U.S. patent application number 10/179048 was filed with the patent office on 2003-03-20 for inverse chromatography methods and apparatus having a reference configuration for evaluation of interactions between modifying agents and receptors.
Invention is credited to Nielsen, Ralph B., Petro, Miroslav, Regan, Jacqueline M..
Application Number | 20030054568 10/179048 |
Document ID | / |
Family ID | 23159740 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030054568 |
Kind Code |
A1 |
Petro, Miroslav ; et
al. |
March 20, 2003 |
Inverse chromatography methods and apparatus having a reference
configuration for evaluation of interactions between modifying
agents and receptors
Abstract
Methods and apparatus for the evaluation of interactions between
substances using inverse chromatography are disclosed. Preferably,
interactions are evaluated between a liquid test sample and a solid
phase comprising a receptor in the presence of a liquid carrier.
Preferably, one of the modifying agent or receptor are members of a
combinatorial library.
Inventors: |
Petro, Miroslav; (San Jose,
CA) ; Nielsen, Ralph B.; (San Jose, CA) ;
Regan, Jacqueline M.; (Santa Clara, CA) |
Correspondence
Address: |
SYMYX TECHNOLOGIES INC
LEGAL DEPARTMENT
3100 CENTRAL EXPRESS
SANTA CLARA
CA
95051
|
Family ID: |
23159740 |
Appl. No.: |
10/179048 |
Filed: |
June 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60300589 |
Jun 22, 2001 |
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Current U.S.
Class: |
436/514 ;
435/7.1 |
Current CPC
Class: |
C40B 30/04 20130101;
B01J 2219/00317 20130101; B01J 2219/00274 20130101; B01J 2219/00747
20130101; G01N 30/89 20130101; B01J 2219/00707 20130101; B01J
2219/00752 20130101; G01N 30/89 20130101; G01N 30/88 20130101; G01N
33/558 20130101; B01J 2219/00315 20130101; B01J 2219/00722
20130101; B01J 2219/00725 20130101; B01J 2219/00736 20130101; B01J
2219/00585 20130101; G01N 30/466 20130101; B01J 2219/00731
20130101; B01J 2219/00743 20130101; B01J 2219/00599 20130101; G01N
30/466 20130101; G01N 33/5085 20130101 |
Class at
Publication: |
436/514 ;
435/7.1 |
International
Class: |
G01N 033/53; G01N
033/558 |
Claims
We claim:
1. A method of inverse chromatography for evaluating the
interaction between a receptor and a modifying agent in the
presence of a liquid carrier, the method comprising a test
procedure comprising (a) injecting a liquid test sample into a
stream of the liquid carrier, the test sample comprising the
modifying agent, and the injection being carried out over a limited
time so that a distinct test section of the stream contains the
test sample, (b) passing the stream of liquid carrier containing
the test sample over a solid phase in a test chamber, the solid
phase comprising the receptor, (c) passing the stream of liquid
carrier leaving the test chamber through a detector to ascertain a
property of the modifying agent which remains in a distinct
evaluation section of the stream corresponding to the test section,
(d) injecting a liquid reference sample into a stream of the liquid
carrier, the composition of the second sample being substantially
identical to the composition of the test sample, and the injection
being carried out over a limited time so that only a distinct
reference section of the stream contains the reference sample, (e)
passing the stream of liquid carrier containing the reference
sample through a reference chamber which is free of any substance
which interacts with the modifying agent; (f) examining the stream
of liquid carrier leaving the reference chamber to ascertain a
property of the modifying agent remaining in the stream, and (g)
comparing the results obtained in steps (c) and (f) to evaluate the
interaction between the receptor and the modifying agent.
2. A method of inverse chromatography for evaluating the
interaction between a receptor and a modifying agent in the
presence of a liquid carrier, the method comprising providing the
receptor and the modifying agent, one of the receptor and modifying
agent being a member of a combinatorial library having at least
four members, and conducting at least four test procedures with the
receptor, modifying agent and liquid carrier being the same in each
of the test procedures except that in each test procedure a
different member of the combinatorial library is used, each of the
test procedures comprising (a) injecting a liquid test sample
comprising the modifying agent into a stream of the liquid carrier,
the injection being carried out over a limited time so that a
distinct test section of the stream contains the test sample, (b)
passing the stream of liquid carrier containing the test sample
over a solid phase in a test chamber, the solid phase comprising
the receptor, and (c) examining the stream of liquid carrier
leaving the test chamber to ascertain a property of the modifying
agent which remains in a distinct evaluation section of the stream
corresponding to the test section, (d) injecting a liquid reference
sample into a stream of the liquid carrier, the composition of the
second sample being substantially identical to the composition of
the test sample, and the injection being carried out over a limited
time so that only a distinct reference section of the stream
contains the reference sample, (e) passing the stream of liquid
carrier containing the reference sample through a reference chamber
which is free of any substance which interacts with the modifying
agent, (f) examining the stream of liquid carrier leaving the
reference chamber to ascertain a property of the modifying agent
remaining in the stream, (g) comparing the results obtained in
steps (c) and (f) to evaluate the interaction between the receptor
and the modifying agent.
3. A method of inverse chromatography for evaluating the
interaction between a receptor and a modifying agent comprising a
non-biological polymer in the presence of a liquid carrier, the
method comprising providing the receptor and the modifying agent
comprising a non-biological polymer, the modifying agent being a
member of a combinatorial library having at least four members, and
conducting at least four test procedures with the receptor and
liquid carrier being the same in each of the test procedures, and
with the a different member of the combinatorial library being used
in each of the at least four test procedures, each of the test
procedures comprising (a) injecting a liquid test sample comprising
the modifying agent into a stream of the liquid carrier, the
injection being carried out over a limited time so that a distinct
test section of the stream contains the test sample, (b) passing
the stream of liquid carrier containing the test sample over a
solid phase in a test chamber, the solid phase comprising the
receptor, and (c) examining the stream of liquid carrier leaving
the test chamber to ascertain a property of the modifying agent
which remains in a distinct evaluation section of the stream
corresponding to the test section, (d) injecting a liquid reference
sample into a stream of the liquid carrier, the composition of the
second sample being substantially identical to the composition of
the test sample, and the injection being carried out over a limited
time so that only a distinct reference section of the stream
contains the reference sample, (e) passing the stream of liquid
carrier containing the reference sample through a reference chamber
which is free of any substance which interacts with the modifying
agent, (f) examining the stream of liquid carrier leaving the
reference chamber to ascertain a property of the modifying agent
remaining in the stream, and (g) comparing the results obtained in
steps (c) and (f) to evaluate the interaction between the receptor
and the modifying agent.
4. The method according to claims 1, 2 or 3 wherein each of steps
(c) and (f) comprises passing the stream of liquid carrier through
a detector which measures a property which depends on the
concentration of the modifying agent, the measurement being carried
out without removing anything from the stream and at intervals
which make it possible to ascertain the amount of the modifying
agent which remains in the stream.
5. The method according to claims 1, 2 or 3 wherein, in each of the
test procedures, the test sample comprises an initial test quantity
of the modifying agent and step (c) consists essentially of
ascertaining the proportion of the initial test quantity which
remains in the evaluation section; and the reference sample
contains an initial reference quantity of the modifying agent and
step (f) consists essentially of ascertaining the proportion of the
initial reference quantity which remains in the reference
section.
6. The method according to claims 1, 2 or 3 wherein, in each of the
test procedures, the first liquid stream containing the test
section and the second liquid stream containing the reference
section are obtained by injecting into the stream of liquid carrier
a liquid unit whose composition is the same as the composition of
the test and reference samples and whose size is equal to the sum
of the sizes of the test and reference samples, the injection being
carried out over a limited time so that only a distinct section of
the liquid stream contains the liquid unit, and splitting the
liquid stream containing the liquid unit into a first sub-stream
which passes through the test chamber and includes the test section
and a second sub-stream which passes through the reference chamber
and includes the reference section.
7. The method according to claim 6 wherein the liquid stream
containing the liquid unit is split into the first and second
sub-streams by a stream-splitting junction.
8. The method according to claim 7 further comprising recombining
the first and second sub-streams after the test sample and
reference sample leave the test chamber and the reference chamber,
respectively, and passing the recombined first and second
sub-streams through a common detector for ascertaining the property
of the modifying agent remaining in the evaluation section and the
property of the modifying agent remaining in the reference
section.
9. The method according to claim 6 wherein the liquid stream
containing the liquid unit is split into the first and second
sub-streams by a switching valve.
10. The method according to claim 9 further comprising recombining
the first and second sub-streams after the test sample and
reference sample leave the test chamber and the reference chamber,
respectively, and passing the recombined first and second
sub-streams through a common detector for ascertaining the property
of the modifying agent remaining in the evaluation section and the
property of the modifying agent remaining in the reference
section.
11. The method according to claims 1, 2 or 3 wherein in each of the
test procedures comprising steps (d) to (g), the liquid stream
containing the test section and the liquid stream containing the
reference section are obtained by injecting the liquid test sample
into a first stream of the liquid carrier, the first stream being
contained in a first line in fluid communication with an inlet of
the test chamber so that the first stream containing the test
section passes through the test chamber, injecting the reference
sample into a second stream of the liquid carrier, the second
stream being contained in a second line in fluid communication with
an inlet of the reference chamber so that the second stream
containing the reference section passes through the reference
chamber.
12. The method according to claim 11 wherein an outlet of the test
chamber is in fluid communication with a first detector, so that
the first stream containing the evaluation section passes through
the first detector for ascertaining the property of the modifying
agent remaining in the evaluation section, and an outlet of the
reference chamber is in fluid communication with a second detector,
so that the second stream containing the reference section passes
through the second detector for ascertaining the property of the
modifying agent remaining in the reference section.
13. The method according to claim 2 wherein the library is a
library of receptors.
14. The method according to claim 2 wherein the library is a
library of receptors, each of the members of the library comprising
a different material.
15. The method according to claim 2 wherein the library is a
library of receptors, each of the members of the library comprising
a same common material that has been pretreated with a different
non-biological material.
16. The method according to claim 2 wherein the library is a
library of modifying agents.
17. The method according to claim 2 wherein the library is a
library of modifying agents, each of the at least four members of
the library comprising a non-biological polymer.
18. A method according to claim 2 wherein the receptor comprises a
fabric, the modifying agents are non-biological organic polymers,
and the liquid carrier contains a detergent.
19. The method according to claims 1, 2 or 3 wherein the liquid
carrier is water or an aqueous solution.
20. The method according to claims 1, 2 or 3 wherein the liquid
carrier is a non-aqueous polar solvent.
21. The method according to claims 1, 2 or 3 wherein the liquid
carrier is a non-polar solvent.
22. The method according to claims 2 or 3 further comprising (d)
using the results from the steps (c) of the test procedures to
select at least one member of the library for further testing; and
(e) subjecting the member or members of the library selected in
step (d) to further testing.
23. The method according to claims 2 or 3 wherein the at least four
test procedures are conducted in parallel.
24. The method according to claims 1, 2 or 3 wherein in each of the
test procedures, the efficiency of the test chamber is less than 50
theoretical plates.
25. The method according to claims 1, 2 or 3 wherein in each of the
test procedures, the efficiency of the test chamber is less than 10
theoretical plates.
26. The method according to claims 1, 2 or 3 wherein in each of the
test procedures, the test sample comprises an initial quantity of
the modifying agent, and at least 10% of the initial quantity of
the modifying agent is irreversibly retained on the solid phase in
the test chamber under test conditions of the procedures.
27. The method according to claims 1, 2 or 3 wherein in each of the
test procedures, the test sample comprises an initial quantity of
the modifying agent, and 30% to 50% of the initial quantity of the
modifying agent is irreversibly retained on the solid phase in the
test chamber under test conditions of the procedures.
28. The method according to claims 2 or 3 wherein in each of the
test procedures, the test sample comprises an initial quantity of
the modifying agent, a percentage of the initial quantity of the
modifying agent is irreversibly retained on the solid phase in the
test chamber under test conditions of the procedures, and the
difference between the lowest and highest percentages of the
initial quantity of modifying agent retained in the test chamber is
at least 10% as compared between the at least four test
procedures.
29. The method according to claims 1, 2 or 3 wherein in each of the
test procedures, the receptor is not saturated by the modifying
agent.
30. The method according to claims 1, 2 or 3 wherein in each of the
test procedures, a plot of time against concentration of the
modifying agent in the stream of liquid carrier leaving the test
chamber has only a single peak, with the slope of the plot being
positive at all points on one side of the peak and negative at all
points after the peak.
31. The method according to claims 1, 2 or 3 wherein the receptor
comprises at least 50% by weight of particles having an aspect
ratio of at least 2 and at least one dimension which is greater
than 100 micron.
32. Apparatus for inverse chromatography for evaluating the
interaction between a receptor and a modifying agent, the apparatus
comprising a test chamber having an inlet and an outlet and
containing a solid phase comprising the receptor, a reference
chamber having an inlet and an outlet and being free of any
substance which interacts with the modifying agent, a reservoir for
containing a liquid carrier, a main passageway providing fluid
communication from the reservoir to a splitter, the splitter being
a stream-splitting junction or a switching valve, the junction or
valve being in fluid communication with a first branch passageway
and a third branch passageway, a pump for extracting a stream of
the liquid carrier from the reservoir and passing it through the
main passageway, a first branch passageway connected to the main
passageway at the junction or valve and providing fluid
communication from the main passageway to the inlet of the test
chamber, such that a first substream of the stream of the liquid
carrier is provided to the test chamber, a second branch passageway
providing fluid communication from the outlet of the test chamber
to a common exit passageway, a third branch passageway connected to
the main passageway at the junction or valve and providing fluid
communication from the main passageway to the inlet of the
reference chamber, such that a second substream of the stream of
the liquid carrier is provided to the reference chamber, a fourth
branch passageway providing fluid communication from the outlet of
the reference chamber to the common exit passageway, the common
exit passageway adapted to combine the second and fourth branch
passageways, such that the first and second substreams of the
liquid carrier are recombined to form a common exit carrier stream,
an injector adapted for injecting a liquid unit containing an
initial quantity of the modifying agent into the stream of liquid
carrier, before the stream passes from the main passageway to the
first and third branch passageways, such that the liquid unit is
injected over a limited time so that only a distinct section of the
liquid carrier stream contains the unit, and such that the liquid
unit is divided at the junction or valve into the first and second
substreams with the first substream containing a first sample of
the liquid unit and passing through the test chamber and a second
substream containing a second sample of the liquid unit and passing
through the reference chamber; and a detector in fluid
communication with the common exit passageway for examining the
common exit carrier stream passing out of the exit passageway and
for ascertaining the proportion of the modifying agent remaining
therein.
33. The apparatus according to claim 32 wherein the splitter is a
stream-splitting junction.
34. The apparatus according to claim 32 wherein the splitter is a
switching valve.
35. The apparatus according to claim 32 wherein a first loop is
defined by the first branch passageway, the test chamber and the
second branch passageway, a second loop is defined by the third
branch passageway, the reference chamber and the fourth branch
passageway, each of the first and second loops having a void
volume, the void volume of the first loop and the second loop being
sufficiently different that, when the first and second sub-streams
are recombined, the detector can ascertain the amounts of the
modifying agent remaining in the evaluation section and the
reference section of the recombined stream, which correspond to the
evaluation section and reference section of the first and second
sub-streams, respectively.
Description
[0001] This application is related to, and claims priority under 35
U.S.C. Sec. 119(e) to co-owned, co-pending U.S. provisional
application Ser. No. 60/300,589 entitled "Evaluation of
Interactions Between Substances" filed Jun. 22, 2001 by Petro et
al.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to the evaluation of interactions
between substances using inverse chromatography methods and
apparatus. The term "substance" is used herein, i.e. throughout
this specification, to mean a single element or compound, or a
mixture of elements and/or compounds. The invention is particularly
(but not exclusively) useful in the field of combinatorial
science.
[0004] 2. Introduction to the Invention
[0005] In the field of combinatorial science, there is a continuing
need for improved methods for characterizing the members of
libraries of substances.
[0006] The techniques of chromatography are well-known. In one
widely used technique, often referred to as liquid solid
chromatography, a liquid carrier (the "mobile phase") is passed
over a solid (the "stationary phase") in a column. A sample is
injected into the mobile phase before the mobile phase enters the
column. The sample comprises at least one substance which interacts
with the solid. The rate at which the substance migrates through
the column depends on its relative affinity for the mobile and
stationary phases. The technique can be used to separate two
substances with different relative affinities for the phases, to
characterize an unknown sample (using one or more known solids), or
(in the process sometimes referred to as inverse chromatography) to
characterize a known solid (using one or more known samples).
Reference may made for example to U.S. Pat. No. 4,869,093
(Gilbert); J. Liquid Chromatography, 3 (3), (1983), 403-417, Schram
et al.; Org. Geochem. (1989), 14 (2), 157-164, Antworth et al;
Chromatographia 36 (1993), 259-262, Jerebak et al.; Sekitan Kagaku
Kaigi Happyo Ronbunshu (1993), 30th, 122-125 (Morino et al.); Conf.
Proc. Int. Conf. Coal Sci, 7th (1993, 1, 419 -422, Takanohashi et
al.; Energy Fuels (1996), 10 (4), 1012-1016, Morino et al.; Energy
Fuels (1996), 10 (4), 1017-1021, Kaneko et al.; Anal. Biochem. 242
(1996), 104-111; Brissova et al; and Reactive Polymers 23 (1994),
173-182 Petro et al. Reference may also be made to U.S. Pat. Nos.
6,175,409, 6,406,632, 6,265,226, 6,260,407, 6,294,388 and PCT
Publication WO 99/51980. The entire disclosure of each of the
patents, publications and patent applications referred to above is
incorporated herein by reference for all purposes.
SUMMARY OF INVENTION
[0007] This invention relates to methods and apparatus which are
useful for evaluating the interaction between a receptor and a
modifying agent in the presence of a liquid carrier, and which make
use of novel and inventive variations of the known techniques of
liquid solid chromatography. In the terminology often used in
chromatography, the invention includes (but is not limited to)
methods in which the receptor is the probe and the modifying agent
is the sample, and methods in which the modifying agent is the
probe and the receptor is the sample.
[0008] In a first aspect of the invention, one of the receptor,
modifying agent and liquid carrier is a member of a library,
preferably either the receptor or modifying agent is a member of a
library, and in either case, the method includes evaluating members
of the library in a number of test procedures, and using the
results of test procedures to select at least one substance for
further testing. The substance selected for further testing will
generally be a member of the library, but may be a substance having
a known relationship to a member of the library.
[0009] In other aspects, the invention provides various methods
which are useful as test procedures in the first aspect of the
invention, but which are also useful in other contexts, for example
for evaluation of interaction between substances by inverse
chromatography, and for the rapid testing of a sample withdrawn
from a continuous process. In yet further aspects, the, invention
provides apparatus suitable for carrying out the methods of the
invention.
[0010] The term "evaluating" is used herein in a broad sense to
denote any useful assessment of the interaction between the
receptor and the modifying agent. For example, in the first aspect
of the invention, the term includes any assessment which makes it
possible to select at least one substance for further testing. The
term "receptor" is used herein to denote a substance which can form
at least part of a solid phase. Thus, the receptor, alone or in
combination with a solid, corresponds generally to the stationary
phase in conventional liquid solid chromatography. The term "liquid
carrier" is used herein to denote any liquid which can be passed
continuously over the solid comprising the receptor while the solid
is confined in a test chamber. Thus, the liquid carrier corresponds
generally to the mobile phase in conventional liquid solid
chromatography. The term "modifying agent" is used herein to denote
a substance which can form at least part of a liquid sample which
can be injected into a stream of the liquid carrier before the
stream is passed over the solid in the test chamber. The term
"library" is used herein in a broad sense to denote a plurality of
identified substances, typically an associated collection of
identified substances. The library is preferably a spatially
determinate array of identified substances formed at or in or
situated on a common substrate. Preferably, but not necessarily,
the members of the library differ from each other in some
systematic way, so that the variation of the results of the test
procedures can be correlated with those differences. For example,
the members can differ from each other in one or more quantified
ways. Such differences can be quantified by measurements carried
out during preparation of the substances, or by measurements
carried out on the substances themselves. The-differences can be
ascertained before or after members of the library have been
selected in accordance with the present invention.
[0011] The method of the first aspect of the invention comprises
conducting a plurality of test procedures, each of the test
procedures comprising the steps of
[0012] (a) injecting a liquid test sample comprising the modifying
agent into a stream of the liquid carrier, the injection being
carried out over a limited time so that a distinct test section of
the stream contains the test sample;
[0013] (b) passing the stream of liquid carrier containing the test
sample over a solid phase in a test chamber, the solid phase
comprising the receptor; and
[0014] (c) examining the stream of liquid carrier leaving the test
chamber to ascertain a variable which reflects the interaction of
the modifying agent and the receptor in the presence of the liquid
carrier.
[0015] Step (c) of the method will often comprise examining a
distinct evaluation section of the stream corresponding to the test
section in order to ascertain a property of the modifying agent.
The term "to ascertain a property of the modifying agent" is used
herein to include ascertaining a property of the modifying agent
itself and/or ascertaining a property of a substance which is
produced by the interaction of the modifying agent and the receptor
and/or ascertaining a property of part only of the modifying
agent.
[0016] The results obtained in the steps (c) of the test procedures
can optionally be used to select at least one substance for further
testing. The further testing can for example involve the
interaction between (i) a selected member or members of the library
and (ii) the other two of the receptor, modifying agent and liquid
carrier used in the test procedures. The further testing can
otherwise be unrelated to the original evaluation.
[0017] The term "examining the stream of liquid carrier leaving the
test chamber to ascertain a variable which reflects the interaction
of the modifying agent and the receptor in the presence of the
liquid carrier" is used herein in a broad sense to denote any
examination which enables the desired evaluation to be made. For
example, as further explained below, it may be sufficient to
compare the results of the test procedures without quantifying any
property of the modifying agent. Preferably (but not necessarily)
step (c), alone or in combination with other steps, makes it
possible to calculate the proportion of the modifying agent, or a
component of the modifying agent, retained in the test chamber.
[0018] The objective of the known chromatographic procedures is (i)
to achieve at least some separation of the individual components of
the sample, followed by collection of at least one separated
product, and/or (ii) to achieve a substantial degree of
equilibration between the sample and the stationary phase in order
to provide detailed characterizing data. The known procedures use,
therefore, combinations of stationary and mobile phases, processing
times, and columns which achieve these objectives. By contrast, it
is not an objective of the present invention to recover a separated
product or to achieve equilibrium between the sample and the solid
phase. Furthermore, in the first aspect of the invention, the
library is preferably examined as quickly as is consistent with
selection of a limited number of members for further testing and
under dynamic conditions which simulate an ultimate end-use. For
example, the objective might be to identify the members of a
library having the potential to be additives which produce a
desired effect on a fabric being laundered or dry cleaned, or on
photographic film being developed, or in a paint being applied to a
building material.
[0019] Because of these differences in objectives, the test
procedures used in the first aspect of the invention often have at
least one (i.e. one or more) of the following characteristics which
distinguish them from known chromatographic procedures.
[0020] (I) The receptor is a substance which has not been used as
the stationary phase in liquid solid chromatography. For example,
the receptor comprises
[0021] (1) a fabric,
[0022] (2) at least 50% by weight of particles having an aspect
ratio of at least 2 and at least one dimension which is greater
than 100 micron,
[0023] (3) a substantially pore- free sheet of an organic
polymer,
[0024] (4) a foodstuff,
[0025] (5) a natural or artificial mammalian tissue,
[0026] (6) a polysaccharide, a protein or a nucleic acid,
[0027] (7) a catalyst,
[0028] (8) an elemental metal or metal alloy,
[0029] (9) wood,
[0030] (10) concrete,
[0031] (11) natural or artificial stone,
[0032] (12) a semiconductor substance, or
[0033] (13) a DNA chip, a molecular recognition chip, or a
separation chip.
[0034] The receptor material preferably includes mammalian tissue,
either artificial or natural. Other preferred receptor materials
are selected from the group consisting of a fabric, a substantially
pore-free sheet of an organic polymer, a foodstuff, wood, concrete,
natural or artificial stone, and a semiconductor substance. Within
this group, receptor materials selected from a fabric, a foodstuff
and a semiconductor substance are particularly preferred.
[0035] (II) The test chamber is less efficient than columns used in
liquid solid chromatography. For example, the efficiency of the
test chamber is less than 80, preferably less than 50, particularly
less than 10, theoretical plates.
[0036] (III) The test procedures are carried out rapidly and under
dynamic conditions. These dynamic conditions are not intended to
promote separation of the modifying agent into component parts or
to ensure that all of the modifying agent passes through the test
chamber. Furthermore, the extent of the interaction between the
receptor and the modifying agent can vary widely in the different
test procedures. As a result, one or more of the following
conditions are often fulfilled.
[0037] (1) In each of the test procedures, the test sample
comprises an initial quantity of the modifying agent, and in at
least one of the test procedures at least 1%, often at least 10%,
e.g. 20-70% or 30-50%, of the initial quantity of the modifying
agent is retained in the test chamber. Substantially all of the
modifying agent retained until the end of a test procedure remains
retained on the solid phase during subsequent test procedures, and
thus can be regarded as being irreversibly retained on the solid
phase under the test conditions.
[0038] (2) In each of the test procedures, the test sample
comprises an initial quantity of the modifying agent, and the
difference between (i) the lowest percentage of the initial
quantity of modifying agent retained in the test chamber in any of
the test procedures and (ii) the highest percentage of the initial
quantity of modifying agent retained in the test chamber in any of
the test procedures, is at least 10, for example 10-70, often at
least 25, for example 30-60.
[0039] (3) In at least one, preferably in each, of the test
procedures, the receptor is not saturated by the modifying agent.
The extent to which saturation is being approached can be assessed
by repeating one of the test procedures. Preferably, in such a
repeated test procedure, the same proportion of the modifying agent
is retained by the receptor, indicating that the receptor is far
from saturation. If a lesser proportion is retained, this indicates
that the receptor is approaching saturation; and when none of the
modifying agent is retained, the receptor is saturated.
[0040] (4) In at least one, preferably in each, of the test
procedures, a plot of time against concentration of the modifying
agent in the stream of liquid carrier leaving the test chamber has
only a single peak, with the slope of the plot being positive at
all points on one side of the peak and negative at all points on
the other side of the peak.
[0041] (5) In at least one, preferably in each, of the test
procedures, the stream of liquid leaving the test chamber is passed
through a detector, and the time taken for the evaluation section
to pass through the detector is less than five times the time taken
for the test section containing the modifying agent to pass through
the entrance to the test chamber.
[0042] (IV) The desired evaluation is carried out merely by
ascertaining the proportion of the modifying agent which is
retained in the test chamber (i.e. it is unnecessary to know, in
absolute terms, the quantity of modifying agent initially present
in the sample). This makes it possible to carry out the desired
evaluation by comparing (a) the results of passing the
sample-carrying liquid stream through the test chamber and (b) the
results of a reference procedure in which a similar sample-carrying
liquid stream is passed through a reference chamber which (i) is
free of any substance which interacts with the modifying agent, or
(ii) contains a known solid substance. In some cases, satisfactory
results can be obtained by comparing the results of each of the
test procedures with a single reference procedure or with two or
more reference procedures which are carried out at appropriate
intervals. However, this requires a uniformity between the
different procedures which is not always easy to attain. It is
preferred, therefore, that each of the test procedures should
itself incorporate the reference procedure. In this case, each of
the test procedures further comprises
[0043] (d) injecting a liquid reference sample into a second stream
of the liquid carrier, the composition of the second sample being
substantially identical to the composition of the test sample, and
the injection being carried-out over a limited time so that only a
distinct reference section of the stream contains the reference
sample;
[0044] (e) passing the second stream of liquid carrier containing
the reference sample through a reference chamber which is free of
any substance which interacts with the modifying agent;
[0045] (f) examining the stream of liquid carrier leaving the
reference chamber to ascertain a property of the modifying agent
remaining in the stream; and
[0046] (g) comparing the results obtained in steps (c) and (f) to
evaluate the interaction between the receptor and the modifying
agent.
[0047] In the aforementioned protocol, the second stream into which
the reference sample is injected can be the same stream into which
the test sample was injected (or alternatively, can be a separate
and independent stream (i.e., a separate line)).
[0048] Preferably, therefore, the first liquid stream containing
the test section and second liquid stream containing the reference
section are obtained by
[0049] (i) injecting into the stream of liquid carrier a liquid
unit whose composition is the same as the composition of the test
and evaluation samples and whose size is equal to the sum of the
sizes of the test and evaluation samples, the injection being
carried out over a limited time so that only a distinct section of
the liquid stream contains the liquid unit, and
[0050] (ii) splitting the liquid stream containing the liquid unit
into a first sub-stream which passes through the test chamber and
includes the test section and a second sub-stream which passes
through the reference chamber and includes the reference
section.
[0051] In the methods of the first aspect of the invention, the
test procedures can be carried out in series or in parallel or
both.
[0052] In some embodiments, it is particularly preferably to use
parallel (simultaneous) procedures, especially with respect to
applications in combinatorial materials science, in which either
the library or receptors are members of a combinatorial library
comprising at least four members, such that members are evaluated
for interaction between a receptor member of the combinatorial
library and a modifying agent, or alternatively for interaction
between a modifying agent member of a combinatorial library and a
receptor, in each case in the presence of a liquid carrier.
Preferably each of the at least four members of the combinatorial
library comprise a different non-biological polymer. The method
includes conducting at least four test procedures in parallel under
a common set of test conditions, with the receptor, modifying agent
and liquid carrier being the same in each of the test procedures
except that in each test procedure a different member of the
combinatorial library is used.
[0053] The parallel testing/evaluation can include multiple flow
channels with simultaneous contacting of test samples (comprising a
modifying agent) and solid phases (comprising a receptor) as
compared between different channels. The injection into such
parallel-configured carrier streams, each having its own dedicated
test chamber, can be accomplished in a sequential manner, as
described for example in co-owned U.S. Pat. No. 6,296,771.
Alternatively, the injection into such parallel-configured carrier
streams can also be accomplished in parallel, using parallel
injection systems such as are disclosed in co-owned, co-pending
U.S. applications, Ser. No. 09/641,442 filed Aug. 2, 2002 by
Freitag et al. and Ser. No. 10/092,035 filed Mar. 6, 2002 by Bergh
et al. In either case, the detection (examining step) is preferably
done in parallel, but can also be sequential. In a preferred
protocol, the at least four parallel test procedures can
comprise:
[0054] (a) simultaneously injecting at least four liquid test
samples comprising an initial quantity of the modifying agent into
at least four separate and distinct streams of the liquid carrier,
respectively, the injections being carried out over a limited time
so that distinct test sections of the at least four streams contain
the test sample,
[0055] (b) simultaneously passing the at least four streams of
liquid carrier containing the test samples over a solid phase in at
least four separate and distinct test chambers, respectively, the
solid phase comprising the receptor,
[0056] (c) retaining at least 10% of the initial quantity of the
modifying agents on the solid phase in each of the at least four
test chambers, the retained modifying agents being irreversibly
retained under the test conditions of the procedures, and
[0057] (d) simultaneously examining the at least four streams of
liquid carrier leaving the test chamber to ascertain, for each of
the at least four streams, a variable which reflects the
interaction of the modifying agent and the receptor in the presence
of the liquid carrier for that respective stream, and
[0058] (e) comparing the interaction of the modifying agent and the
receptor for each of the at least four streams to determine a
relative ranking of the members of the combinatorial library with
respect to such interaction.
[0059] It is specifically contemplated that such parallel methods,
and in particular the above-detailed preferred parallel methods for
evaluation of combinatorial libraries can be used in connection
with each and every other embodiment disclosed herein (e.g.,
evaluating interaction between a modifying agent and a receptor
comprising natural or artificial mammalian tissue, or other types
of materials (as listed above), evaluation protocols involving a
reference chamber, etc.)--such as test procedures (I) to (IV) as
outlined above.
[0060] Individual test procedures having one or more of
characteristics (I) to (IV) set out above are in themselves novel
and inventive, and form part of the present invention. Thus, in
second to fifth aspects, the present invention provides methods
which are suitable for evaluating the interaction between a
receptor and a modifying agent in the presence of a liquid carrier
and which respectively have one of characteristics (I) to (IV) set
out above.
[0061] In a sixth aspect, the invention provides apparatus suitable
for carrying out the method of the first aspect of the invention
wherein the modifying agent is one of the library and each of the
test procedures has characteristic (I) above, the apparatus
comprising
[0062] (A) a test chamber for the solid phase comprising a receptor
as defined in characteristic (I) above;
[0063] (B) a reservoir for the liquid carrier;
[0064] (C) a pump for continuously extracting a stream of the
liquid carrier from the reservoir and passing the stream through
the test chamber;
[0065] (D) an autodilution and sampling robot for sequentially
injecting into the stream of liquid carrier, before the stream
passes through the chamber, a plurality of liquid test samples,
each sample containing an initial quantity of one of the library of
modifying agents and each sample being injected over a limited time
so that only a distinct test section of the liquid stream contains
the sample; and
[0066] (E) a detector for examining the stream of liquid carrier
leaving the test chamber without removing anything from the stream
and for determining for each sample the proportion of the initial
quantity of the modifying agent remaining therein.
[0067] In a seventh aspect, the invention provides apparatus
suitable for carrying out the method of the first aspect of the
invention wherein the method has characteristic (II) above, the
apparatus comprising a test chamber which contains the solid phase
comprising the receptor and which has an efficiency of less than
80, preferably less than 50, especially less than 10, theoretical
plates; and a reservoir, pump, autodilution and sampling robot and
detector as in the apparatus of the seventh aspect of the
invention.
[0068] In an eighth aspect, the invention provides apparatus which
is suitable for carrying out the method of the first aspect of the
invention wherein the method has characteristic (IV) as defined
above, and which comprises
[0069] (A) a test chamber containing the solid phase comprising the
receptor;
[0070] (B) a reference chamber which is free of any substance which
interacts with the modifying agent;
[0071] (C) a reservoir for the liquid carrier;
[0072] (D) a main passageway from the reservoir;
[0073] (E) a first branch passageway which is connected to the main
passageway has a junction and which leads from the main passageway
to the test chamber;
[0074] (F) a second branch passageway which leaves from the test
chamber;
[0075] (G) a third branch passageway which is connected to the main
passageway at the junction and which leads from the main passageway
to the reference chamber;
[0076] (H) a fourth branch passageway which leaves from the
reference chamber;
[0077] (I) an exit passageway which combines the second and fourth
branch passageways;
[0078] (J) a pump for extracting a stream of the liquid carrier
from the reservoir and passing it through the main passageway;
[0079] (K) an injector for injecting into the stream of liquid
carrier, before the stream passes from the main passageway to the
first and third branch passageways, a liquid unit containing an
initial quantity of the modifying agent, the unit being injected
over a limited time so that only a distinct section of the liquid
stream contains the unit, and the sample being divided into a first
sub-stream which contains a first sample of liquid unit and which
passes through the test chamber and a second sub-stream which
contains a second sample of the liquid unit and which passes
through the reference chamber; and
[0080] (L) a detector for examining the stream of liquid carrier
passing out of the exit passageway and for ascertaining the
proportion of the modifying agent remaining therein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0081] The invention is illustrated in the accompanying drawings,
which are described briefly as follows.
[0082] FIG. 1 is a diagrammatic illustration of an apparatus for
injecting a plurality of liquid units into a stream of liquid
carrier;
[0083] FIGS. 2, 4, 6, 9, 10 and 11 are diagrammatic illustrations
of apparatus for evaluating the interaction between a receptor and
a library of modifying agents, by passing a stream containing
successive samples of the library through a chamber containing the
receptor, and examining the stream leaving the chamber with a
detector, the apparatus of FIG. 11 also being suitable for
evaluating the interaction between a modifying agent and a library
of receptors; and
[0084] FIGS. 3, 5, 7 and 8 are plots of time against the amount of
modifying agent remaining in the stream leaving the chamber, as
recorded by the detectors in FIGS. 2, 4, 6, 9, 10 and 11.
DETAILED DESCRIPTION OF THE INVENTION
[0085] In the Summary of the Invention above, the Detailed
Description of the Invention, the Examples, and the Statements
below, and the accompanying drawings, reference is made to
particular features of the invention. It is to be understood that
the disclosure of the invention in this specification includes all
appropriate combinations of such particular features. For example,
where a particular feature is disclosed in the context of a
particular embodiment, a particular statement, or a particular
Figure, that feature can also be used, to the extent appropriate,
in the context of other particular embodiments, Statements and
Figures, and in the invention generally.
[0086] The detailed description below is directed to each aspect of
the invention, considered individually and in various combinations.
Aspects of the detailed description are chiefly directed to the
first aspect of the invention, in which libraries of compositions
are evaluated, and to apparatus for use in the first aspect of the
invention. It is to be understood, however, that insofar as the
detailed description is concerned with test procedures having one
or more of the characteristics (I) to (IV) above, it is also
applicable to the second to sixth aspects of the invention.
[0087] Receptors
[0088] The receptor can be any element, compound or composition
which is a solid or which can be immobilized on a solid. Receptors
that are suitable, and indeed in some embodiments preferred,
include the following.
[0089] (1) Fabrics (i.e., a fabric material), including woven,
knitted and nonwoven fabrics (including papers), made from natural
or artificial (including regenerated natural) sources, such as
continuous filament or staple fiber yams, for example polyamide,
polyester, polyolefin, polyurethane, cellulosic, cotton, silk and
wool yams. The particular form of the fabric material is not
narrowly critical, and can include sheets of fabric materials, bulk
fabric materials, stranded fabric materials (e.g. yarns), etc.
[0090] (2) Particulate materials, including particulate materials
which comprise at least 50%, preferably at least 75%, e.g. 75-100%,
by weight of particles having an aspect ratio of at least 2,
preferably at least 5, particularly at least 10, especially at
least 50, e.g. at least 100, and optionally having at least one
dimension greater than 100 micron, preferably greater than 500
micron.
[0091] (3) Continuous sheets of natural or synthetic (including
regenerated natural) organic polymers, including such sheets which
are free of open pores and preferably are substantially free of all
pores.
[0092] (4) Foodstuffs, including coffee beans (green and roasted),
ground coffee, meats, poultry, beans (e.g., soybeans), grains,
cereals, rice, pastas, fruits and vegetables.
[0093] (5) Natural and artificial mammalian tissues, including for
example human or animal epidermis, epithelium, bone and hair. More
generally, the receptors of the invention can comprise biological
tissue, including both natural and artificial biological tissue. A
more specific description of the mammalian tissue and/or more
generally, the biological tissue of the invention is set forth as
follows.
[0094] (5a) The biological tissue (especially mammalian tissue) can
include both natural and non-natural model biological tissue, as
well as both hard biological tissue and soft biological tissue.
Details discussed hereinafter with regard to biological tissue, is
particularly intended to refer to mammalian tissues. In general,
biological tissue can be naturally occurring animal biological
tissue or non-natural biological tissue (including both non-natural
materials and natural materials that are not natural biological
tissues). Also, in general, it is preferred that the biological
tissues used in the arrays and methods of the present invention are
soft biological tissues and/or bard biological tissues. These
tissues should preferably, in each case, fulfill the following
requirements: (i) they should be chemically and physically similar
to human tissues (e.g., with respect to properties of interest,
such as polymer adsorptivity), and (ii) and they should be suitable
for high-throughput screening (e.g., with respect to availability
in sufficient quantities and reproducability to allow for
comparison between experiments of the screen).
[0095] (5b) A natural biological tissue can be an animal biological
tissue, and in many cases, is preferably a human biological tissue,
but can also be an animal biological tissue such as bovine, porcine
(pig), etc. Exemplary natural oral tissues include tissues from the
tongue, teeth, heart, lungs, liver, kidney, spleen, brain vein,
skin, blood, muscle, hair, etc. of animals, especially mammals such
as humans and non-human mammals, and especially of non-human
mammals such as non-human primates, porcine (pig), bovine, rabbit,
and mice, among others. For oral care applications, for example,
exemplary natural oral tissues include human teeth (as an exemplary
natural hard oral tissue), or tongue, such as porcine (pig) tongue
(as an exemplary natural soft oral tissue), or gum, such as bovine
or porcine gum (as an exemplary natural soft oral tissue) or cheek
lining, such as bovine cheek lining (as another exemplary soft oral
tissue).
[0096] (5c) A model biological tissue can include both non-natural
materials, and natural materials that are not natural biological
tissues. Model biological tissues preferably can have substantially
the same chemical properties as natural human biological tissue,
particularly in an environment that is representative of the
in-vivo conditions for the biological tissue of interest. In
particular, the model biological tissue is preferably effective for
emulating natural animal biological tissue (e.g., natural human
biological tissue or natural bovine biological tissue or natural
porcine biological tissue) for polymer adsorption to the biological
tissue, typically in the presence of a composition or atmosphere
that is representative of, or emulates the natural composition or
atmosphere in which the biological tissue is found. Especially, the
model biological tissue is preferably effective for emulating
natural oral tissue for polymers already known in the art as having
a certain degree of interaction, such as adsorptivity, with that
biological tissue. For example, in oral care applications, the
model oral tissue is preferably effective for emulating natural
oral tissue for polyoxyalkylene polymer adsorption to the tissue in
the presence of natural or artificial saliva. Preferably, as
determined using the evaluation protocol of choice (e.g., as
described above), the degree of adsorptivity between the model
biological tissue and the polymer of choice in the presence of the
conditions that emulate the in vivo conditions for the tissue, can
be comparable within at least about 50%, preferably at least about
30%, more preferably at least about 10%, and most preferably at
least about 5%, as compared to the degree of adsorptivity between
polymer and the natural animal biological tissue in the presence of
such conditions. Exemplary model biological tissues include porous
man-made polymeric materials (as an example of non-natural model
biological tissues), mineral-based materials (as an example of
natural or non-natural model hard tissues), epidermal (skin) or
epithelium cells or other cells or soft tissue (as an example of a
natural material that is not a natural oral tissue for a model soft
oral tissue). More specific examples, for oral care applications,
of model oral tissues include hydroxyapatite (as an example of a
model hard oral tissue) and reconstructed epithelium (as an example
of a model soft oral tissue).
[0097] (5d) For oral care applications, the oral tissue used
according to the present invention is preferably selected from the
group, consisting of a soft oral tissue, such as an animal tongue,
especially a pig tongue (emulating human tongue), a reconstructed
epithelium (emulating human soft tissue such as cheek lining or
gum), and a hard oral tissue, such as animal teeth, especially
human teeth, and hydroxyapatite (emulating human teeth). Pig tongue
provides reproducible results in high-throughput screening
experiments and is a reliable substrate for high-throughput
screening purposes. The surface area of pig tongue is high enough
to provide sufficient sensitivity to distinguish between
adsorptivity of various polymers. Epithelium reconstructed from
human cells is commercially available from SkinEthics (France).
Reconstructed epithelium also provides reproducible results in
high-throughput screening experiments. The reconstructed epithelium
may be used in the form of small pieces provided on the bottom of
the wells of a microtiter plate, or in the form of strips of a
larger piece of epithelium. Human teeth are available from dental
offices or other dental supply sources, or from kids having ages
ranging from about 4 to about 10 years, preferably from about 5 to
about 7 years. If the array of the present invention comprises
hydroxyapatite (HAP), the form of the HAP is not narrowly critical,
and can be varied depending on the application of interest. For
example, the HAP can be in the form of a sheet, disc, or powder.
HAP powder or HAP in the form of discs is commercially available
(Clarkson Chromatography Products, Inc.; South Williamsport, Pa.).
HAP powder is preferred for some applications, and can comprise
particles having a size ranging from about 1 pm to about 100 pm,
preferably from about 5 pm to about 50 pm, and in general, can more
preferably consist essentially of particles having a size ranging
in these same ranges. A preferred HAP powder comprises or consists
essentially of porous HAP particles having a particle size of from
10 to 30 pm, preferably about 20 pm. HAP powder has a high surface
area of about 7 to 8 m.sup.2/g (measured in accordance with the BET
method and mercury porosimetry) and is characterized by the ability
to average differences among the individual HAP particles in a
certain volume of the powder. Moreover, HAP powder does not show a
significant fluorescent background. Therefore, quantitatively
strong signals may be obtained. HAP powder is preferably used in
the present invention. HAP discs have a thin porous layer at the
surface only and, therefore, are practically impermeable. The
surface area of HAP discs is about 0.1 m.sup.2/g.
[0098] (6) Compositions comprising a polysaccharide, protein (i.e.
an amino acid polymer, including peptides, enzymes, biologically
functional proteins, etc.) or nucleic acid (i.e., a nucleic acid
polymer, including deoxyribonucleic acid, ribonucleic acid,
oligonucleotides and biologically functional nucleic acid
polymers). In particular, such compositions are preferred in
combination with one or non-biological polymers (e.g. provided as
supports, linkers, brushes or otherwise. For example, the receptor
can comprise a polysaccharide, protein (i.e., amino acid polymer)
or nucleic acid (i.e., nucleic acid polymer) as a biological probe,
where the probes are presented using specifically designed
non-biological polymer brushes (e.g., polymer-coated surfaces
prepared for example through living free radical polymerization
techniques). Such biological compositions presented on polymer
brushes are described in co-owned PCT application WO 01/02452, and
in the corresponding co-owned U.S. applications, Ser. Nos.
09/347,606, 09/347,607, 09/347,608 and 09/347,609, each of which is
incorporated herein by reference for all purposes.
[0099] (7) Catalyst, including catalyst comprising a metal, e.g. a
transition metal or a Group IV metal, or a metal compound, or a
protein, optionally supported by an in the support, e.g. a support
composed of a silicate, a zeolite, alumina or another metal
oxide.
[0100] (8) Elemental metals and metal alloys.
[0101] (9) Building materials including wood, concrete, natural
stone and artificial stone.
[0102] (10) Semiconductors.
[0103] (11) DNA chips, molecular recognition chips, and separation
chips. DNA chips, as used herein, refer to an array of different
deoxyribonucleic acid polymer molecules formed on, residing on, or
supported by or in a common substrate. Molecular recognition chips
refer to an array of different compounds or compositions that are
selective for (i.e., selectively recognize) a particular chemical
entity of interest, with such compounds or compositions being
formed on, residing on or supported by or in a common substrate. An
example of a molecular recognition chip can include an arrays of
monoclonal antibodies formed on a common substrate. Separation
chips refer to array of different compounds or compositions that
are functional for separating (e.g., by size, by charge, by
chemical composition, etc.) components of a mixture (e.g.
components of a test sample), with such compounds or compositions
being formed on, residing on or supported by or in a common
substrate.
[0104] Modifying Agents
[0105] The modifying agent can be any composition which can form at
least part of a liquid sample and which will interact with the
receptor. The sample generally comprises a liquid in which a solid
or fluid modifying agent is dissolved, dispersed or emulsified. The
liquid in the sample preferably does not interact with the
receptor, and is preferably the same as the liquid carrier. The
concentration of the modifying agent in the sample is generally
0.001 to 10% by weight, preferably 0.05 to 0.5% by weight.
[0106] Often the interaction between the receptor and the modifying
agent will be merely physical adsorption of the modifying agent on
the surface of the receptor. However, the interaction can
alternatively or additionally involve other types of interaction,
including for example Lewis acid/Lewis Base interaction, hydrogen
bonding, occlusion, and clathration. In the first aspect of the
invention, the objective is generally to identify the combinations
of receptor, modifying agent and liquid carrier which result in the
greatest retention of the modifying agent in the test chamber. It
is preferred, therefore, that in the least one of the test
procedures, at least 10%, particular least 20%, e.g. 20-70%, or
30-50%, of the initial quantity of the modifying agent is retained
in the test chamber. The variation in the results of the test
procedures may be for example such that there is a difference of
least 10, for example 10-70, often at least 25, for example 30-60,
between (i) the percentage of the initial quantity of the modifying
agent retained in the test chamber in one of the test procedures
and (ii) the percentage of the initial quantity of the modifying
agent retained in the test chamber in another of the test
procedures.
[0107] Organic polymers, including non-biological polymers and
synthetic polymers, are preferred modifying agents. The polymer can
be, for example, crystalline or non-crystalline, a thermoplastic or
an elastomer, (including a thermoplastic elastomer), and a
homopolymer or copolymer, for example a polyamide, a polyester, a
polyurethane, a polyether, a polyurea, or a polymer comprising
units derived from at least one ethylenically unsaturated monomer,
e.g. an olefin, a vinyl monomer, or acrylic or methacrylic acid or
an ester or amide thereof. Suitable polymers include polymers
containing polar groups, e.g. a hydroxyl group, a primary,
secondary or tertiary amino group (substituted or unsubstituted),
or a carboxyl, sulfonyl or other acidic group or a salt or ester
thereof.
[0108] The modifying agents of the invention can also include
therapeutic agents and/or diagnostic agents, themselves, and/or in
combination with non-biological polymers (i.e., non-biological
organic polymers). As used herein, therapeutic agents includes
prophylactic agents. Hence, the modiflying agents can be chemical
or biological entities that provide a diagnostic benefit for
identifying or determining a particular state or condition such as
a particular malady, medical concern or health concern, that
provide a therapeutic benefit for a particular malady or medical
concern or health concern, and/or that provide a prophylactic
benefit, to avoid a particular malady or medical concern or health
concern. When such therapeutic agents or diagnostic agents are used
in combination with non-biological polymers, either the agents or
the non-biological polymers or both can be members of a
combinatorial library. In some embodiments, different
non-biological polymers are preferably members of a combinatorial
library being evaluated for interaction with a receptor, for
example, such as a mammalian tissue, or other biological
tissue.
[0109] When the modifying agent contains two or more compounds,
including polymer molecules of different molecular weights, there
may be, some separation of those compounds as they pass through the
test chamber. Such separation is not generally desired or useful
for the purposes of the present invention and preferably does not
take place. If such separation does take place, and the detector
measures both the separated compounds, a plot of time against the
concentration of the modifying agent in the stream leaving the test
chamber will evidence that separation. In some cases, the plot will
show more than one peak, each peak corresponding to one of the
separated components of the modifying agent. Preferably the plot
has a principal peak which, when the plot is subjected to
valley-to-valley integration, amounts to at least 70%, preferably
at least 80%, of the area under the curve. It is particularly
preferred that the plot has only a single peak, with the slope of
the plot being positive at all points on one side of the peak and
negative at all points on the other side of the peak.
[0110] Liquid Samples
[0111] The liquid samples are preferably small in size, so that
they can be injected in a very short time, e.g. a fraction of the
second, into the stream of liquid carrier. For example, the volume
of the liquid sample may be, for example from 1 to 500 microliters,
preferably from 5 to 50 microliters. The samples are generally
injected at a time interval of not more than 10 minutes, preferably
one every 0.15 to 3 minute.
[0112] Liquid Carriers
[0113] The liquid carrier can be any liquid which can be passed
over the solid phase in the test chamber and which permits
satisfactory ascertainment of a property of the modifying agent.
Generally, the liquid carrier can include water, aqueous solvents,
non-aqueous polar solvents and/or non-polar solvents. Preferably,
there will be substantially no interaction between the liquid
carrier and the solid phase. The liquid carrier can be aqueous,
organic or a mixture of miscible aqueous and organic liquids.
Preferred liquid carriers are aqueous solutions of inorganic salts
optionally containing surface active agents, e.g. laundry
additives, particularly solutions of the kind found in clothes
washing machines and dishwashers. Other liquid carriers are organic
solvents of the kind used in the dry cleaning of clothes,
optionally containing surface active agents. When the library is a
library of liquid carriers, the liquid carriers can for example be
aqueous solutions which differ from each other in pH or temperature
or both.
[0114] Libraries
[0115] In each of the test procedures in the methods of the first
aspect of the invention, one of the receptor, modifying agent and
liquid carrier is a member of a library. In preferred embodiments,
the library members are either receptors or modifying agents, such
that members are evaluated for interaction between a receptor
member of the combinatorial library and a modifying agent, or
alternatively for interaction between a modifying agent member of a
combinatorial library and a receptor, in each case in the presence
of a liquid carrier. Regardless of the characterization of the
library members, the library generally contains at least four
members and may contain many more, for example up to 96 members.
Sometimes all the members of the library are tested, but this is
not necessary. Generally at least four test procedures are carried
out, using a different member of the library in each test
procedure. If not all the members of a library are tested, the
tested members will generally be taken from different sections of
the library. If not all members of a library are tested, the
results of the steps (c) can be used to select one or more members
of the library for further testing. The selected member(s) may be
member(s) which were tested and/or member(s) which were not tested.
Different library members may be screened simultaneously using the
same test procedures (ie., screened under a common set of
conditions using common protocols), and/or the different library
members may be screened sequentially.
[0116] The invention is particularly useful when the library is a
library of modifying agents, because it is then possible to use a
conventional autodilution and sampling robot to inject the
different test samples into the stream of liquid carrier (e.g., in
sequential fashion). Furthermore, when, as is preferred, the
surface area of the solid phase is relatively large by comparison
with the amount of the modifying agent, the members of the library
can be tested in series, with the stream of liquid carrier,
containing the different samples in sequence, being passed through
the same test chamber. This is particularly advantageous where each
sample does not saturate the solid phase receptor. In this way, a
rapid rate of testing can be maintained, and moreover, can be
multiplied particularly if at least two, e.g. four or more,
substantially identical test chambers are used in parallel. By
contrast, when the library is a library of receptors, a plurality
of test chambers, each containing a different receptor, can be
individually prepared, and they are preferably tested in parallel.
Alternatively, the plurality of different individual receptors can
be presented for testing in a single flow-channel system by
alternately and successively replacing one test chamber (comprising
a solid phase having a receptor comprising one member of the
library) with the next test chamber (comprising a solid phase
having another receptor comprising another member of the library).
When the library is a library of liquid carriers, they too can be
tested in parallel, or with proper hardware configuration, in
series.
[0117] When the library is a library of modifying agents, any
appropriate diversity element can form the basis of the library.
For example, the library of modifying agents can comprise four or
more different therapeutic agents or can comprise four or more
different diagnostic agents, especially for screening with
biological tissue such as mammalian tissue. Other diversity
elements are well known in the art, especially for small organic
molecules, enantiomeric molecules and for molecules having
biological and/or pharmaceutical activity. The library of modifying
agents can, in any case, further comprise non-biological polymers.
The same common non-biological polymers can be used in connection
with the library (e.g., when a different chemical entity such as a
therapeutic or diagnostic agent is employed as the diversity
element of the library). Alternatively, and in one particularly
preferred embodiment, the non-biological polymers can be the
diversity element of the library, and can preferably (but not
necessarily) be applied to a same common agent such as a same
common diagnostic agent or therapeutic agent. The library of
modifying agents can also have more than one diversity elements,
including for example at least four members that vary with respect
to two or more features (e.g. different non-biological polymer and
different therapeutic and/or diagnostic agent).
[0118] The combinatorial library can be also be a library of
receptors. The library of receptors can include different receptor
materials. Hence, when the receptors comprise mammalian tissue, the
library can comprise different natural or artificial mammalian
tissue. The variation in the receptors as compared between members
of such a library can include the specific nature of the tissue,
the source of the tissue, the pretreatment history of the tissue,
etc. The different receptor materials can, in a preferred
embodiment, include different non-biological polymers to create
diversity between members of the library. For example, a same
common receptor material (e.g., mammalian tissue, fabric materials,
etc.) can be pretreated (e.g. by coating, immersion, etc.) with a
different non-biological organic polymer. In this way, various
treatment agents and protocols for a given receptor (e.g. mammalian
tissue, fabric materials) can be investigated to determine a
preferred non-biological treatment agent and protocol. A specific
example of such application relates to skin care and/or wound care,
in which the receptor can comprise skin cells (e.g., epidermal
cells, epithelium cells) that have been pretreated with various
different non-biological polymers to form a library of different
receptor materials. The different receptor materials can then be
screened by exposure to a same common modifying agent to determine
the interaction between the modifying agent and the different
receptor materials.
[0119] Detectors
[0120] The term "detector " is used herein to denote any instrument
which can be used to ascertain a property of the modifying agent in
the stream of liquid carrier (including, as noted above, a property
of part of the modifying agent and a property of a substance which
is produced by the interaction of the modifying agent and the
receptor). Hence, the detector can be a flow detector as known in
the chromatographic arts. Preferably the detector operates without
removing anything from the stream (i.e. is non-destructive). Such
detectors measure, for example, (i) the adsorption by the liquid
stream of radiation whose wavelength is absorbed to by the modifier
but not by the liquid carrier, (ii) the fluorescence of the stream,
or (iii) the refractory index of the stream. The detector often
makes a series of measurements at spaced intervals of time, and
(via transducers and amplifiers) produces a concentration profile
from which, for example, the total quantity of the modifying agent
in the stream and the peak value of the concentration of the
modifying agent can be determined. The detector may measure the
presence or absence of a particular ingredient, and may produce
results in the form of a graph having a maximum or a minimum, each
of which is referred to herein as a "peak". Such detectors are
well-known to those skilled in the art. Additional useful
information may be available from the shape of the concentration
profile. For example the more symmetrical the peak (i.e. the
smaller the size of the tail representing material which is
extracted from the solid phase by the subsequent stream of liquid
carrier), the less reversible the interaction between the modifying
agent and the receptor. The center of gravity of the concentration
profile (i.e. the time at which half of the total detected amount
of the modifying agent has passed through the detector) shows the
average retention time of the modifying agent in the test chamber.
FIGS. 3, 5, 7 and 8 are typical plots of time against a measured
value which is directly proportional to the amount of the modifying
agent in the stream leaving the test or reference chamber.
[0121] Test Chambers
[0122] The test chamber can be any structure which will contain the
solid phase so that the stream of liquid carrier can be passed over
it. Hence, the test chamber typically has at least one inlet and at
least one outlet. The volume of test chamber will often be from
0.01 to 50 mL, preferably 0.2 to 5 mL. The test chamber will often
be cylindrical, with a diameter of 0.1 to 30 mm, preferably 1 to 8
mm, and a length of 1 to 150 mm, preferably 3 to 30 mm. However,
chambers of other shapes can be used, typically with the same
cross-sectional areas and lengths. The solid phase can be retained
within the test chamber in any way. Often the solid phase is packed
within the chamber so that the stream of liquid carrier flows
through the chamber at a linear velocity of 0.01 to 10, preferably
0.5 to 2, cm/min and/or a flow rate of 0.01 to 50, preferably 0.5
to 5, mL/min. The efficiency of the test chamber, with the solid
phase therein, is generally less than 80 theoretical plates,
preferably less than 50 theoretical plates, particularly less than
10 theoretical plates, and in many cases is 2-5, for example 3-4,
theoretical plates.
[0123] Reference Chambers
[0124] In some embodiments of the invention, the need to determine
absolute amounts of the modifying agent is eliminated by passing a
reference sample through a reference chamber. Hence, the reference
chamber has at least one inlet and at least one outlet. The
reference chamber is free of any substance which interacts with the
modifying agent, and in some cases is simply empty. That is, the
reference chamber has an essential absence of interferring solid
phase media--especially solid phase media that would interfere
substantially with the evaluation of the reference section. Hence,
the reference chamber can comprise materials that are interactively
inert with respect to the test sample, and particularly, with
respect to the modifying agent in the test sample. Examination of
the stream leaving the reference chamber provides a reference
standard from which the proportion of the modifying agent retained
in the test chamber can be ascertained. When the streams leaving
the test and reference chambers are examined by different
detectors, their void volumes are preferably substantially the same
(for example, from 0.8 to 1.2 times the arithmetic average of their
void volumes). When the liquid stream is split, and the two
resulting sub-streams are passed through the test and reference
chambers, recombined and examined by the same detector, the void
volumes of the loops containing the test and reference chambers can
be sufficiently different that the detector can distinguish between
the sections which have passed through the different chambers. The
sections need not be entirely distinct, provided that the amount of
modifying agent in each can be ascertained with sufficient accuracy
for the purposes of the invention. For example, the smaller of the
void volumes can be 0.5 to 0.8 times the larger of void volumes.
The void volume of a chamber is its total volume less the volume
occupied by any solid material therein.
[0125] Test Procedures
[0126] Generally the temperature, pressure, flow rates, sample
sizes, and other test conditions are the same in each of the test
procedures and the receptor, modifying agent and liquid carrier are
the same in each of the test procedures, except that in each test
procedure a different member of the library is used. Often all the
test procedures are substantially identical except for the use of
the different member of the library. However, this is not
necessary. For example, the detector can be programmed simply to
report that the test was negative if the peak height of the
concentration profile is over a particular value; or there can be a
feedback loop to change the rate of flow of the liquid carrier in
response to the results in earlier tests. In addition, the
invention includes the possibility that at least some of the test
procedures (i) differ only in the test conditions, or (ii) differ
in the test conditions and otherwise differ only in that each test
procedure uses a different member of the library, or (iii) use the
same conditions in the test procedures and otherwise differ in that
more than one of the receptor, modifying agent and liquid carrier
are changed (for example, when more than one of the receptor,
modifying agent and liquid carrier are selected from libraries,
different members are selected from two or three of the
libraries).
[0127] The flow rate of liquid carrier through the test chamber is
generally 1-10, preferably 2-6, especially 3-5, mL/min.
[0128] The conditions in the test chamber can be such that the
change in the pressure within the test chamber changes, or does not
change, the flow rate of liquid carrier. A graph of the flow rate
of the liquid carrier against the pressure in the test chamber
generally reaches a plateau at a pressure of less than 5000,
preferably less than 1000, particular less than 500, especially
less than 100, psi.
DESCRIPTION OF THE DRAWINGS
[0129] FIG. 1 is a diagrammatic illustration of an apparatus for
injecting a plurality of liquid units into a stream of a liquid
carrier, each unit comprising a modifying agent which is one of a
library. In FIG. 1, a liquid carrier 10 is stored in reservoir 11.
Pump 12 conveys liquid carrier from the reservoir 11 to an
injection port 13. Microtiter plate 14 and injection needle 15 are
part of a conventional autodilution and sampling robot. Members of
the library are retained separately on the microtiter plate 14 and
are sequentially removed by the injection needle 15 and diluted
with carrier liquid from the reservoir 11 to form units containing
one of the members of the library. The units are then transported
to the injection port 13, where they are injected sequentially into
the stream of liquid carrier. After passing through filter 16, the
stream of liquid carrier, carrying the units in distinct sections,
is conveyed down line 17.
[0130] FIG. 2 is a diagrammatic illustration of a first apparatus
for evaluating the interaction between a solid phase comprising a
receptor and a library of modifying agent. A stream of liquid
carrier is fed to the apparatus through line 17 (for example from
apparatus as illustrated in FIG. 1). The stream contains liquid
units in distinct sections, and each unit comprises a modifying
agent which is one of the library of second materials. The stream
passes through a test chamber 22 containing a solid phase
comprising the receptor, and then through a detector 24, before
being discarded. The test chamber has an inlet for receiving the
liquid carrier, and the liquid test sample in the distinct test
section thereof, in fluid communication with a an upstream source
of the carrier liquid. The test chamber also has an outlet for
discharging the carrier liquid after it has passed through the test
chamber, the outlet being in fluid communication with a detector
24. The detector 24 ascertains the amount of the modifying agent
remaining in the stream.
[0131] FIG. 3 is a typical plot of time against the response of the
detector 24 in FIG. 2, for a section of the stream containing a
modifying agent which is partially adsorbed by the receptor. In
FIG. 3 (and in the other Figures showing the response of the
detector), there is a base level which represents the response of
the detector to the liquid carrier alone (or to the liquid carrier
containing a very small amount of modifying agent which results
from the extraction of modifying agent which was deposited on the
solid phase in earlier test procedures). The amount of modifying
agent in the stream can be calculated from the area under the
curve, above the base level. The height of the peak and the shape
of the curve can also provide other information about the
interaction between the modifying agent and the first solid
material. If the amount of the modifying agent originally present
in the unit is known, then the amount retained in the test chamber
can be calculated from a plot of the type shown in FIG. 3.
[0132] FIG. 4 is a diagrammatic illustration of a second apparatus
for evaluating the interaction between a receptor and a library of
modifying agents. This apparatus makes it possible to determine the
percentage of the modifying agent retained by the receptor and is
not dependent on the amount of modifying agent originally present
in the sample. A stream of liquid carrier is fed to the apparatus
through a main passageway, such as line 17 (for example from
apparatus as illustrated in FIG. 1). The stream contains liquid
units in distinct sections and each unit comprises a modifying
agent which is one of the library. The main passageway provides
fluid communication between a liquid carrier source (e.g., such as
reservoir 11 as shown in FIG. 1) and a flow splitter, such that the
liquid unit is divided by the valve 41 into first and second
substreams. The splitter is illustrated in FIG. 4 as switching
valve 41, which itself is in fluid communication with two or more
branch passageways. As illustrated, switching valve 41 is in fluid
communication with a first loop defined by a first branch
passageway providing fluid communication to an inlet of the test
chamber 42, the test chamber 42 itself, and second branch
passageway providing fluid communication from an outlet of the test
chamber 42 to a common exit passageway. Switching valve 41 is also
in fluid communication with a second loop defined by a third branch
passageway providing fluid communication to an inlet of a reference
chamber 43, the reference chamber 43 itself, and a fourth branch
passageway providing fluid communication from an outlet of the
reference chamber 43 to the common exit passageway. As each
unit-carrying section of the liquid stream reaches the switching
valve 41, the valve passes a first part of the section containing
half of the modifying agent through the first loop containing test
chamber 42 and a second part of the section containing the other
half of the modifying agent through the second loop containing
reference chamber 43. The order in which this is done is not
important. (That is, for example, the first part of each
unit-carrying section could be directed to the reference chamber
loop, and the second part of each unit-carrying section could be
directed to the test chamber loop. The test chamber 42 contains a
solid phase comprising the receptor. The reference chamber 43 is
empty or contains a material which does not interact with the
modifying agent (that is, does not not interact to a degree having
a substantial effect on the comparison). The void volumes of the
first and second loops are preferably substantially the same, but
can also be different in some embodiments. For example, the void
volumes of the loops containing the chambers 42 and 43 can be
sufficiently different that, when the first and second sub-streams
are recombined, the detector 44 can ascertain, for each section,
the amounts of the modifying agent remaining in the parts of the
recombined stream which correspond to the first and second
sub-streams. Preferably, the first and second parts of the stream
return sequentially from the chambers 42 and 43 respectively to the
valve 41 and are recombined in a common exit passageway to form a
common exit carrier stream, and are then passed sequentially
through a detector 44, before being discarded. The detector 44 is
in fluid communication with the common exit passageway and can be
used to ascertain the amounts of the modifying agent remaining in
the first and second parts of the stream respectively.
[0133] FIG. 5 is a typical plot of time against the concentration
of the modifying agent, as measured by the detector 44 in FIG. 4,
for a section of the stream containing a modifying agent which is
partially adsorbed by the receptor. In this plot, the first curve,
containing the sharper and higher peak, is for the modifying agent
which passed through the reference chamber, and the second curve,
containing the broader and lower peak, is for the modifying agent
which passed through the test chamber. If the area under the first
curve is A5a, and the area under the second curve is A5b, the
percentage of the modifying agent retained in the test chamber is
(A5a-A5b)/A5a. The height of the peak and the shape of the second
curve can also provide other information about the interaction
between the modifying agent and the receptor.
[0134] FIG. 6 is a diagrammatic illustration of a third apparatus
for evaluating the interaction between a receptor and a library of
modifying agents. Like the apparatus shown in FIG. 4, this
apparatus makes it possible to determine the percentage of the
modifying agent retained in the test chamber and is not dependent
on the amount of modifying agent originally present in the sample.
Furthermore, unlike the apparatus of FIG. 4, the flow splitter is a
stream-splitting junction 61 rather than a switching valve (41,
FIG. 4). The junction 61 will consistently direct one half of the
modifying agent through the test chamber and the other half through
the reference chamber. A stream of liquid carrier is fed to the
apparatus of FIG. 6 through a main passageway, such as for example
line 17 (for example from apparatus as illustrated in FIG. 1). The
liquid carrier carries liquid units in distinct sections, and each
unit comprises a modifying agent which can be one of the library
members. The main passageway provides fluid communication between a
liquid carrier source (e.g., such as reservoir 11 as shown in FIG.
1) and a flow splitter, such that the liquid unit is divided by the
stream-splitting junction 61 into first and second substreams.
Junction 61 is itself is in fluid communication with two or more
branch passageways. Specifically, as illustrated, when the liquid
stream reaches junction 61, the stream splits into a first
sub-stream and a second sub-stream. Junction 61 is in fluid
communication with a first loop defined by a first branch
passageway providing fluid communication to an inlet of the test
chamber 62, the test chamber 62 itself, and second branch
passageway providing fluid communication from an outlet of the test
chamber 62 to a common exit passageway. Junction 61 is also in
fluid communication with a second loop defined by a third branch
passageway providing fluid communication to an inlet of a reference
chamber 63, the reference chamber 63 itself, and a fourth branch
passageway providing fluid communication from an outlet of the
reference chamber 63 to the common exit passageway. In operation,
the first sub-stream passes through the first loop containing test
chamber 62 and the second sub-stream passes through the second loop
containing reference chamber 63. The test chamber 62 contains a
solid phase comprising the receptor. The reference chamber 63 is
empty or contains a material which does not interact with the
modifying agent. The first and second sub-streams are recombined
after leaving the chambers 62 and 63 respectively, into a common
exit passageway in fluid communication with a detector 64. The
recombined stream is passed through the detector 64, before being
discarded. The void volumes of the loops containing the chambers 62
and 63 can be the same, but are preferably sufficiently different
that, when the first and second sub-streams are recombined, the
detector 64 can ascertain, for each section, the amounts of the
modifying agent remaining in the parts of the recombined stream
which correspond to the first and second sub-streams.
[0135] FIG. 7 is a typical plot of time against the concentration
of the modifying agent, as measured by the detector 64 in FIG. 6,
for a section of the stream containing a liquid sample in which the
modifying agent is partially adsorbed by the receptor. In this
plot, the first curve, containing the sharper and higher peak, is
for the modifying agent remaining in the part of the stream which
passed through the empty chamber, and the second curve, containing
the broader and lower peak, is for the modifying agent remaining in
the part of the stream which passed through the chamber containing
the first solid material. In order to ascertain from FIG. 7 the
percentage of the modifying agent retained in the test chamber, it
is necessary to know the flow splitting ratio between the test
chamber and the reference chamber. The flow splitting ratio can be
ascertained by replacing the library material by an additive which
does not interact with the solid phase in the test chamber.
[0136] FIG. 8 is a typical plot of the concentration of such an
additive, as measured by the detector 64. In this plot, the first
curve, containing the sharper and higher peak, is for the additive
which passed through the empty chamber, and the second curve,
containing the broader and lower peak, is for the additive which
passed through the chamber containing the first solid material. The
flow splitting ratio (FSR) is the ratio of the area under the first
curve to the area under the second curve. If the area under the
first curve in FIG. 7 is A7a, the area under the second curve in
FIG. 7 is A7b, the area under the first curve in FIG. 8 is A8a, and
the area under the second curve in FIG. 8 is A8b, the percentage of
the modifying agent retained in the test chamber is
100-[100.times.A7a/A7b .times.A8a/A8b]. The shape of the second
curve can also provide information about the rate of interaction
between the modifying agent and the first solid material.
[0137] FIG. 9 is a diagrammatic illustration of a fourth apparatus
for evaluating the interaction between a receptor and a library of
modifying agents. In FIG. 9, a first stream of carrier liquid is
fed through line 17a (for example from apparatus as illustrated in
FIG. 1). The stream contains liquid samples in distinct sections
and each sample comprises a modifying agent which is one of the
library. The stream passes through a test chamber 92 which contains
a solid phase comprising the receptor, and then through a detector
94a, before being discarded. A second stream, identical to the
first stream, is fed through line 17b (for example from apparatus
as illustrated in FIG. 1), through a reference chamber 93 which is
empty or contains a material which does not interact with the
modifying agent, and then through a detector 94b, before being
discarded.
[0138] FIG. 10 is a diagrammatic illustration of a fifth apparatus
for evaluating the interaction between a receptor and a library of
modifying agents. In FIG. 10, a stream of carrier liquid is fed
through line 17 (for example from apparatus as illustrated in FIG.
1). The stream contains liquid samples in distinct sections and
each sample comprises a modifying agent which is one of the
library. The stream passes consecutively through a reference
chamber 103 which is empty or contains a material which does not
interact with the modifying agent, a first detector 104b, a test
chamber 102 which contains a solid phase comprising the receptor,
and second detector 104a, before being discarded.
[0139] The proportion of modifying agent retained in the test
chamber in FIGS. 9 and 10 can be determined in the way described
above for FIGS. 4 and 5.
[0140] FIG. 11 is a diagrammatic illustration of a sixth apparatus
for evaluating the interaction between a receptor and a library of
modifying agents. In a stream carrier liquid is fed through line 17
(for example from apparatus as illustrated in FIG. 1). The stream
contains liquid samples in distinct sections and each sample
comprises a modifying agent which is one of the library. The
sample-carrying stream is fed to a valve 111 which sequentially
directs successive sub-streams, each sub-stream containing a single
sample, to a loop containing a reference chamber 113 and to one of
a plurality of loops each containing a test chamber 112 a,b,c . . .
Four test chambers are shown in FIG. 11, but any appropriate
number, for example from 2 to 64, could be used. Each of the test
chambers contains a solid phase comprising the receptor. The
reference chamber is empty or contains a material which does not
interact with the modifying agent. The liquid sub-streams, after
passing through the reference or test chamber, are recombined into
a single combined stream containing substantially separate
evaluation sections corresponding to the samples. The combined
stream is passed through a detector 114, which detects the amount
of modifying agent in each of the evaluation sections in turn.
[0141] The apparatus of the type shown in FIG. 11 can also be used
to evaluate the interaction between a modifying agent and a library
of receptors. In this case, the liquid samples in the stream are
identical, and the solid phase in each of the test chambers
comprises one of the library of receptors. In apparatus of the type
shown in FIG. 11, the single detector 114 could be replaced by a
plurality of detectors, each examining a single sub-stream or a
suitable proportion of the sub-streams.
EXAMPLES
[0142] The invention as illustrated in the following Examples.
Example 1
[0143] Apparatus of the type shown in FIGS. 1 and 4 was used to
evaluate the interaction between (i) a copolymer of
N-(3-(dimethylamino)propyl)met- hacrylamide, styrene and acrylic
acid, in a molar ratio of 98.3:0.1:0.12, and (ii) a cotton fabric.
The cotton fabric was a plain white untreated cotton sheet, about
50 mm wide, rolled tightly and inserted into the column. The pump
was a high performance liquid chromatography (HPLC) pump sold by
Waters under the tradename Model 515. The injection port was a
two-position injection valve sold by Valco under the tradename
Model EHMA. The valve was equipped with two 50 microliter injection
loops. The multiposition switching valve was a valve sold by Valco
under the tradename Model EMTMA-CE.
[0144] The liquid carrier was a 1% by weight solution of ammonium
acetate in deionized distilled water to which had been added
sufficient ammonium hydroxide to bring the pH to 10.5 and 1% by
weight of a stock detergent solution. The stock detergent solution
was composed of ionized distilled water to which had been added 0.6
g/L of a proprietary linear alkane sulfonate, 0.4 g/L. of a
proprietary non-ionic surfactant, 1.25 g/L. of sodium carbonate,
1.1 g/L. of pentasodium triphosphate, 1.0 g/L. sodium chloride and
0.0882 g/L. of calcium chloride dihydrate. The flow rate of the
liquid carrier was about 4 mL/min.
[0145] The liquid sample contained 2 mg/mL of the copolymer
dissolved in the liquid carrier. The time between the injections
was 2 minutes.
[0146] The test chamber was a stainless steel column having a
height of 50 mm and an internal diameter of 7.5 mm, with the cotton
fabric tightly rolled to completely fill the column. The reference
chamber was a proprietary on-line pre-column HPLC metal filter sold
by Valco Instruments. The passageways connected to the first and
second chambers were sized to insure that the void volumes of the
first and second loops were substantially the same.
[0147] The detector was an evaporative light scattering detector
sold by Polymer Laboratories under the tradename Model ELSD-1000.
The results obtained by the detector are shown in FIG. 5.
Example 2
[0148] Apparatus of the type shown in FIGS. 1 and 6 was used to
evaluate the interaction between (i) the cotton fabric used in
Example 1 and (ii) (a) the copolymer used in Example 1, and (b) a
library of 96 different polymers each composed of units derived
from three monomers. Details of the polymers in the library are
shown in Table 1 below, which gives the proportions by weight of
the units derived from each of the three monomers, the first figure
being for units derived from 4-acetoyloxymethyl) styrene, the
second figure being for units derived from 2-hydroxyethyl
methacrylate, and the third figure being for units derived from
2-(dimethylamino) ethyl methacrylate.
[0149] The autodilution and sampling robot, the pump, the injection
port and the detector were as described in Example 1. The test
chamber and the reference chamber were as described in Example 1,
but the passageways to and from them were sized so as to insure
that, in the combined stream entering the detector, the samples
from the respective chambers were substantially separated so that
they could be ascertained seperately by the detector.
[0150] The liquid carrier, and its flow rate, were as in Example
1.
[0151] The liquid sample contained about 2 mg/mL of the polymer
dissolved in a liquid carrier, and each sample was about 50
microliters. The time between the injections was 3 minutes.
[0152] The results obtained by the detector are shown in FIG.
5.
1 TABLE 1 1 2 3 4 5 6 7 8 9 10 11 12 1 30 30 30 30 30 30 30 30 30
30 30 30 69 64 58 53 47 41 36 30 25 19 13 41 1 6 12 18 23 29 34 40
46 51 57 29 2 25 25 25 25 25 25 25 25 25 25 25 25 74 68 62 56 50 44
38 32 26 20 14 44 1 7 13 19 25 31 37 43 49 55 61 31 3 20 20 20 20
20 20 20 20 20 20 20 20 79 73 66 60 54 47 41 34 28 22 15 47 1 7 14
20 26 33 39 46 52 58 65 33 4 15 15 15 15 15 15 15 15 15 15 15 15 84
77 71 64 57 50 43 37 30 23 16 50 1 8 14 21 28 35 42 48 55 62 69 35
5 10 10 10 10 10 10 10 10 10 10 10 10 89 82 75 68 60 53 46 39 32 24
17 53 1 8 15 23 30 37 44 51 59 66 73 37 6 8 8 8 8 8 8 8 8 8 8 8 8
92 84 77 69 62 55 47 40 32 25 18 55 1 8 16 23 31 38 45 53 60 68 75
38 7 5 5 5 5 5 5 5 5 5 5 5 5 94 86 79 71 64 56 48 41 33 26 18 56 1
9 16 24 31 39 47 54 62 69 77 39 8 3 3 3 3 3 3 3 3 3 3 3 3 97 89 81
73 65 58 50 42 34 26 19 58 1 9 17 24 32 40 48 56 63 71 79 40
[0153] In order to determine the flow splitting ratio of the
apparatus, a sample of poly(N-vinylpyrrolidone), a polymer which
does not interact with the cotton fabric, is passed through the
apparatus. FIG. 8 shows the results recorded by the detector. The
area A3 under the narrow, higher peak, from the stainless-steel
filter, was about 43,000. The area A4 under the broad, lower peak,
from the first chamber containing the cotton fabic, was about
62,800. Thus the flow splitting ratio was 0.685.
[0154] A sample of the copolymer used in Example 1 was then passed
through the apparatus. FIG. 7 shows the results recorded by the
detector. The area A1 under the narrow, higher peak, from the
stainless-steel filter, was about 65,900. The area A2 under the
broader, lower peak, from the first chamber containing the cotton
fabric, was about 70,500. The percentage of the copolymer remaining
in the liquid stream which had passed through the first chamber
was, therefore, about 73%, i.e. (70500/65900).times.0.685, and the
percentage that had been retained by the cotton fabric was about
27%.
[0155] Samples of each of the 96 members of the library were then
passed through the apparatus and the percentage of each retained by
the cotton fabric was calculated. The results are shown in Table 2
below.
2 TABLE 2 1 2 3 4 5 6 7 8 9 10 11 12 1 0 10 18 3 38 40 41 46 59 45
32 0 2 0 0 23 48 47 46 54 52 58 45 50 0 3 0 16 13 45 39 41 58 48 56
44 28 0 4 0 13 9 20 31 36 27 15 32 36 16 4 5 0 1 5 0 2 16 6 8 19 21
16 19 6 0 0 0 0 0 0 4 5 7 9 24 0 7 0 0 0 0 0 0 3 0 15 17 22 46 8 0
0 8 0 0 0 4 6 5 18 23 0
[0156] In light of the detailed description of the invention and
the examples presented above, it can be appreciated that the
several objects of the invention are achieved.
[0157] The explanations and illustrations presented herein are
intended to acquaint others skilled in the art with the invention,
its principles, and its practical application. Those skilled in the
art may adapt and apply the invention in its numerous forms, as may
be best suited to the requirements of a particular use.
Accordingly, the specific embodiments of the present invention as
set forth are not intended as being exhaustive or limiting of the
invention.
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