International Patent Application
No. WO 96/04547 to Ramsey et al., as well as U.S. Ser. No.
08/761,575 by Parce et al.
[0154] In brief, these fluidic control systems typically include
electrodes disposed within reservoirs that are placed in fluid
connection with the channels fabricated into the surface of the
substrate. The materials stored in the reservoirs are transported
through the channel system delivering appropriate volumes of the
various materials to one or more regions on the substrate in order
to carry out a desired screening assay.
[0155] Fluid transport and direction is accomplished through
electroosmosis or electrokinesis. In brief, when an appropriate
fluid is placed in a channel or other fluid conduit having
functional groups present at the surface, those groups can ionize.
For example, where the surface of the channel includes hydroxyl
functional groups at the surface, protons can leave the surface of
the channel and enter the fluid. Under such conditions, the surface
will possess a net negative charge, whereas the fluid will possess
an excess of protons or positive charge, particularly localized
near the interface between the channel surface and the fluid. By
applying an electric field along the length of the channel, cations
will flow toward the negative electrode. Movement of the positively
charged species in the fluid pulls the solvent with them.
[0156] To provide appropriate electric fields, the system generally
includes a voltage controller that is capable of applying
selectable voltage levels, simultaneously, to each of the
reservoirs, including ground. Such a voltage controller can be
implemented using multiple voltage dividers and multiple relays to
obtain the selectable voltage levels. Alternatively, multiple,
independent voltage sources may be used. The voltage controller is
electrically connected to each of the reservoirs via an electrode
positioned or fabricated within each of the plurality of
reservoirs. In one embodiment, multiple electrodes are positioned
to provide for switching of the electric field direction in a
microchannel, thereby causing the analytes to travel a longer
distance than the physical length of the microchannel.
[0157] Substrate materials are also selected to produce channels
having a desired surface charge. In the case of glass substrates,
the etched channels will possess a net negative charge resulting
from the ionized hydroxyls naturally present at the surface.
Alternatively, surface modifications may be employed to provide an
appropriate surface charge, e.g., coatings, derivatization, e.g.,
silanation, or impregnation of the surface to provide appropriately
charged groups on the surface. Examples of such treatments are
described in, e.g., Provisional Patent Application Serial No.
60/015,498, filed Apr. 16, 1996 (Attorney Docket No.
017646-002600). See also, Attorney Docket Number 17646-002610,
filed Apr. 14, 1997.
[0158] Modulating voltages are then concomitantly applied to the
various reservoirs to affect a desired fluid flow characteristic,
e.g., continuous or discontinuous (e.g., a regularly pulsed field
causing the flow to oscillate direction of travel) flow of
receptor/enzyme, ligand/substrate toward the waste reservoir with
the periodic introduction of test compounds. Particularly,
modulation of the voltages applied at the various reservoirs can
move and direct fluid flow through the interconnected channel
structure of the device in a controlled manner to effect the fluid
flow for the desired screening assay and apparatus.
[0159] Detectors and Labels
[0160] A "label" is any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means. Useful labels in the present invention include
fluorescent dyes (e.g., fluorescein isothiocyanate, texas red,
rhodamine, and the like), radiolabels (e.g., .sup.3H, .sup.125I,
.sup.35S, .sup.14C, .sup.32P, .sup.33P, etc.), enzymes (e.g.,
horse-radish peroxidase, alkaline phosphatase etc.) colorimetric
labels such as colloidal gold or colored glass or plastic (e.g.
polystyrene, polypropylene, latex, etc.) beads. The label may be
coupled directly or indirectly to the a component of the assay
according to methods well known in the art. As indicated above, a
wide variety of labels may be used, with the choice of label
depending on sensitivity required, ease of conjugation with the
compound, stability requirements, available instrumentation, and
disposal provisions. Non-radioactive labels are often attached by
indirect means. Generally, a ligand molecule (e.g., biotin) is
covalently bound to the molecule. The ligand then binds to an
anti-ligand (e.g., streptavidin) molecule which is either
inherently detectable or covalently bound to a signal system, such
as a detectable enzyme, a fluorescent compound, or a
chemiluminescent compound. A number of ligands and anti-ligands can
be used. Where a ligand has a natural anti-ligand, for example,
biotin, thyroxine, or cortisol, it can be used in conjunction with
the labeled, naturally occurring anti-ligands. Alternatively, any
haptenic or antigenic compound can be used in combination with an
antibody (see, e.g., Coligan (1991) Current Protocols in Immunology
Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A
Laboratory Manual Cold Spring Harbor Press, NY for a general
discussion of how to make and use antibodies). The molecules can
also be conjugated directly to signal generating compounds, e.g.,
by conjugation with an enzyme or fluorophore. Enzymes of interest
as labels will primarily be hydrolases, particularly phosphatases,
esterases and glycosidases, or oxidoreductases, particularly
peroxidases. Fluorescent compounds include fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone,
etc. Chemiluminescent compounds include luciferin, and
2,3-dihydrophthalazinediones, e.g., luminol.
[0161] In some embodiments, a first and second label on the same or
different components interact when in proximity (e.g., due to
fluorescence resonance transfer), and the relative proximity of the
first and second labels is determined by measuring a change in the
intrinsic fluorescence of the first or second label. For example,
the emission of a first label is sometimes quenched by proximity of
the second label. Many appropriate interactive labels are known.
For example, fluorescent labels, dyes, enzymatic labels, and
antibody labels are all appropriate. Examples of interactive
fluorescent label pairs include terbium chelate and TRITC
(tetrarhodamine isothiocyanate), europium cryptate and
Allophycocyanin, DABCYL and EDANS and many others known to one of
skill. Similarly, two colorimetric labels can result in
combinations which yield a third color, e.g., a blue emission in
proximity to a yellow emission provides an observed green emission.
With regard to preferred fluorescent pairs, there are a number of
fluorophores which are known to quench one another. Fluorescence
quenching is a bimolecular process that reduces the fluorescence
quantum yield, typically without changing the fluorescence emission
spectrum. Quenching can result from transient excited state
interactions, (collisional quenching) or, e.g., from the formation
of nonfluorescent ground state species. Self quenching is the
quenching of one fluorophore by another; it tends to occur when
high concentrations, labeling densities, or proximity of labels
occurs. Fluorescent resonance energy transfer (FRET) is a distance
dependent excited state interaction in which emission of one
fluorophore is coupled to the excitation of another which is in
proximity (close enough for an observable change in emissions to
occur). Some excited fluorophores interact to form excimers, which
are excited state dimers that exhibit altered emission spectra
(e.g., phospholipid analogs with pyrene sn-2 acyl chains); see,
Haugland (1996) Handbook of Fluorescent Probes and Research
Chemicals Published by Molecular Probes, Inc., Eugene, Oreg. e.g.,
at chapter 13).
[0162] Detectors for detecting labeled compounds are known to those
of skill in the art. Thus, for example, where the label is a
radioactive label, means for detection include a scintillation
counter or photographic film as in autoradiography. Where the label
is a fluorescent label, it may be detected by exciting the
fluorochrome with the appropriate wavelength of light and detecting
the resulting fluorescence. The fluorescence may be detected
visually, by means of photographic film, by the use of electronic
detectors such as charge coupled devices (CCDs) or
photomultipliers, phototubes, photodiodes or the like. Similarly,
enzymatic labels are detected by providing the appropriate
substrates for the enzyme and detecting the resulting reaction
product. Finally simple calorimetric labels may be detected simply
by observing the color associated with the label. This is done
using a spectrographic device, e.g., having an appropriate grating,
filter or the like allowing passage of a particular wavelength of
light, and a photodiode, or other detector for converting light to
an electronic signal, or for enhancing visual detection.
[0163] The substrate includes a detection window or zone at which a
signal is monitored. For example, reactants or assay components are
contacted in a microfluidic channel in a first region, and
subsequently flowed into a second channel region comprising a
detection window or region. The first and second channel region are
optionally part of a single channel, but can also be separate
channels, e.g., which are in fluid connection. This detection
window or region typically includes a light or radiation
transparent cover allowing visual or optical observation and
detection of the assay results, e.g., observation of a
colorometric, fluorometric or radioactive response, or a change in
the velocity of colorometric, fluorometric or radioactive
component. Detectors detect a labeled compound. Example detectors
include spectrophotometers, photodiodes, microscopes, scintillation
counters, cameras, film and the like, as well as combinations
thereof. Examples of suitable detectors are widely available from a
variety of commercial sources known to persons of skill.
[0164] In one aspect, monitoring of the signals at the detection
window is achieved using an optical detection system. For example,
fluorescence based signals are typically monitored using, e.g., in
laser activated fluorescence detection systems which employ a laser
light source at an appropriate wavelength for activating the
fluorescent indicator within the system. Fluorescence is then
detected using an appropriate detector element, e.g., a
photomultiplier tube (PMT). Similarly, for screens employing
colorometric signals, spectrophotometric detection systems may be
employed which detect a light source at the sample and provide a
measurement of absorbance or transmissivity of the sample. See
also, The Photonics Design and Applications Handbook, books 1, 2, 3
and 4, published annually by Laurin Publishing Co., Berkshire
Common, P.O. Box 1146, Pittsfield, Mass. for common sources for
optical components.
[0165] In alternative aspects, the detection system comprises
non-optical detectors or sensors for detecting a particular
characteristic of the system disposed within detection window 116.
Such sensors may include temperature (useful, e.g., when a reaction
produces or absorbs heat), conductivity, potentiometric (pH, ions),
amperometric (for compounds that may be oxidized or reduced, e.g.,
O.sub.2, H.sub.2O.sub.2, I.sub.2, oxidizable/reducible organic
compounds, and the like).
[0166] Alternatively, schemes similar to those employed for the
enzymatic system may be employed, where there is a signal that
reflects the interaction of the receptor with its ligand. For
example, pH indicators which indicate pH effects of receptor-ligand
binding may be incorporated into the device along with the
biochemical system, i.e., in the form of encapsulated cells,
whereby slight pH changes resulting from binding can be detected.
See Weaver, et al., Bio/Technology (1988) 6:1084-1089.
Additionally, one can monitor activation of enzymes resulting from
receptor ligand binding, e.g., activation of kinases, or detect
conformational changes in such enzymes upon activation, e.g.,
through incorporation of a fluorophore which is activated or
quenched by the conformational change to the enzyme upon
activation.
[0167] One conventional system carries light from a specimen field
to a cooled charge-coupled device (CCD) camera. A CCD camera
includes an array of picture elements (pixels). The light from the
specimen is imaged on the CCD. Particular pixels corresponding to
regions of the substrate are sampled to obtain light intensity
readings for each position. Multiple positions are processed in
parallel and the time required for inquiring as to the intensity of
light from each position is reduced. Many other suitable detection
systems are known to one of skill.
[0168] Assays
[0169] In the assays of the invention, a first reactant or assay
component is contacted to a second reactant or product, typically
to form a product. The reactants or components can be elements of
essentially any assay which is adaptable to a flowing format; thus,
while often described in terms of enzyme-substrate or
receptor-ligand interactions, it will be understood that the
reactants or components herein can comprise a moiety derived from
any of a wide variety of components, including, antibodies,
antigens, ligands, receptors, enzymes, enzyme substrates, amino
acids, peptides, proteins, nucleosides, nucleotides, nucleic acids,
fluorophores, chromophores, biotin, avidin, organic molecules,
monomers, polymers, drugs, polysaccharides, lipids, liposomes,
micelles, toxins, biopolymers, therapeutically active compounds,
molecules from biological sources, blood constituents, cells or the
like. No attempt is made herein to describe how known assays
utilizing these components are practiced. A wide variety of
microfluidic assays are practiced using these components. See,
e.g., U.S. Ser. No. 08/761,575 entitled "High Throughput Screening
Assay Systems in Microscale Fluidic Devices" by Parce et al. (see
also U.S. Ser. No. 08/881,696).
[0170] As used herein, the phrase "biochemical system" generally
refers to a chemical interaction that involves molecules of the
type generally found within living organisms. Such interactions
include the full range of catabolic and anabolic reactions which
occur in living systems including enzymatic, binding, signalling
and other reactions. Further, biochemical systems, as defined
herein, also include model systems which are mimetic of a
particular biochemical interaction. Examples of biochemical systems
of particular interest in practicing the present invention include,
e.g., receptor-ligand interactions, enzyme-substrate interactions,
cellular signaling pathways, transport reactions involving model
barrier systems (e.g., cells or membrane fractions) for
bioavailability screening, and a variety of other general systems.
Cellular or organismal viability or activity may also be screened
using the methods and apparatuses of the present invention, e.g.,
in toxicology studies. Biological materials which are assayed
include, but are not limited to, cells, cellular fractions
(membranes, cytosol preparations, etc.), agonists and antagonists
of cell membrane receptors (e.g., cell receptor-ligand interactions
such as e.g., transferrin, c-kit, viral receptor ligands (e.g.,
CD4-HIV), cytokine receptors, chemokine receptors, interleukin
receptors, immunoglobulin receptors and antibodies, the cadherein
family, the integrin family, the selectin family, and the like;
see, e.g., Pigott and Power (1993) The Adhesion Molecule FactsBook
Academic Press New York and Hulme (ed) Receptor Ligand Interactions
A Practical Approach Rickwood and Hames (series editors) IRL Press
at Oxford Press NY), toxins and venoms, viral epitopes, hormones
(e.g., opiates, steroids, etc.), intracellular receptors (e.g.
which mediate the effects of various small ligands, including
steroids, thyroid hormone, retinoids and vitamin D; for reviews
see, e.g., Evans (1988) Science, 240:889-895; Ham and Parker (1989)
Curr. Opin. Cell Biol, 1:503-511; Burnstein et al. (1989), Ann.
Rev. Physiol., 51:683-699; Truss and Beato (1993) Endocr. Rev.,
14:459-479), peptides, retro-inverso peptides, polymers of
.alpha.-, .beta.-, or .omega.- amino acids (D- or L-), enzymes,
enzyme substrates, cofactors, drugs, lectins, sugars, nucleic acids
(both linear and cyclic polymer configurations), oligosaccharides,
proteins, phospholipids and antibodies. Synthetic polymers such as
heteropolymers in which a known drug is covalently bound to any of
the above, such as polyurethanes, polyesters, polycarbonates,
polyureas, polyamides, polyethyleneimines, polyarylene sulfides,
polysiloxanes, polyimides, and polyacetates are also assayed. Other
polymers are also assayed using the systems described herein, as
would be apparent to one of skill upon review of this disclosure.
One of skill will be generally familiar with the biological
literature. For a general introduction to biological systems, see,
Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods
in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.
(Berger); Sambrook et al. (1989) Molecular Cloning--A Laboratory
Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold
Spring Harbor Press, NY, (Sambrook); Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (through 1998 Supplement) (Ausubel); Watson et
al. (1987) Molecular Biology of the Gene, Fourth Edition The
Benjamin/Cummings Publishing Co., Menlo Park, Calif.; Watson et al.
(1992) Recombinant DNA Second Edition Scientific American Books,
NY; Alberts et al. (1989) Molecular Biology of the Cell Second
Edition Garland Publishing, NY; Pattison (1994) Principles and
Practice of Clinical Virology; Darnell et al., (1990) Molecular
Cell Biology second edition, Scientific American Books, W. H.
Freeman and Company; Berkow (ed.) The Merck Manual of Diagnosis and
Therapy, Merck & Co., Rahway, N.J.; Harrison's Principles of
Internal Medicine, Thirteenth Edition, Isselbacher et al. (eds).
(1994) Lewin Genes, 5th Ed., Oxford University Press (1994); The
"Practical Approach" Series of Books (Rickwood and Hames (series
eds.) by IRL Press at Oxford University Press, NY; The "FactsBook
Series" of books from Academic Press, NY, ; Product information
from manufacturers of biological reagents and experimental
equipment also provide information useful in assaying biological
systems. Such manufacturers include, e.g., the SIGMA chemical
company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.),
Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH
Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich
Chemical Company (Milwaukee, Wis.), Glen Research,
Inc..sup..cndot., GIBCO BRL Life Technologies, Inc. (Gaithersberg,
Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs,
Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems
(Foster City, Calif.), as well as many other commercial sources
known to one of skill.
[0171] In order to provide methods and devices for screening
compounds for effects on biochemical systems, the present invention
generally incorporates model in vitro systems which mimic a given
biochemical system in vivo for which effector compounds are
desired. The range of systems against which compounds can be
screened and for which effector compounds are desired, is
extensive. For example, compounds are optionally screened for
effects in blocking, slowing or otherwise inhibiting key events
associated with biochemical systems whose effect is undesirable. As
described supra, the effects of velocity of the components are
corrected for to provide accurate determinations of the rates of
these key events.
[0172] For example, assay compounds are optionally screened for
their ability to block systems that are responsible, at least in
part, for the onset of disease or for the occurrence of particular
symptoms of diseases, including, e.g., hereditary diseases, cancer,
bacterial or viral infections and the like. Compounds which show
promising results in these screening assay methods can then be
subjected to further testing to identify effective pharmacological
agents for the treatment of disease or symptoms of a disease. Using
the data correction methods described herein, the effects of assay
compounds on biochemical systems is properly determined. For
example, the binding properties of a test molecule to a target, or
the effects of an enzyme modulator are easily determined using the
methods herein.
[0173] Alternatively, compounds can be screened for their ability
to stimulate, enhance or otherwise induce biochemical systems whose
function is believed to be desirable, e.g., to remedy existing
deficiencies in a patient. Furthermore, as described extensively
supra, enzyme activity levels (which can be diagnostic of diseases)
are correctly determined using the methods herein.
[0174] Once a model system is selected, batteries of test compounds
can be applied against these model systems. By identifying those
test compounds that have an effect on the particular biochemical
system, in vitro, one can identify potential effectors of that
system, in vivo.
[0175] In one form, the biochemical system models employed in the
methods and apparatuses of the present invention will screen for an
effect of a an assay compound on an interaction between two or more
components of a biochemical system, e.g., receptor-ligand
interaction, enzyme-substrate interaction, and the like. In this
form, the biochemical system model will typically include the two
normally interacting components of the system for which an effector
is sought, e.g., the receptor and its ligand or the enzyme and its
substrate.
[0176] Determining whether a test compound has an effect on this
interaction then involves contacting the system with an assay
compound and assaying for the functioning of the system, e.g.,
receptor-ligand binding or substrate turnover. The assayed function
is then compared to a control, e.g., the same reaction in the
absence of the test compound or in the presence of a known
effector, taking proper steps to correct for velocity of components
as described supra. Typically, such assays involve the measurement
of a parameter of the biochemical system. By "parameter of the
biochemical system" is meant some measurable evidence of the
system's functioning, e.g., the presence or absence of a labeled
group or a change in molecular weight (e.g., in binding reactions,
transport screens), the presence or absence of a reaction product
or substrate (in substrate turnover measurements), or an alteration
in electrophoretic mobility (detected, e.g., by a change in signal
from a detector in the system).
[0177] Although described in terms of two-component biochemical
systems, the methods and apparatuses may also be used to screen for
effectors of much more complex systems, where the result or end
product of the system is known and assayable at some level, e.g.,
enzymatic pathways, cell signaling pathways and the like.
Alternatively, the methods and apparatuses described herein are
optionally used to screen for compounds that interact with a single
component of a biochemical system, e.g., compounds that
specifically bind to a particular biochemical compound, e.g., a
receptor, ligand, enzyme, nucleic acid, structural macromolecule,
etc. In all of these instances, the ability to correctly measure
binding reactions, product production rates, assay component
concentrations and the like, using the methods herein, makes the
assay more predictive and representative.
[0178] Biochemical system models are also embodied in whole cell
systems. For example, where one is seeking to screen test compounds
for an effect on a cellular response, whole cells are optionally
utilized. Modified cell systems are employed in the systems
encompassed herein. For example, chimeric reporter systems are
optionally employed as indicators of an effect of a test compound
on a particular biochemical system. Chimeric reporter systems
typically incorporate a heterogenous reporter system integrated
into a signaling pathway which signals the binding of a receptor to
its ligand. For example, a receptor is fused to a heterologous
protein, e.g., an enzyme whose activity is readily assayable.
Activation of the receptor by ligand binding then activates the
heterologous protein, which then allows for detection. Thus, the
surrogate reporter system produces an event or signal which is
readily detectable, thereby providing an assay for receptor/ligand
binding. Examples of such chimeric reporter systems have been
previously described in the art. An example is the common
chloramphenicol acetyl transferase (CAT) assay.
[0179] Additionally, where one is screening for bioavailability,
e.g., transport, biological barriers are optionally included. The
term "biological barriers" generally refers to cellular or
membranous layers within biological systems, or synthetic models
thereof. Examples of such biological barriers include the
epithelial and endothelial layers, e.g. vascular endothelia and the
like.
[0180] Biological responses are often triggered and/or controlled
by the binding of a receptor to its ligand. For example,
interaction of growth factors, e.g., EGF, FGF, PDGF, etc., with
their receptors stimulates a wide variety of biological responses
including, e.g., cell proliferation and differentiation, activation
of mediating enzymes, stimulation of messenger turnover,
alterations in ion fluxes, activation of enzymes, changes in cell
shape and the alteration in genetic expression levels. Accordingly,
control of the interaction of the receptor and its ligand may offer
control of the biological responses caused by that interaction.
[0181] Accordingly, in one aspect, the present invention will be
useful in screening for, or testing the activity of, compounds that
affect an interaction between a receptor molecule and its ligands.
As used herein, the term "receptor" generally refers to one member
of a pair of compounds which specifically recognize and bind to
each other. The other member of the pair is termed a "ligand."
Thus, a receptor/ligand pair may include a typical protein
receptor, usually membrane associated, and its natural ligand,
e.g., another protein or small molecule. Receptor/ligand pairs can
include antibody/antigen binding pairs, complementary nucleic
acids, nucleic acid associating proteins and their nucleic acid
ligands. A large number of specifically associating biochemical
compounds are well known in the art and can be utilized in
practicing the present invention.
[0182] Traditionally, methods for screening for effectors of a
receptor/ligand interaction have involved incubating a
receptor/ligand binding pair in the presence of a test compound.
The level of binding of the receptor/ligand pair is then compared
to negative and/or positive controls. Where a decrease in normal
binding is seen, the test compound is determined to be an inhibitor
of the receptor/ligand binding. Where an increase in that binding
is seen, the test compound is determined to be an enhancer or
inducer of the interaction. The methods of correcting for velocity
and other effects as noted herein provide for correct determination
of these parameters.
[0183] Typically, effectors of an enzyme's activity toward its
substrate are screened by contacting the enzyme with a substrate in
the presence and absence of the compound to be screened and under
conditions optimal for detecting changes in the enzyme's activity.
After a set time for reaction, the mixture is assayed for the
presence of reaction products or a decrease in the amount of
substrate. The amount of substrate that has been catalyzed is them
compared to a control, i.e., enzyme contacted with substrate in the
absence of test compound or presence of a known effector. As above,
a compound that reduces the enzymes activity toward its substrate
is termed an "inhibitor," whereas a compound that accentuates that
activity is termed an "inducer." Again, using the data correction
methods herein, a correct determination of whether a component is
an inhibitor, an inducer, or irrelevant to the system can more
easily be determined.
[0184] The various methods encompassed by the present invention
optionally involve the serial or parallel introduction of one or a
plurality of assay components into a microfluidic device. Once in
the device, the assay component is screened for effect on a
biological or chemical system using a serial or parallel assay
format.
[0185] Assay components are optionally screened for their ability
to affect a particular biochemical or chemical system. Assay
components can include a wide variety of different compounds,
including chemical compounds, mixtures of chemical compounds, e.g.,
polysaccharides, small organic or inorganic molecules, biological
macromolecules, e.g., peptides, proteins, nucleic acids, or an
extract made from biological materials such as bacteria, plants,
fungi, or animal cells or tissues, naturally occurring or synthetic
compositions. Depending upon the particular embodiment being
practiced, the assay components are provided from a source of assay
components, e.g., injected, free in solution, optionally attached
to a carrier, a solid support, e.g., beads or the like. A number of
suitable supports are employed for immobilization of the assay
components. Examples of suitable solid supports include agarose,
cellulose, dextran (commercially available as, i.e., Sephadex,
Sepharose) carboxymethyl cellulose, polystyrene, polyethylene
glycol (PEG), filter paper, nitrocellulose, ion exchange resins,
plastic films, glass beads, polyaminemethylvinylether maleic acid
copolymer, amino acid copolymer, ethylene-maleic acid copolymer,
nylon, silk, etc. Additionally, for the methods and apparatuses
described herein, test compounds are screened individually, or in
groups. Group screening is particularly useful where hit rates for
effective test compounds are expected to be low such that one would
not expect more than one positive result for a given group.
Alternatively, such group screening is used where the effects of
different test compounds are differentially detected in a single
system, e.g., through electrophoretic separation of the effects, or
differential labelling which enables separate detection.
[0186] Assay components are commercially available, or derived from
any of a variety of biological sources apparent to one of skill and
as described, supra. In one aspect, a tissue homogenate or blood
sample from a patient is tested in the assay systems of the
invention. For example, in one aspect, blood is tested for the
presence or activity of a biologically relevant molecule. For
example, the presence and activity level of an enzyme are detected
by supplying and enzyme substrate to the biological sample and
detecting the formation of a product using an assay systems of the
invention. Similarly, the presence of infectious pathogens
(viruses, bacteria, fungi, or the like) or cancerous tumors can be
tested by monitoring binding of a labeled ligand to the pathogen or
tumor cells, or a component of the pathogen or tumor such as a
protein, cell membrane, cell extract or the like, or alternatively,
by monitoring the presence of an antibody against the pathogen or
tumor in the patient's blood. For example, the binding of an
antibody from a patient's blood to a viral protein such as an HIV
protein is a common test for monitoring patient exposure to the
virus. Many assays for detecting pathogen infection are well known,
and are adapted to the assay systems of the present invention.
[0187] Biological samples are derived from patients using well
known techniques such as venipuncture or tissue biopsy. Where the
biological material is derived from non-human animals, such as
commercially relevant livestock, blood and tissue samples are
conveniently obtained from livestock processing plants. Similarly,
plant material used in the assays of the invention are conveniently
derived from agricultural or horticultural sources. Alternatively,
a biological sample can be from a cell or blood bank where tissue
and/or blood are stored, or from an in vitro source such as a
culture of cells. Techniques and methods for establishing a culture
of cells for use as a source for biological materials are well
known to those of skill in the art. Freshney Culture of Animal
Cells, a Manual of Basic Technique, Third Edition Wiley-Liss, New
York (1994) provides a general introduction to cell culture.
[0188] In addition to biological systems, the apparatus and methods
of the invention are adaptable to chemical synthetic approaches.
For example chemical synthetic methods for making proteins, nucleic
acids, amino acids, polymers, organic compounds and the like are
well known. In general, most chemical synthetic protocols employ
fluid mixing to mix reactants, reagents and the like. As applied to
the present invention, a source of reactants, reagents or the like
is fluidly coupled to a microfluidic channel. The reactants or
reagents, which optionally comprise labels, are mixed in a
microchannel. After mixing, reaction rates, product concentrations,
reactant concentrations or the like are easily determined using the
methods described herein. Representative mixtures can be aliquoted
from one channel into a different channel for subsequent analysis,
e.g., using the time-gated methods described supra. No attempt is
made to describe all of the possible reactants, reactions or
products which can be employed in the methods and devices of the
invention; it is presumed that one of skill is generally familiar
with such known methods, and that, upon review of this disclosure,
could adapt these known assays to the present system.
[0189] As described above, the screening methods of the present
invention are generally carried out in microfluidic devices or
"microlaboratory systems," which allow for integration of the
elements required for performing the assay, automation, and minimal
environmental effects on the assay system, e.g., evaporation,
contamination, human error, or the like. A number of devices for
carrying out the assay methods of the invention are described in
substantial detail herein. However, it will be recognized that the
specific configuration of these devices will generally vary
depending upon the type of assay and/or assay orientation desired.
For example, in some embodiments, the screening methods of the
invention can be carried out using a microfluidic device having two
intersecting channels. For more complex assays or assay
orientations, multichannel/intersection devices are optionally
employed. The small scale, integratability and self-contained
nature of these devices allows for virtually any assay orientation
to be realized within the context of the microlaboratory system. In
addition, it will be realized that the data correction methods
herein are applicable to flowing systems generally, and not simply
in microfluidic systems.
[0190] Computers
[0191] Typically, when using a detection device such as that
described herein, data thus obtained is stored and analyzed using a
computer. This may be accomplished by digitizing an image from the
detection device and storing the image on a computer-readable
medium. This is normally accomplished by storing the data
representing the digitized image in a database, spreadsheet file,
or similar storage vehicle on a computer's storage media. A
computer operably linked to the analyte detector is therefore
provided. The computer is coupled to the microfluidic device using
cables to connect the computer to the data detection device.
Alternatively, the data may be recorded on a data collection device
and transported (e.g., on a computer-readable storage medium) to
the computer for processing. Software on the computer determines
the rate of formation of the analyte, correcting for the effects of
the motion of the analyte. This is done, for example, by
determining or collating the velocities of one or more components
and the concentrations of one or more components and calculating
the rate of formation of one or more components, while correcting
for each components' velocity.
[0192] A variety of commercially available hardware and software is
available for digitizing, storing, and analyzing a signal or image
such as that generated by the microfluidic device described herein.
Typically, a computer commonly used to transform signals from the
detection device into reaction rates will be a PC-compatible
computer (e.g., having a central processing unit (CPU) compatible
with x86 CPUs, and running an operating system such as DOS.TM.,
OS/2 Warp.TM., WINDOWS/NT.TM., or WINDOWS 95.TM.), a Macintosh.TM.
(running MacOS.TM.), or a UNIX workstation (e.g., a SUN.TM.
workstation running a version of the Solaris.TM. operating system,
or PowerPC.TM. workstation) are all commercially common, and known
to one of skill in the art. Data analysis software on the computer
is then employed to determine the rate of formation of the analyte
in motion. Software for determining reaction rates is available, or
can easily be constructed by one of skill using a standard
programming language such as Visual Basic, Fortran, Basic, Java, or
the like. The software is designed to determine velocities,
concentrations, flux relationships and the like, as described
herein.
[0193] In general, software designed to perform data manipulations
will include several common steps. FIG. 4B illustrates the steps
performed in calculating a concentration profile along a
microfluidic channel for a continuous flow binding assay as a
function of time for a given association constant (K.sub.a) The
process illustrated by FIG. 4B begins at step 400 with the
acquisition of the data from the detection device. Data thus
acquired is then stored in a database, spreadsheet file, or similar
construct (step 405). As noted, these steps may be carried out
remotely from the computer system used to analyze the acquired
data, with the acquired data being transferred to the computer
system using removable media, network, or other such mechanism. The
structures used to store the data (e.g., arrays) are then
initialized (step 410). This includes calculating row indices and
initializing the time index, and zeroing-out the concentration
arrays. Test parameters are then read in from storage on the
computer (step 415). This includes ranges for the variables,
including the time increment between measurements. These operations
need not be performed in this order, as they merely set up the
variables considered in performing the calculations outlined supra.
Next equilibrium concentrations are calculated (step 420). The
concentration profile information generated by this step is then
output to the database (step 425). The timing signal value
corresponding to the concentration profile information is also
output to the database (step 430).
[0194] Next, at step 435, flow conditions are used to calculate
motion of the various chemical species involved in the test being
analyzed. This, in effect, corresponds to the motion of the various
chemical species down the microfluidic channel. The changes are
reflected in the variable representing the concentrations of each
of the chemical species. At step 436, new equilibrium
concentrations are calculated for each of the chemical species.
Again, concentration profile information and corresponding timing
signal information generated by the equilibrium calculations are
output to the database (steps 437 and 438, respectively). As noted,
each test is broken up into time increments. Analysis of the test
finishes when the number of time increments equals the duration of
the test (step 440). Otherwise, the index representing the time
elapsed is incremented (also represented by step 440) and steps
435-438 repeated, as illustrated in FIG. 4B.
[0195] FIG. 4C illustrates an alternative set of steps according to
the present invention for calculating a concentration profile along
a microfluidic channel for a continuous flow binding assay as a
function of time for a given association constant (K.sub.a). The
process illustrated by FIG. 4C begins at step 445 with the
acquisition of the data from the detection device. Data thus
acquired is then stored in a database, spreadsheet file, or similar
construct (step 450). As noted, these steps may be carried out
remotely from the computer system used to analyze the acquired
data, with the acquired data being transferred to the computer
system using removable media, network, or other such mechanism.
Test parameters are then read in from storage on the computer (step
455), including ranges for the considered variables, including the
time increment between measurements. The structures used to store
the data (e.g., arrays) are then initialized (step 460), including
calculating row indices and initializing the time index, and
zeroing-out the concentration arrays. As before, these operations
need not be performed in this order. Next, initial concentration
profile information is output to the database (step 465). The
timing signal value corresponding to the concentration profile
information is also output to the database (step 470).
[0196] Next, at step 475, motion of the chemical species is
calculated, corresponding to the motion of the various chemical
species down the microfluidic channel. These changes are reflected
in the variable representing the concentrations of each of the
chemical species. At step 476, enzyme reactions are calculated.
Again, concentration profile information and corresponding timing
signal information generated by the equilibrium calculations are
output to the database (steps 477 and 478, respectively). As noted,
each test is broken up into time increments. Analysis of the test
finishes when the number of time increments equals the duration of
the test (step 480). Otherwise, the index representing the time
elapsed is incremented (also represented by step 480) and steps
475-478 repeated, as illustrated in FIG. 4C.
[0197] Exemplary spreadsheet macro software is provided in Appendix
A and Appendix B.
EXAMPLES
[0198] The following examples are offered to illustrate, but not to
limit the present invention.
Example 1
[0199] Monitoring Flux in a Microchannel
[0200] In a given microchannel of a microfluidic device, the flux
(J), with units of molecules/(cross sectional area.times.time), is
equal to the velocity of the molecules under consideration (U)
times the concentration of molecules (C); J=U.times.C. Flux is
conserved in the microchannel under consideration. In other words,
the sum of the number of analyte molecules (enzymes, substrates and
products, or ligands and ligand partners) times the velocity of the
components is constant.
[0201] Enzyme-Substrate Assay
[0202] For example, in the following chemical system, a substrate
and an enzyme are mixed at point M, and travel along a microchannel
with length L to a detection point. The detector at the detection
point can observe product molecules formed from the substrate,
and/or substrate molecules and/or enzyme molecules as described in
FIG. 4A. The enzyme (E) and substrate (S) are mixed and react to
convert a small portion of the substrate into a product (P). In a
preferred embodiment, the product is florescent, and easily
detectable, e.g., using a photodiode, photomultiplier, a
spectrometer, or the like.
[0203] In the case in which P and S have the same mobility, or in a
stationary system, a concentration balance for the reacted and
unreacted components is described by a simple concentration
balance.
[0204] [E].times.T.sub.LS.times.k=[S].sub.converted=C.sub.P, where
[E], [S] and C.sub.P are enzyme, substrate and product
concentrations, respectively, in units of molecules per volume;
T.sub.LS is the transit time of substrate between mixing and
detection points or the reaction time, which is equivalent to the
length for reaction divided by the velocity of substrate,
L/U.sub.s. The reaction constant, k, has units of molecules of
product per molecules of enzyme per time.
[0205] Analyzing with the flux being conserved in a system where
the product velocity and substrate velocity are not necessarily
identical results in: Flux
(J)=[E].times.T.sub.LS.times.k.times.U.sub.s=[S].sub.con-
verted.times.U.sub.s=U.sub.p.times.C.sub.P, where U.sub.p is the
product velocity. Rearranging and writing transit time of substrate
as L/U, results in:
[E].times.L/U.sub.s.times.k.times.U.sub.s=U.sub.p.times.C.sub- .P.
Then: [E]/U.sub.p.times.L.times.k=C.sub.P. Substituting transit
time, T.sub.LP for product gives the non-intuitive result that
product concentration is proportional to the transit time of the
product, not substrate as might be extrapolated from the stationary
or non-mobility changing case above:
[E].times.T.sub.LP.times.k=C.sub.P.
[0206] Joined Reactants Assay
[0207] In a binding assay where the binding of two molecules in a
reaction system results in a product with a change in mobility, a
similar analysis can be undertaken. For example when streptavidin
(SA), a large molecule, binds to biotin, it changes the mobility of
the labeled biotin. In one embodiment, spacer molecules (T10) are
placed between the B and SA molecules to prevent quenching of B
when SA is bound. Thus, both B and product molecules (B-SA) are
fluorescent. The simple device depicted in FIG. 5 can be used for
mixing and detection of the substrates and products, optionally
further including a detector, computer, or the like.
[0208] With flux being conserved, the concentration of detected (in
this case fluorescent) species changes as a result of a change in
velocity. As the label does not change upon conversion of B into
B-SA, the number of labeled molecules in the system remains
constant. Where B molecules are converted to B-SA molecules, taking
the principle of the conservation of flux into account:
[B].times.U.sub.B=[B-SA].times.U.sub.B-SA
[0209] where [B] is the concentration of B-T10-Fl and U.sub.B is
the velocity of the same molecule in the system; U.sub.B is
relatively slow. [B-SA] is the concentration of the complexed
molecule and U.sub.B-SA is the velocity of the complexed molecule,
which is relatively fast. Recognition of this relationship allows
quantification of the amount of streptavidin present in the system
by detecting downstream fluorescence. The relationship between the
concentrations of B-T10-Fl bound to streptavidin (B-SA) and unbound
to streptavidin (B) is proportional to their mobilities:
[B-SA]=[B].times.U.sub.B/U.sub.B-SA.
[0210] At intermediate amounts of SA, where a portion of B is bound
to SA the concentration is proportional to the fraction (Y.sub.b)
of B that is bound to SA:
[Fl]=(1-Y.sub.b)[B]+Y.sub.B([B]U.sub.B/U.sub.B-SA.
[0211] Without the knowledge that concentration changes as velocity
changes, the assay is much more complicated. For example, one could
sample the mixture into a separation column which separated reacted
and unreacted molecules, and detected florescence. The amount of
material coming off of the column per unit time is optionally
detected as depicted in FIG. 6.
[0212] However, assuming conservation of flux, much simpler
arrangements are possible. For instance, an electrokinetic
substrate with one channel and one electrode driving fluid flow in
an electrokinetic device is optionally used to monitor formation of
reaction products.
Example 2
[0213] Non-fluorogenic Biotin-streptavidin Binding
[0214] The binding reaction of biotin and streptavidin was chosen
as a model assay to validate the concepts of mobility shift and
flux conservation as a means to detect non-fluorogenic assays in a
continuous flow mode. The labeled biotin was a 5'-biotin,
3'-fluorescein derivatized short oligonucleotide, containing 10
thymidine residues (B-T.sub.10-F). The thymidine residues act as a
spacer to prevent changes in the quantum efficiency of fluorescence
upon the binding of streptavidin to biotin. Experimentally, it was
confirmed by fluorometry (using a Perkin Elmer Luminescence
Spectrometer LS50B) that the quantum yield of B-T.sub.10-F was
indeed unaffected by the binding reaction to streptavidin.
Unlabeled biotin (Sigma B-4501, Lot 37H1389) was also used in this
study as a competitive reactant for BT.sub.10-F in the binding
reaction with streptavidin (Sigma S-4762, Lot 44H6890).
[0215] The buffers used for the reagents contain 100 mM Hepes at pH
7.0 and 1M NDSB-195 (a non-detergent sulfobetaine,
Calbiochem-Novabiochem), filtered with 0.2 .mu.m filters. To vary
the electroosmotic mobility of the buffer solution, a buffer was
prepared without added salt and one with 50 mM NaCl. A neutral dye,
Rhodamine B, was used to measure the electroosmotic mobility of the
buffers.
[0216] All on-chip experiments for this example were performed on a
Caliper technologies "7A" chip design; its channel and reagent well
layout is illustrated in FIG. 7. In this design, each reagent well
(1, 2, and 7) is paired with a buffer well (8, 3, and 6) for
on-chip dilution of reagent concentration. The microfluidic
channels, 70 .mu.m wide and 10 .mu.m deep, were etched in a soda
lime glass substrate and then sealed via thermal bonding with a top
glass plate containing eight 3-mm diameter holes serving as reagent
wells. The electrical currents and voltages of the 8 electrodes in
contact with the wells were controlled by a Caliper 3180
LabChip.TM. controller and Caliper's unified 1 software.
[0217] The fluorescence signals were measured in the
epifluorescence mode using a Nikon microscope (Nikon Eclipse TE300)
equipped with a photomultiplier tube (PTI D104 Microscope
Photometer) and a 50 W tungsten/halogen light source. A dichroic
filter, High Q FITC Filter Set (#41001, Chroma Technology Corp.),
was used for selecting the excitation and emission wavelengths for
B-T.sub.10-F. A High Q TRITC Filter Set (#41002, Chroma Technology
Corp.) was used for Rhodamine B.
[0218] The electroosmotic mobility of the buffers was measured on
the 7A chip using Rhodamine B as a neutral dye marker. The
electrophoretic mobility of B-T.sub.10-F and B-T.sub.10-F bound to
streptavidin (SA-B-T.sub.10-F) was measured directly on the 7A chip
using Hepes buffer without NaCl. For the .mu..sub.ep measurements,
the concentrations of B-T.sub.10-F and SA-B-T.sub.10-F were 3.1
.mu.m and 0.88 .mu.m, respectively. The measured electrokinetic
mobilities are tabulated in Table 1. As can be seen from these
measurements, B-T.sub.10-F has an electrophoretic mobility in the
opposite direction relative to the electroosmotic flow of the
buffers due to its negative charge at pH 7.0. After the binding
reaction, .mu..sub.ep of the product decreases in magnitude due to
a decrease in the charge-to-mass ratio. Thus, the resulting
electrokinetic mobility of B-T.sub.10-F is lower than that of
SA-B-T.sub.10-F, as in the case described in FIG. 1.
1TABLE 1 Electrokinetic Parameters of Buffers and Reagents
Buffer/Reagent .mu..sub.eo (cm.sup.2/v .multidot. s) .mu..sub.ep
(cm.sup.2/v .multidot. s) 100 mM Hepes + 1 M NDSB 5.5 .times.
10.sup.-4 -- 100 mM Hepes + 1M NDSB + 50 3.7 .times. 10.sup.-4 --
mMNACL B-T.sub.10-F -- -2.0 .times. 10.sup.-4 SA-B-T.sub.10-F --
-0.7 .times. 10.sup.-4
[0219] A series of experiments was first performed to determine the
concentration of reagents for the binding and competitive binding
assays such that the signal-to-noise ratio was high and the
fluorescence was still linear as a function of concentration. The
optical setup was also varied to ensure that the light intensity
and the iris size chosen did not cause a significant photobleaching
of the fluorescent dye. Furthermore, based on model calculations,
the buffer with salt gives better separation conditions for
distinguishing the bound and free B-T.sub.10-F within the geometric
and electrical parameters used in our experiments on the chip.
Therefore, the results reported below were performed with the Hepes
buffer containing 50 mM NaCl.
[0220] FIG. 8 illustrates the measured fluorescence signal (solid
curve) of the non-fluorogenic binding assay of B-T.sub.10-F with
streptavidin in the continuous flow mode. The concentrations of
B-T.sub.10-F and streptavidin were 3.1 .mu.m and 78 nM. Since a
streptavidin molecule has 4 biotin binding sites, the stoichiometry
of B-T.sub.10-F to streptavidin is 10:1. In this experiment, the
injection time of streptavidin was varied from 2.5 s, 5 s, 10 s,
and 15 s. The characteristic signature of a peak followed. by a
valley can be seen in all cases. For injection times of 10 and 15
s, the plateau region is also clearly exhibited. In this plot, the
time domain model calculations using the measured electrokinetic
mobilities as input parameters are depicted by the dashed curve. It
should be noted that the actual injection pulse shapes were used in
the model instead of an assumed square pulse shape. For
quantitative comparison, both the measured fluorescence and model
prediction were first normalized by the background fluorescence
level when the channel contained only B-T.sub.10-F. Furthermore,
the magnitude of the model calculations were adjusted by one
multiplicative factor to give the best fit to the measured signals
in arbitrary fluorescence units. Thus, the model has one adjustable
parameter in the y-axis and no adjustable parameter in the time
axis. As can be seen in this comparison, the agreement between the
model and experimental data is quite good.
[0221] In another experiment, the competitive binding reaction
between B-T.sub.10-F and unlabeled biotin with streptavidin was
studied. FIG. 9 shows the measured fluorescence signal (solid
curve) of the non-fluorogenic competitive binding assay results in
the continuous flow mode. The streptavidin injection time was 12 s.
The concentrations of B-T.sub.10-F and streptavidin were 3.1 .mu.m
and 78 nM. The concentration of biotin was varied at 5 levels: 0,
0.78, 1.6, 2.3, and 3.1 .mu.M. The dashed curve, denoting model
calculations based on the actual injection pulse profiles, was
again fitted to the data using one adjustable parameter in the
y-axis as in FIG. 8. As expected, the magnitudes of the peaks and
valleys decrease proportionately as biotin is titrated into the
binding assay to compete with B-T.sub.10-F. Once again, the
agreement between model calculations and measured data is good.
[0222] The data in FIG. 9 is further analyzed by plotting the
magnitude of the peak, plateau, and valley fluorescence level
versus the reciprocal of the sum of the labeled and unlabeled
biotin concentration. A linear relationship is expected for each
set of data for a competitive binding assay, which was exhibited
experimentally as shown in FIG. 10. Any one of these features can
be used as a calibration curve to determine the free biotin
concentration in an assay.
[0223] In summary, on-chip data of binding assays of biotin and
streptavidin validated the use of mobility shift to detect
non-fluorogenic assays in a continuous flow mode. The need for
product concentration correction using conservation of flux to
analyze assays performed in microchannels of a flowing system is
also definitively demonstrated by a quantitative comparison of data
to model calculations.
Example 3
[0224] Applications to Additional Non-Fluorogenic Assays
[0225] The continuous flow, non-fluorogenic assay format can be
applied directly to binding assays such as of antigen-antibody and
receptor ligand binding. It is also readily applicable to other
biochemical assays such kinase enzyme assays and hybridization of
PNA and a complimentary peptide nucleic acid (PNA). FIG. 11 shows a
plot of some data on a protein kinase A (PKA) enzyme assay
(Promega, V5340) in a continuous flow mode using a Caliper 7A chip.
In this assay, the phosphorylation of the substrate alters the
peptide's net charge from +1 to -1. Qualitatively, the data (solid
curve) shows the expected valley appearing before the peak, with a
plateau region in between. A model calculation using estimated
electrokinetic mobilities, enzyme kinetic parameters from the
literature, and estimated applied voltage values in an Excel
spatial domain model (dashed curve) predicted the qualitative
features of the fluorescent signal. The Macro program listing of
the spatial domain model for non-fluorogenic assay is included as
Appendix B.
[0226] In the binding assay of biotin and streptavidin presented
above, the labeled biotin is a small molecule (244 dalton) whereas
the unlabeled streptavidin is large (65,000 dalton). As such, the
reaction produced a labeled product with a significantly different
electrokinetic mobility compared to the labeled reactant, and this
large difference makes the detection of the binding reaction quite
straight forward. In the opposite case when the labeled reactant is
large (such as a protein receptor) and the unlabeled reactant is
small (such as a ligand), the induced mobility shift due to binding
could be very small due to a small change in the mass. In this
case, it is more difficult to detect the onset of reaction using
the continuous flow, non-fluorogenic assay format as described
here. However, methods to enhance the detection of non-fluorogenic
assays on chips for small mobility shifts are available as
described above. One approach is to inject the reaction mixture
into a planar cyclic capillary electrophoresis channel to separate
products from reactants. In this case, the separation time can be
made very long by continuously cycling the voltage around the
cyclic structure. Another method is to use the concept of
interference of concentration waves in channels to enhance to the
magnitude of peaks and valleys in the non-fluorogenic assay
fluorescence signal.
Example 4
[0227] High Throughput Systems
[0228] The present invention relates to the performance of assays,
and particularly, high-throughput assays, within microfluidic
devices. The performance of high-throughput assays within
microfluidic devices has been described in great detail in commonly
owned published International Application No. WO 98/00231, as well
as supra. Apparatus and methods for introducing large numbers of
different compounds into the microfluidic devices are described in
commonly owned published International Application No. WO 98/00705,
which is also incorporated herein by reference in its entirety for
all purposes.
[0229] In many cases, the biochemical system that is being assayed
can be selected or engineered to have an easily detectable result.
For example, assaying enzyme function is typically made simple by
utilizing fluorogenic substrates for the enzyme, e.g.,
non-fluorescent substrates which yield fluorescent products. Such
assays are readily incorporated into microfluidic devices for
performance of assays to identify compounds that may effect normal
enzyme activity. In one embodiment, using a 7A chip as described
supra (see, e.g., FIG. 7), one continuously flows enzyme and
fluorogenic substrate through a channel of the device. This
continuous flow of enzyme and substrate produces a steady state
fluorescent signal from the fluorescent product. Enzyme inhibitor
(or, e.g., compounds' for which one wishes to test inhibitory
activity) are periodically introduced into the main channel. These
inhibitors then reduce the amount of product produced within the
main channel resulting in a deviation from the steady state signal.
See also, Examples of specific assays and their results are shown
in the figures attached herewith. Specifically, both phosphatase
assays and protease assays were performed using a 7A chip.
[0230] The phosphatase assay utilizes a fluorogenic substrate dFMU,
which produces a fluorescent signal upon dephosphorylation. The
reaction is shown schematically in FIG. 12. FIG. 13 shows typical
data obtained from the on-chip phosphatase assay. In this
experiment, the running buffer was 1 M NDSB-195 in 25 mM HEPES, pH
7.9. Reagent concentrations were 125 nM LAR, 50 .mu.M dFMUP and 200
.mu.M peptide inhibitor in wells 6, 8 and 2 respectively. Each
reagent well was paired with a well containing running buffer. The
system was programmed to repeatedly run a sixteen-step loop of
experiments. The sixteen steps were a series of controls followed
by the enzyme plus substrate experiment. Each step of the loop
conserved the total current flux in the main reaction channel. The
total flux remained constant during each step of the loop by
maintaining a constant sum of currents from the wells. The
proportion of that overall flux from each reagent and buffer well
was selected to provide the desired final reagent concentration in
the main reaction channel. The fluorescence response was monitored
in each of the sixteen experimental steps where the continuous flow
stream alternated between buffer, substrate, buffer, substrate plus
enzyme at four different substrate concentrations. An example of a
controller program is shown below, Table 2.
2TABLE 2 Controller Software Program Channel 1 2 3 4 5 6 7 8 time
State: .mu.A .mu.A .mu.A V .mu.A .mu.A .mu.A .mu.A sec 1 5 0 5 1000
0 0 5 0 15 Buffer 2 0 0 5 1000 0 0 5 5 15 Substrate 3 5 0 5 1000 0
0 5 0 15 Buffer 4 0 0 5 1000 0 5 0 5 15 Substrate + Enzyme
[0231] The substrate concentration was varied for each sequence of
three controls followed by the enzyme reaction. The concentrations
of the reagents in the main channel can be calculated from the
ratio of currents used to pump the reagents. The concentrations in
the reaction channel are simply the concentration in the well
multiplied by the ratio of current applied at that well, divided by
the total current. Here the reaction mixture was 62.5 .mu.M LAR,
and either 5, 12.5, 17 or 25 .mu.M dFMUP. The raw fluorescence data
is plotted as a function of time. The purpose of this experiment
was to demonstrate the increase in enzymatic signal as a function
of increasing substrate concentration in a controlled system. Rise
times for the enzyme/substrate signal are less than 5 seconds. The
background signal remained low over the course of many experimental
cycles.
[0232] The raw data for the K.sub.m, V.sub.max, k.sub.cat, and
K.sub.i determinations are plotted in FIG. 14. Each trace
represents a set of experiments performed in seven step cycles. The
enzyme solution was pumped continuously, providing a final
concentration of 83 nM LAR in 1 M NDSB-195, 50 mM HEPES, pH 7.5 in
the reaction channel, while the signal at various substrate
concentrations was recorded. The first step of the cycle is an
enzyme only control. Steps two through six contain different levels
of substrate up to an including 17 .mu.M dFMUP. The final step of
the cycle is a substrate only control, 17 .mu.M dFMUP with no
enzyme. The entire experiment, (no peptide inhibitor), was repeated
at two inhibitor concentrations, 35 uM and 69 uM peptide.
[0233] The blank subtracted signals were averaged for triplicate
measurements and transformed into the reciprocal form of the
Lineweaver-Burke equation:
1/v=K.sub.m/V.sub.max.times.1/[S]+1/V.sub.max, where v is the
reaction rate in RFU/s, K.sub.m is the Michaelis Menton constant
for LAR and dFMUP, V.sub.max is the rate of maximum enzyme
turnover, and S is the dFMUP concentration. The double reciprocal
plot for the range of substrate concentrations, 0-20 .mu.M dFMUP,
in the absence of inhibitor, gives K.sub.m and V.sub.max. The rates
were evaluated as a change in fluorescent product signal over a
fixed time. The change in fluorescence is the difference in signal
for a given substrate and enzyme concentration minus the substrate
only control. The fixed time is the time it takes for the product,
dFMU, to travel from the point of mixing of substrate and enzyme to
the detector. The time for the product to flow was measured
directly. dFMU was placed in well 6, the well in which enzyme
typically resides; the time for the product to flow to the detector
poised 8 mm from the source of dFMU in the reaction channel was
monitored. The slope of a calibration curve of the signal generated
as a function of dFMU concentration was used to convert the
fluorescent signals to dFMU concentrations such that the rates
could be expressed as a change in product concentration per unit
time.
[0234] A least squares fit of the three straight lines: no
inhibitor, 35 .mu.M and 69 .mu.M peptide, was performed with the
constraint that they meet at a common intercept on the y axis,
1/V.sub.max FIG. 15. This fit produced a V.sub.max of 6.71 .mu.M
dFMU/s. k.sub.cat could then be calculated from the ratio of
V.sub.max to the enzyme concentration. The k.sub.cat is 4.74
.mu.mol/min nmol LAR. The parallel analysis performed on the
spectrophotometer in the same running buffer yielded a k.sub.cat of
6 .mu.mol/min nmol LAR. The K.sub.i for 35 and 69 .mu.M peptide
were 155 and 147 .mu.M, respectively. The same analysis performed
in a cuvette experiment with 1 mg/ml BSA in the running buffer gave
167 .mu.M peptide. The data are summarized in Table 3.
3TABLE 3 Summary of LAR/dFMUP Kinetic Constants Km Ki kcat
LAR/dFMUP Peptide .mu.mol/min .mu.M .mu.M mmolLAR Conditions
Cuvette 23.2 167 6 1 M NDSB-195/50 mM Hepes, pH 7.5, 0.1 mg/ ml BSA
Chip 18.7 151 4.74 1 M NDSB-195/50 mM Hepes, pH 7.5
[0235] In addition to the above kinetic studies, rate as a function
of substrate concentration data was collected on three separate
chips in order to consider interchip reproducibility for K.sub.m
analyses. The combined data were used to prepare a double
reciprocal plot. The ratio of the slope to the intercept of the
best fit line for these points, (R.sup.2=0.999), produced a K.sub.m
of 18.2 .mu.M. The average of the three on-chip K.sub.m values is
18.7 .mu.M.+-.4.44 (23.8%), n=3. This is in excellent agreement
with cuvette experiments performed on the spectrophotometer where
dFMU was detected at 360 nm in a temperature controlled cuvette at
25_C. The cuvette experiments gave an average K.sub.m of 23.25
.mu.M.+-.5.25 (22.6%), for four separate K.sub.m
determinations.
[0236] A continuous flow experiment was performed to assess the
chip lifetime for the enzyme inhibitor assay. In this experiment 42
nM LAR was continuously pumped through the reaction channel.
Alternately, 6.25 nM dFMUP or 6.25 nM dFMUP and 41.6 .mu.M peptide
inhibitor were pumped into the flow stream. The reagents were
loaded into reagent wells on the chip, the controller was initiated
and the script was allowed to run for eight hours. The raw data for
the third hour of the experiment is shown in FIG. 16. The entire
experiment is summarized by FIG. 17. Note that although both the
uninhibited and the inhibited signals drift with time, the percent
inhibition remained constant for the entire experiment. The average
percent inhibition is 32.45.+-.1.73 (5.3%). From the flow rate and
the cross sectional area of the capillary it is estimated that
approximately 18 .mu.l total reagent volume was consumed during the
eight hours.
[0237] HCV protease was used in a similar fluorogenic assay to LAR
phosphatase; however, the peptide substrate incorporates a
fluorescence resonance energy transfer (FRET) label (FIG. 18). In
order to verify that the depsipeptide/protease reaction was well
behaved and the reaction parameters are in the range we expect, a
continuous flow enzyme experiment with substrate titration was
performed. FIG. 19 shows the fluorescence generated in a constant
flow stream of 2.14 .mu.M protease when various levels of
depsipeptide are introduced, 0 to 250 .mu.M depsipeptide. The
product fluorescence is proportional to the amount of cleaved
substrate. The height of the product signal is proportional to the
rate of enzyme turnover for that substrate concentration. The rate
of fluorescence generation can be assessed as the fluorescence
signal per mixing time of substrate and enzyme in the reaction
channel. That mixing time is determined by the mean residence time
of the fluorescent product in the reaction channel as it is
electrokinetically pumped from the source of the mixing to the
detector. K.sub.m was determined from the Michaelis Menton
equation.
[0238] Due to the chemistry of this FRET quenching reaction,
several considerations for accurate measurement of K.sub.m on the
Labchip.TM. exist. (1.) There is not an accurate calibration curve
for the EDANS labeled product. (2) Accurate determination of the
substrate concentration in the Labchip.TM. reagent well by a simple
spectrophotometric measurement is not performed. (3) A gross
approximation about the fluorescent efficiency of the EDANS-labeled
peptide product was made relative to EDANS.
[0239] Despite these considerations, the data from FIG. 19 was
converted to rate information and plotted as a function of the
estimated depsipeptide concentration. The rate values were well
behaved and the corresponding double reciprocal plot is shown in
FIG. 20. In a Lineweaver-Burke plot, the slope of the line is
K.sub.m/V.sub.max, the y-intercept is 1/V.sub.max and the
extrapolated -x intercept is -1/K.sub.m. Values for K.sub.m and
k.sub.cat derived from a least squares regression analysis of the
points shown in FIG. 20 are summarized in Table 4 along with
constants obtained using conventional analysis.
4TABLE 4 Michaelis-Menten Constants Measured on a Labchip .TM. and
in cuvette for HCV protease and LAR Phosphatase K.sub.m V.sub.max
k.sub.cat mM mM/s min.sup.-1 HCV Protease/Depsipeptide Kinetics
Chip 0.11 0.086 1.8 25 mM TRIS/HCL, pH 8.5, 0.1% Triton X-100, 10
mM DTT, 1 M NDSB-195 Cuvette 46 50 mM TRIS/HCL, pH 7.5, 1.0% Triton
X-100, 10 mM DTT, 1 *mm EDTA, 10 mM NaCl LAR/dFMUP Kinetics chip
0.020-0.40 0.011 3000-5000 50 mM HEPES, pH 7.5, 10 mM DTT, 0.5 M
NDSB-195
[0240] Note the buffer conditions for the Labchip.TM. analysis and
the traditional analysis is different. Specifically, pH, surfactant
concentration, and the presence of NDSB are known to influence the
enzyme kinetics. Despite this, the agreement between the cuvette
values and the Labchip.TM. kinetic constants is reasonable.
Moreover, a comparison of the protease k.sub.cat with the
phosphatase kinetic constants reveals the broad range of reaction
rates we can expect to accommodate on the Lab-chip. It is possible
to study the reaction kinetics of enzymes with three orders of
magnitude difference in turnover rate on the same Labchip.TM..
[0241] A continuous flow experiment was performed to assess the
Labchip.TM. lifetime for the enzyme assay for applications to high
throughput screening. Since the sensitivity of the in vitro enzyme
assay is depends on the enzyme concentration employed, in this
experiment 0.63 .mu.M HCV protease was continuously pumped through
the reaction channel. Alternately, buffer for 40 seconds or 220
.mu.M depsipeptide for 20 seconds was pumped into the flow stream
such that the cycle time for each experiment was one minute. Every
other substrate injection also contained 267 mM inhibitor. The
reagents were loaded into reagent wells on the chip, the controller
was initiated and the script was allowed to run uninterrupted for
more than 12 hours. The raw data for the third hour of the
experiment is shown in FIG. 21. As expected the inhibited response
can be distinguished from the uninhibited substrate generated
signal, and the peaks are separated by well behaved enzyme only
blanks.
[0242] The first 1000 seconds for each hour of the first nine hours
of data is shown in FIG. 22. The background signal is very stable
for this period of time. Note however that this well controlled
background fluorescence is not the substrate only background. The
extent of substrate hydrolysis over time could not be measured in
the continuous flow analysis where enzyme was pumped throughout the
course of the experiments. After nine hours the background
increases and the assay no longer behaves reproducibly. The
inhibition reaction is clearly seen for hours one through nine
after which time the attenuation of the fluorescence response is
not as great. Also the reproducible peak shapes for hours one
through nine start to change after nine hours. The gradual delay in
on time of inhibited and uninhibited peaks for each period of data
is likely due to deterioration in EO flow. Enzyme adsorbing to the
surface of the capillary can retard the electroosmotic flow,
thereby increasing the incubation time of substrate and enzyme.
This produces both the larger signals and longer mean response
times observed here.
[0243] The signals and percent inhibition for hours one through
nine are summarized in FIG. 23. The chip was operational in that
fluid was flowing for more than 12 hours of continuous
electrokinetic pumping; however, the inhibition response was
reproducible for seven hours. The total reagent volume consumed in
the experiment can be calculated from the cross sectional area of
the capillary and the total current. For a 70 mm.times.20 mm
channel and I.sub.total equal to 1.5 mA, the reagent volume
consumed is 2.8 ml/hour or 33 ml in 12 hours. No effort was made to
maximize the number of experiments in this time. Despite this fact,
assuming each measurement is an individual experiment, a total of
1680 experiments were performed in seven hours. The average percent
inhibition response was calculated for the first three inhibited
and uninhibited signals at the start of each hour. The percent
inhibition was 24.+-.2% for the first seven hours of data.
[0244] An example of a non-fluorogenic enzyme assay is depicted in
FIG. 24. Here a protein kinase reaction is represented in which
substrate is converted to product with differing mobility. Both
substrate and product are fluorescently labeled and we rely on the
separation of substrate and product following conversion to monitor
the extent of reaction in a chip designed for mixing and incubation
followed by separation, e.g. FIG. 25.
[0245] Similar to the phosphatase and protease, the kinase
reactivity can by monitored in the microchip for kinetic analyses
and applications to high throughput screening. FIG. 26 show the
separated peaks due to substrate, dye marker, and product as a
function of substrate concentration. The separation occurs
following incubation of substrate and enzyme via a gated injection
where the flux of substrate and product entering the separation
channel is expected to accurately reflect the homogeneous reaction
kinetics. The reaction conditions were 138 nM PKA in 100 mM Hepes,
pH 7.5, 10 mM DTT, 5 mM MgCl2, 1M NDSB-195. The double reciprocal
transformation is represented in a Lineweaver Burke plot, FIG. 26
and a Km of 12 uM is derived.
[0246] Non-fluorogenic assays can be designed in various other
modes of operation. Among the strategies available are assays that
modulate the enzyme concentration in a reaction channel containing
a constant stream of fluorescent substrate. FIG. 27 contains the
trace resulting from a constant stream of rhodamine-labeled-peptide
injected with PKA for 40, 30, 30 and 10 second periods. Because the
product mobility is faster than the substrate mobility under this
particular set of conditions, the trace shows a decrease in
substrate concentration due to enzymatic consumption, followed by
an increase in signal of concomitant area due to an accelerated
rate of product generation. Displace substrate is turned over to
product and appears as a peak in the fluorescent trace.
[0247] In a similar way utilizing a fluorogenic reaction, here the
protease reaction, constant fluorogenesis can be interrogated with
pulses of inhibitor. An example is the protease and peptide
substrate reaction. This is particularly relevant to high
throughput screening systems in which continuously flowing enzyme
and substrate are electrokinetically pumped through the reaction
channel of the sipper chip and plugs of potential inhibitory
compounds are injected. A decrease in the fluorescence signal
should indicate inhibition for the compounds of interest. In an
effort to simulate the high through put experiment on a planar
chip, a constant fluorescence experiment was conducted. The
reaction channel was continuously flowing 1.8 .mu.M HCV Protease
and 94 .mu.M depsipeptide. Upon observation of the steady state
fluorescence, inhibitor was injected into the flow stream at 75
.mu.M and 37.5 .mu.M for 20 s. The total cycle time for injections
of two concentrations of inhibitor was 240 s. FIG. 28 shows the
fluorescence trace for about 25 minutes.
[0248] Superimposed on the constant fluorescence signal is the
inhibitor signature at two inhibitor concentrations. The higher
inhibitor concentration gives rise to the larger dip followed by a
peak. The lower inhibitor concentration yields a smaller dip
followed by a comparable size peak. The dip and peak pairs are of
similar area. We can rationalize these fluorescence responses.
[0249] The depsipeptide has six minus charges while the EDANS
labeled product contains only two. Therefore we expected, based
simply on the difference in charge, that the substrate should move
more slowly in the flow stream than the product. During the time
the enzyme "sees" inhibitor in the flow stream, the amount of
fluorogenic substrate consumed is less than that during the
uninhibited trace. If the slow moving substrate lags behind the
inhibited response, an increase in the effective substrate
concentration down stream from the inhibition will occur in the
reaction channel. That higher substrate concentration can in turn
generate a higher product concentration such that superimposed on
the steady state fluorescence signal is a product peak. The similar
area of dip and peak for each inhibitor concentration supports this
rationale. The inhibitor concentration dependence of the signatures
also supports this thinking. In light of the constant fluorescence
in the absence of inhibitor it is likely that a similar experiment
may be performed with shortened inhibitor injection times.
[0250] Modifications can be made to the method and apparatus as
hereinbefore described without departing from the spirit or scope
of the invention as claimed, and the invention can be put to a
number of different uses, including:
[0251] The use of an integrated microfluidic system to test the
effect of each of a plurality of reaction, assay or components test
compounds in a biochemical or non-biochemical system, the system
including data correction elements as described herein.
[0252] The use of a microfluidic system as hereinbefore described,
wherein said biochemical system flows through one of said channels
substantially continuously, enabling sequential testing of said
plurality of test compounds, wherein the system includes provisions
for data correction as described.
[0253] The use of a microfluidic system as hereinbefore described,
wherein the provision of a plurality of reaction channels in said
first substrate enables parallel exposure of a plurality of test
compounds to at least one biochemical system, wherein the system
includes provisions for data correction as described.
[0254] The use of a substrate carrying intersecting channels in
screening test materials for effect on a biochemical system by
flowing said test materials and biochemical system together using
said channels wherein an apparatus utilizing the substrate includes
provisions for data correction as described.
[0255] The use of a microfluidic substrate as hereinbefore
described, wherein at least one of said channels has at least one
cross-sectional dimension of range 0.1 to 500 .mu.m.
[0256] The use of a system as described herein for nucleic acid
sequencing, wherein the effects of the velocity of labeled
components of a nucleic acid sequencing reaction are corrected
for.
[0257] An assay, kit or system utilizing a use of any one of the
microfluidic components, methods or substrates hereinbefore
described. Kits will optionally additionally comprise instructions
for performing assays or using the devices herein, packaging
materials, one or more containers which contain assay, device or
system components, or the like.
[0258] In an additional aspect, the present invention provides kits
embodying the methods and apparatus herein. Kits of the invention
optionally comprise one or more of the following: (1) an apparatus
or apparatus component as described herein; (2) instructions for
practicing the methods described herein, and/or for operating the
apparatus or apparatus components herein, e.g., for correcting
observed concentration for effects of velocity; (3) one or more
assay component; (4) a container for holding apparatus or assay
components, and, (5) packaging materials.
[0259] In a further aspect, the present invention provides for the
use of any apparatus, apparatus component or kit herein, for the
practice of any method or assay herein, and/or for the use of any
apparatus or kit to practice any assay or method herein.
[0260] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference for all purposes, as if each
reference were specifically indicated to be incorporated by
reference.
* * * * *