U.S. patent application number 13/826198 was filed with the patent office on 2014-09-18 for compositions and methods for performing assays.
This patent application is currently assigned to Palo Alto Research Center Incorporated. The applicant listed for this patent is PALO ALTO RESEARCH CENTER INCORPORATED. Invention is credited to Peter KIESEL, Joerg MARTINI, Michael I. RECHT, Francisco E. TORRES.
Application Number | 20140273009 13/826198 |
Document ID | / |
Family ID | 50343623 |
Filed Date | 2014-09-18 |
United States Patent
Application |
20140273009 |
Kind Code |
A1 |
RECHT; Michael I. ; et
al. |
September 18, 2014 |
COMPOSITIONS AND METHODS FOR PERFORMING ASSAYS
Abstract
The disclosure relates to compositions for use in assays, the
compositions comprising at least one latent fluorophore including
at least one enzyme-reactive quenching group and a conjugative
group; and a support connectable to the latent fluorophore by the
conjugative group. The disclosure further relates to methods of
measuring the presence and/or concentration of an analyte, as well
as methods of measuring the relative activity of at least two
enzymes.
Inventors: |
RECHT; Michael I.; (Mountain
View, CA) ; KIESEL; Peter; (Palo Alto, CA) ;
MARTINI; Joerg; (San Francisco, CA) ; TORRES;
Francisco E.; (San Jose, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PALO ALTO RESEARCH CENTER INCORPORATED |
Palo Alto |
CA |
US |
|
|
Assignee: |
Palo Alto Research Center
Incorporated
Palo Alto
CA
|
Family ID: |
50343623 |
Appl. No.: |
13/826198 |
Filed: |
March 14, 2013 |
Current U.S.
Class: |
435/7.5 ; 435/21;
435/23; 435/26 |
Current CPC
Class: |
G01N 2333/90209
20130101; C12Q 1/00 20130101; G01N 21/6486 20130101; G01N 33/582
20130101 |
Class at
Publication: |
435/7.5 ; 435/26;
435/23; 435/21 |
International
Class: |
G01N 21/64 20060101
G01N021/64 |
Claims
1. A composition comprising: a. at least one latent fluorophore
comprising: i. at least one enzyme-reactive quenching group, and
ii. at least one conjugative group; and b. at least one support
connectable to the latent fluorophore.
2. The composition of claim 1, wherein the at least one support is
connected to the latent fluorophore by the at least one conjugative
group.
3. The composition of claim 1, wherein the at least one support is
chosen from microspheres.
4. The composition of claim 3, wherein the at least one support is
magnetic.
5. The composition of claim 1, wherein the at least one conjugative
group is chosen from bioconjugative groups, PEG linkers, biotin
groups, and combinations thereof.
6. The composition of claim 1, where the at least one latent
fluorophore is chosen from trimethyl lock fluorophores.
7. The composition of claim 6, wherein the trimethyl lock
fluorophore is chosen from compounds of Chemical Formula 1,
Chemical Formula 2, Chemical Formula 3, and combinations thereof:
##STR00005##
8. The composition of claim 1, wherein the at least one latent
fluorophore comprises at least one diaphorase-reactive quenching
group that is releasable from the latent fluorophore by a
diaphorase.
9. A method of detecting and/or measuring the concentration of an
analyte in a sample, the method comprising: a. providing a
composition comprising: i. at least one latent fluorophore
comprising at least one enzyme-reactive quenching group and at
least one conjugative group, and ii. at least one support
connectable to the at least one latent fluorophore by the at least
one conjugative group; b. providing a test sample and a reference
sample; c. contacting the test sample with the composition, at
least one first unquenching enzyme capable of releasing the
enzyme-reactive quenching group from the latent fluorophore, and at
least one second enzyme capable of reacting with the analyte; and
d. contacting the reference sample with the latent fluorophore
composition and the at least one first unquenching enzyme.
10. The method of claim 9, further comprising: e. measuring the
fluorescence signal of the test sample and the fluorescence signal
of the reference sample.
11. The method of claim 10, further comprising: f. comparing the
fluorescence signal of the test sample with the fluorescence signal
of the reference sample.
12. The method of claim 11, further comprising: g. connecting the
latent fluorophore to the support, and h. isolating the latent
fluorophore composition from the sample.
13. The method of claim 9, wherein the at least one support is
chosen from microspheres.
14. The method of claim 13, wherein the at least one support is
magnetic.
15. The method of claim 9, wherein the at least one first
unquenching enzyme and the at least one second enzyme are the
same.
16. The method of claim 9, wherein the at least one second enzyme
is chosen from diaphorase, esterase, phosphatase, pyruvate oxidase,
and combinations thereof.
17. The method of claim 9, wherein the at least one first
unquenching enzyme is capable of generating NADH or NADPH when
reacted with the analyte.
18. The method of claim 9, wherein the at least one first enzyme is
chosen from a dehydrogenase, a protease, a phosphatase, and
combinations thereof.
19. The method of claim 9, wherein the at least one conjugative
group is chosen from bioconjugative groups, PEG linkers, biotin
groups, and combinations thereof.
20. The method of claim 9, wherein the at least one latent
fluorophore is chosen from trimethyl lock fluorophores.
21. The method of claim 20, wherein the trimethyl lock fluorophore
is chosen from compounds of Chemical Formula 1, Chemical Formula 2,
Chemical Formula 3, and combinations thereof: ##STR00006##
22. A method of measuring the activities of at least two enzymes in
a sample, the method comprising: a. providing a first fluorophore
composition comprising: i. at least one first enzyme-reactive
latent fluorophore comprising at least one first enzyme-reactive
quenching group and at least one conjugative group, and ii. at
least one support connectable to the at least one first latent
fluorophore by at least one conjugative group; b. providing a
second fluorophore composition comprising: i. at least one second
enzyme-reactive latent fluorophore comprising at least one second
enzyme-reactive quenching group and at least one conjugative group,
wherein the at least one second enzyme-reactive latent fluorophore
is different from said first enzyme-reactive latent fluorophore in
said first fluorophore composition, and ii. at least one support
connectable to the at least one first latent fluorophore by at
least one conjugative group; c. providing a test sample to be
analyzed and a reference sample to be analyzed; d. contacting the
test sample with the first and second latent fluorophore
compositions, at least one first unquenching enzyme capable of
releasing the enzyme-reactive quenching group from the first latent
fluorophore, and at least one second unquenching enzyme capable of
releasing the enzyme-reactive quenching group from the second
latent fluorophore; e. contacting the reference sample with the
first and second latent fluorophore compositions.
23. The method of claim 22, further comprising: f. measuring the
fluorescence signal of the test sample and the fluorescence signal
of the reference sample.
24. The method of claim 23, further comprising: g. comparing the
fluorescence signal of the test sample with the fluorescence signal
of the reference sample.
25. The method of claim 24, further comprising: h. distinguishing
the fluorescence signal of the at least one first enzyme from the
fluorescence signal of the at least one second enzyme.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to compositions and methods
for performing assays. In various embodiments, the disclosure
relates to latent fluorophores linked to a support, and their use
in assays.
BACKGROUND
[0002] Assays are commonly used for qualitatively assessing or
quantitatively measuring the presence, amount, or functional
activity of a target entity (the analyte). The analyte may, in
various assays, be a drug, biochemical substance, or biological
cell. By way of example, certain medical conditions may utilize an
assay to screen for the presence of a target entity, or measure the
amount of the target entity, in bodily fluids such as urine,
saliva, or blood.
[0003] For example, phenylketonuria (PKU) is a genetic condition
characterized by the inability to metabolize the amino acid
phenylalanine. As a result, individuals exhibiting this condition
must control their intake of phenylalanine, and some use blood
tests (assays) to monitor the amount of phenylalanine (the analyte)
in their blood serum. Likewise, blood serum levels of phenylalanine
may be used to screen for, or diagnose, PKU.
[0004] Methods for laboratory quantitation of phenylalanine levels
in serum include a variety of enzyme assays having colorimetric or
fluorescence signals. For example, latent fluorophores are
compounds with intense fluorescence that is revealed by a
user-designated chemical reaction. Latent fluorophores using a
trimethyl lock mechanism to cloak or quench fluorescence can be
used to detect levels of phenylalanine by coupling the reaction of
phenylalanine dehydrogenase to a diaphorase-activated trimethyl
lock quenched latent fluorophore. The fluorescence intensity of the
sample may be substantially directly proportional to the amount of
phenylalanine present. This technique can provide a broad dynamic
range, but requires several blood sample processing steps to
improve the signal-to-noise ratio, such as isolating the serum from
whole blood and deproteinizing the serum.
[0005] The above-mentioned processing steps require equipment and
techniques that are not ideal for adaptation to at-home testing
devices. At-home phenylalanine testing devices are desirable
though, as they may allow for more frequent and convenient
monitoring of blood phenylalanine levels.
[0006] There is, therefore, a need for at-home phenylalanine
testing devices capable of testing whole blood, and capable of
supplying a measurement signal having a broad dynamic range and a
high signal-to-noise ratio. Accordingly, there is a need for assays
that may be useful in such at-home devices, compositions for use in
the assays, and methods of measuring the concentration of an
analyte, such as phenylalanine, in a sample, such as whole blood.
More generally, there is a need for compositions and methods of
detecting and/or measuring an analyte in a sample.
SUMMARY
[0007] The disclosure relates, in various embodiments, to
compositions for use in assays. The compositions may, in various
exemplary embodiments, comprise at least one latent fluorophore
comprising at least one enzyme-reactive quenching group and at
least one conjugative group; and a support connectable to the
latent fluorophore, for example by at least one conjugative
group.
[0008] Further embodiments of the disclosure relate to methods for
measuring the concentration and/or presence of an analyte in a
sample. In various exemplary embodiments, the methods comprise one
or more steps chosen from:
[0009] a. providing a fluorophore composition comprising: [0010] i.
at least one enzyme-reactive latent fluorophore comprising at least
one enzyme-reactive quenching group and at least one conjugative
group, and [0011] ii. a support connectable to the latent
fluorophore by at least one conjugative group;
[0012] b. providing a test sample to be analyzed and a reference
sample to be analyzed;
[0013] c. contacting the test sample with the latent fluorophore
composition, at least one first unquenching enzyme capable of
releasing the enzyme-reactive quenching group from the latent
fluorophore, and at least one second enzyme capable of reacting
with the analyte;
[0014] d. contacting the reference sample with the latent
fluorophore composition and the at least one first unquenching
enzyme;
[0015] e. measuring the fluorescence signal of the test sample and
the fluorescence signal of the reference sample; and
[0016] f. comparing the fluorescence signal of the test sample with
the fluorescence signal of the reference sample.
[0017] Further embodiments of the disclosure relate to methods for
measuring the activity and/or presence of an enzyme in a test
sample including an analyte. In various exemplary embodiments, the
methods comprise one or more steps chosen from:
[0018] a. providing a fluorophore composition comprising: [0019] i.
at least one enzyme-reactive latent fluorophore comprising at least
one enzyme-reactive quenching group and at least one conjugative
group, and [0020] ii. a support connectable to the latent
fluorophore by at least one conjugative group;
[0021] b. providing a test sample to be analyzed and a reference
sample to be analyzed, wherein the reference sample contains a
known quantity of the analyte;
[0022] c. contacting the test sample with the latent fluorophore
composition, at least one first unquenching enzyme capable of
releasing the enzyme-reactive quenching group from the latent
fluorophore, and at least one second enzyme capable of reacting
with the analyte;
[0023] d. contacting the reference sample with the latent
fluorophore composition and the at least one first unquenching
enzyme;
[0024] e. measuring the fluorescence signal of the test sample and
the fluorescence signal of the reference sample; and
[0025] f. comparing the fluorescence signal of the test sample with
the fluorescence signal of the reference sample.
[0026] Further exemplary embodiments of the disclosure relate to
methods for measuring the activities of at least two enzymes in a
sample, for example a multiplexed assay. In various embodiments,
the methods comprise one or more steps chosen from:
[0027] a. providing a first fluorophore composition comprising:
[0028] i. at least one first enzyme-reactive latent fluorophore
including at least one first enzyme-reactive quenching group and at
least one conjugative group, and [0029] ii. at least one support
connectable to the at least one first latent fluorophore by at
least one conjugative group;
[0030] b. providing a second fluorophore composition comprising:
[0031] i. at least one second enzyme-reactive latent fluorophore
including at least one second enzyme-reactive quenching group and
at least one conjugative group, wherein the at least one second
enzyme-reactive latent fluorophore is different from said first
enzyme-reactive latent fluorophore in said first fluorophore
composition, and [0032] ii. at least one support connectable to the
at least one first latent fluorophore by at least one conjugative
group;
[0033] c. providing a test sample to be analyzed and a reference
sample to be analyzed;
[0034] d. contacting the test sample with the first and second
latent fluorophore compositions, at least one first unquenching
enzyme capable of releasing the enzyme-reactive quenching group
from the first latent fluorophore, and at least one second
unquenching enzyme capable of releasing the enzyme-reactive
quenching group from the second latent fluorophore;
[0035] e. contacting the reference sample with the first and second
latent fluorophore compositions;
[0036] f. measuring the fluorescence signals of the test sample and
the fluorescence signals of the reference sample; and
[0037] g. comparing the fluorescence signals of the test sample
with the fluorescence signals of the reference sample.
[0038] Yet a further exemplary embodiment of the disclosure relates
to a kit comprising a composition, where the composition comprises
at least one latent fluorophore comprising at least one
enzyme-reactive quenching group and at least one conjugative group,
and at least one support connectible to the at least one latent
fluorophore via at least one conjugative group.
[0039] Additional objects and advantages will be set forth in part
in the description which follows, and in part will be obvious from
the description, or may be learned by practice of various
embodiments according to the disclosure.
[0040] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only, and are not restrictive of the invention, as
claimed.
[0041] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several
exemplary embodiments of the disclosure and, together with the
description, serve to explain the principles of the disclosure.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIG. 1 is an exemplary schematic of a coupled assay for
detecting an analyte having a specific dehydrogenase according to
an embodiment of the disclosure;
[0043] FIG. 2 is an exemplary schematic of a coupled assay for
detecting phenylalanine according to an embodiment of the
disclosure;
[0044] FIG. 3 is an exemplary schematic of a direct assay for
detecting an analyte;
[0045] FIG. 4a is an exemplary reaction schematic showing a latent
fluorescent compound revealed by a trimethyl lock reaction
according to an embodiment of the disclosure;
[0046] FIG. 4b is an exemplary reaction schematic showing a latent
fluorescent compound revealed by a trimethyl lock reaction and
connected to a microsphere with a biotin-streptavidin linkage
according to an embodiment of the disclosure;
[0047] FIG. 5 is an exemplary reaction schematic showing a latent
fluorescent compound revealed by a double trimethyl lock reaction
according to an embodiment of the disclosure;
[0048] FIG. 6 is a flowchart showing the steps for a general
dehydrogenase differential assay according to an exemplary
embodiment of the disclosure;
[0049] FIG. 7 is a flowchart showing the steps for a phenylalanine
dehydrogenase differential assay according to an exemplary
embodiment of the disclosure;
[0050] FIG. 8 is an exemplary reaction schematic showing a latent
fluorescent biotin-containing compound revealed by a trimethyl lock
reaction and subsequently linked to a streptavidin-coated
microsphere, according to an embodiment of the disclosure;
[0051] FIG. 9a is a flowchart showing the steps for a general
enzymatic multiplexed differential assay using fluorophores having
different emission properties, according to an exemplary embodiment
of the disclosure;
[0052] FIG. 9b is a flowchart showing the steps for an enzymatic
multiplexed differential assay using fluorescent microspheres
having different emission properties, according to an exemplary
embodiment of the disclosure;
[0053] FIG. 9c is a flowchart showing the steps for an enzymatic
multiplexed differential assay using fluorescent microspheres
having different sizes, according to an exemplary embodiment of the
disclosure;
[0054] FIG. 10a is a flowchart showing the steps for a general
enzymatic multiplexed differential screening assay using
fluorophores having different emission properties, according to an
exemplary embodiment of the disclosure;
[0055] FIG. 10b is a flowchart showing the steps for a general
enzymatic multiplexed differential screening assay using
fluorescent microspheres having different emission properties,
according to an exemplary embodiment of the disclosure; and
[0056] FIG. 10c is a flowchart showing the steps for a general
enzymatic multiplexed differential screening assay using
fluorescent microspheres having different sizes, according to an
exemplary embodiment of the disclosure.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0057] The disclosure relates, in various exemplary embodiments, to
compositions for use in an assay. In various exemplary embodiments,
the measurement from the assay is used to detect levels of
phenylalanine in whole blood.
[0058] In various exemplary embodiments, the compositions comprise
at least one latent fluorophore and at least one support. In
further exemplary embodiments, the composition is an
enzyme-reactive latent fluorophore cloaked or quenched by an
enzyme-reactive group, and the latent fluorophore is linked to the
support. In at least certain exemplary embodiments, the
enzyme-reactive quenching group may be released by a trimethyl lock
mechanism, and the enzyme-reactive latent fluorophore may be
revealed or unquenched by an enzymatic reaction. The resulting
fluorescence intensity of the sample may, in certain embodiments,
be proportional to the amount of enzyme activity. In various
embodiments, the support may be chosen from microspheres,
microbeads, or beads.
[0059] In various exemplary embodiments, the assay using the
composition comprising the at least one latent fluorophore and at
least one support may exhibit a broad dynamic range and high
signal-to-noise ratio, although it should be noted that a broad
dynamic range and high signal-to-noise ratio are not required.
[0060] In at least certain embodiments, the latent fluorophore is
attached to the support with a linking group. By way of
non-limiting example, the linker may be chosen from polyethylene
glycol ("PEG") linkers. In certain embodiments, the latent
fluorophore is attached to the support with at least one conjugate.
In at least one embodiment, the conjugate may be biotin, and the
support may be coated with a suitable complementary functional
group, for example streptavidin.
[0061] According to various embodiments of the disclosure,
compositions comprising the latent fluorophore may be contacted
with at least one enzyme. In various exemplary embodiments, the
latent fluorophore is attached to the support prior to the
enzymatic reaction; in other exemplary embodiments, it is attached
after the enzymatic reaction.
[0062] In at least certain embodiments according to the disclosure,
the fluorescence of the composition used in the analyte assay can
be determined by measuring bulk fluorescence. In various
embodiments, the fluorescence is measured using flow cytometry or
spatially modulated fluorescence detection technology. However, it
should be noted that any method of measuring fluorescence known to
those skilled in the art may be used according to various
embodiments of the disclosure.
Latent Fluorophores
[0063] As used herein, the term "latent fluorophore," and
variations thereof, means a chemical compound capable of exhibiting
fluorescence, which may be revealed by a designated chemical
reaction. As used herein, the terms "quench," "quenched," and
variations thereof mean a suppression or concealment of some, all,
or substantially all fluorescence of a fluorophore by one or more
functional groups, referred to herein as "quenching groups." In its
quenched state, a latent fluorophore exhibits no or substantially
no fluorescence, or a decreased amount of fluorescence compared to
its unquenched state.
[0064] The quenching group may quench or partially quench a latent
fluorophore by various chemical or physical means. In certain
embodiments, the quenching group completes a system of conjugated
double bonds when altered and/or released from the latent
fluorophore, thus allowing fluorescence. The quenching group may be
altered and/or released from the latent fluorophore by various
chemical or physical means. In certain embodiments, for example,
the quenching group may be altered and/or released from the latent
fluorophore by a predetermined chemical reaction. In further
exemplary embodiments, the predetermined chemical reaction may be
catalyzed by an enzyme and/or an enzyme cascade. Nonlimiting
examples of enzymes capable of catalyzing the unquenching or
unlocking of a latent fluorophore include diaphorase and pyruvate
oxidase.
[0065] Among the latent fluorophores that may be used according to
exemplary embodiments of the disclosure include, but are not
limited to, fluorophores quenched by a quenching group releasable
by a trimethyl lock mechanism (trimethyl lock fluorophores), and
fluorophores quenched by an enzyme-activated trimethyl lock
mechanism, such as diaphorase-activated trimethyl lock
fluorophores.
[0066] Latent fluorophores that may be revealed, unquenched, or
unlocked by an enzymatic reaction, referred to herein as
"enzyme-reactive latent fluorophores," comprise an enzyme-reactive
quenching group that may be altered and/or released from the latent
fluorophore upon reaction with or in the presence of the
unquenching enzyme. In certain embodiments, the enzyme-reactive
quenching group is a trimethyl lock. The resulting fluorophore, an
active fluorophore, exhibits fluorescence. Because the alteration
and/or release of the enzyme-reactive group is typically
irreversible, for example due to the relative thermodynamic
stability of the unreacted and reacted quenching group, the
fluorescence signal typically remains throughout any subsequent
assay steps such as collection, rinsing, and measurement.
[0067] According to various exemplary embodiments of the
disclosure, fluorophores may be chosen to have certain specific
excitation and emission properties. By way of example, the
fluorophore may be chosen from fluoresceins and rhodamines. In
certain embodiments, the fluorophore may optionally include amine
groups on either side of the fluorophore.
[0068] The enzyme reactive group may be any moiety having
sensitivity and/or specificity toward an unquenching enzyme. In
certain exemplary embodiments, the enzyme reactive quenching group
activates a trimethyl lock mechanism and is then released from the
enzyme-reactive latent fluorophore. A trimethyl lock is a
functional group including three methyl groups in close proximity.
Steric interactions between these three methyl groups promote a
lactone reaction and liberation of a leaving group. The resulting
cyclic hydrocoumarin group is thermodynamically favored, making the
trimethyl lock mechanism well suited as a fluorophore quenching
group. In various embodiments, the unquenching enzyme reacts
directly with the trimethyl lock to cleave the enzyme reactive
group from the enzyme-reactive latent fluorophore.
[0069] Exemplary and non-limiting trimethyl lock fluorophores
useful in various embodiments may be chosen from, for example,
compounds represented by Chemical Formula 1, Chemical Formula 2,
Chemical Formula 3, and mixtures thereof:
##STR00001##
[0070] According to various exemplary embodiments of the
disclosure, a latent fluorophore quenched by more than one
enzyme-reactive quenching group, for example the compound
represented by Chemical Formula 3, may be chosen, for example to
increase the difference in fluorescence intensity. By way of
non-limiting example, in an embodiment where a compound represented
by Chemical Formula 3 is chosen, the enzyme-reactive latent
fluorophore is quenched by two enzyme-reactive groups. In that
exemplary embodiment, the compound fluoresces at a first level of
intensity when the first enzyme-reactive group is released, and
fluoresces at a higher level of intensity when the second
enzyme-reactive group is released.
[0071] In various exemplary embodiments, more than one quenching
group may be chosen, where different quenching groups have
different properties, for example the ability to be altered and/or
released by reaction with certain enzymes, rates of reaction, and
conditions for reaction. In certain embodiments, multiple latent
fluorophores having different quenching groups, and thus having
different properties, may be chosen for use in multiplexed assays.
In certain embodiments involving different quenching groups,
different unlocking enzymes and/or varying reaction conditions may
optionally be used.
Conjugative Group
[0072] According to various embodiments of the disclosure, the
latent fluorophores may be connected to a support by at least one
linking group, or conjugative group. As used herein, the terms
"connected," "connection," and variations thereof, mean being held
in a set or predetermined spatial relationship, or a set or
predetermined spatial relationship, and the term "connectable" and
variations thereof means capable of being held in a set or
predetermined spatial relationship. As used herein, the terms
"linking group," "linker," "conjugative group," "bioconjugative
group," and variations thereof, which may be used interchangeably,
mean a functional group or agent capable, by itself or in the
presence of a complementary group, of forming a connection between
the latent fluorophore and the support.
[0073] In one embodiment, the linking group is a bioconjugative
group comprising a pyrrole-2,5-dione group.
[0074] It may, in various embodiments, be desirable to choose a
conjugate that has a complementary functional group to which it is
attracted. By way of non-limiting example, according to one
embodiment of the disclosure, the linking group may be chosen from
a biotin group or biotin-containing compound. It is known that the
biotin group preferentially binds to the protein streptavidin to
form a strong protein-ligand interaction, which may be desirable
according to certain embodiments of the disclosure. For example, a
latent fluorophore containing a biotin linking group will form a
connection with a support coated with streptavidin.
[0075] According to various embodiments of the disclosure, the
linking group may include a polymer or oligomer having a degree of
polymerization or polymerization number, n, which indicates the
number of repeated monomer units. The polymerization number may
determine the length of the polymer or oligomer linker. The polymer
or oligomer linker can be of any appropriate length to allow an
enzyme to bind the enzyme-reactive quenching group on the
enzyme-reactive latent fluorophore by providing a suitable space
between the enzyme reactive group and the support. If n is too
large, the polymer or oligomer linker will be too long, and may be
prone to breakage or other instability. Also, if the polymer or
oligomer linker is too long, the fluorophore may form undesirable
aggregates. If the polymer or oligomer linker is too short, it may
be difficult for the enzyme to access the reactive group. The
length of the polymer or oligomer linker can be adjusted so that
the enzyme reactive group is accessible to the enzyme.
[0076] Thus, in various embodiments, n is any positive number. By
way of example, n may range up to about 200, such as up to about
150, up to about 100, up to about 75, up to about 50, up to about
25, or up to about 20, such as about 1 to about 100, about 1 to
about 25, about 1 to about 20, about 3 to about 100, about 4 to
about 100, about 3 to about 25, about 4 to about 25, or about 4 to
about 20. For example, in at least certain non-limiting
embodiments, n ranges from about 1 to about 25. In yet further
exemplary embodiments, n ranges from about 3 to about 20, or about
4 to about 100. A PEG linker having n ranging from about 4 to about
100 may result in a spacer length of approximately 16 to 110
.ANG..
[0077] In certain embodiments, the polymer or oligomer may be water
soluble, biocompatible and/or nonreactive. Biocompatability of the
polymer or oligomer may, in at least certain embodiments,
advantageously decrease the likelihood of interactions between the
linking group and the enzyme, although it is not required. In
various exemplary embodiments, the linking group comprises a PEG
linker or a dextran. In yet further exemplary embodiments, the
linking group comprises a PEG linker represented by Chemical
Formula 4:
H--[O--CH.sub.2--CH.sub.2].sub.n--OH Chemical Formula 4
[0078] According to various exemplary embodiments, the at least one
conjugative group chosen may lead to improved stability and/or
resistance to enzymatic degradation, although it is not
required.
[0079] In various exemplary embodiments, more than one conjugative
group may optionally be chosen, where different conjugative groups
have different properties. For example, multiple conjugative groups
having different properties may be chosen for use in multiplexed
assays. In certain embodiments using multiplexed arrays, different
latent fluorophores may be connected to one or more types of
supports by the same conjugative group.
Support
[0080] Supports useful according to various embodiments of the
disclosure include, by way of non-limiting example, microspheres,
microbeads, and beads, as well as any other support to which the
compounds described herein can be attached. As used herein, the
terms "microsphere," "microbead," and "bead," which may be used
interchangeably herein to denote any particulate support, as well
as variations thereof, mean a particle having a size ranging from
about 0.1 .mu.m to about 1000 .mu.m.
[0081] It may be possible to increase the range of sensitivity in
an assay by modifying properties of the support, and/or by choosing
supports with a specific property or set of properties. For
example, microspheres having larger or smaller diameters may be
chosen, depending on the sensitivity desired. A high surface area
of the support allows a large number of fluorophores to be attached
thereto, which is thought to increase the operating range of the
assay. As such, the number of latent fluorophores connected to the
support, e.g. a microsphere, can be adjusted to achieve a desired
operating range.
[0082] Further, supports having different functionalities,
coatings, fluorescence excitation/emission characteristics, and/or
scattering characteristics, may be chosen. One of skill in the art
will be able to select a support having the appropriate properties,
depending on, for example, the intended application and/or specific
latent fluorophore comprising at least one enzyme-reactive
quenching group and at least one conjugative group to be used.
[0083] In various exemplary embodiments, more than one support may
be chosen, where different supports have different properties. For
example, multiple supports having different properties may be
chosen for use in multiplexed assays. In certain embodiments,
different latent fluorophores may be connected to supports for use
in multiplexed arrays.
[0084] By way of non-limiting example, more than one microbead
having substantially the same properties constitutes a class or
type of microbeads. Different classes or types of microbeads may be
chosen where the microbeads have a variety of functionalities,
fluorophores, and/or coatings. More than one class or type of
microbeads measured simultaneously constitute a multiplexed
array.
[0085] For example, SPHERO.TM. Blue Fluorescent Particle Kits
(Spherotech, Lake Forest, Ill.) are available with carboxyl
functionality for covalent attachment of ligands. These microbeads
are produced with up to 10 different amounts of fluorescent dyes
incorporated in them. Depending on the diameter of the beads, they
are sold in kits of 7 to 10 individual classes of microbeads. The
classes differ in fluorescence intensity in the PE-Cy5,
allophycocyanin ("APC"), and APC-Cy7 channels with minimal
fluorescence in the fluorescein isothiocyanate ("FITC") and
R-phycoerythrin ("PE") channels, allowing identification of the
beads' class. By using enzyme-reactive specific latent fluorophores
which fluoresce in the FITC or PE channel (for example, rhodamine
derivatives), the specific class of enzymatic activity can be
identified by the fluorescence intensity of the bead in the PE-Cy5,
APC, and APC-Cy7 channels.
[0086] As a further non-limiting example, microspheres having
different fluorescence excitation/emission characteristics may be
chosen. Examples include SPHERO.TM. Fluorescent Particles
(Spherotech, Lake Forest, Ill.) and Fluorescent Microspheres (Bangs
Laboratories, Fishers, Ind.).
[0087] A wide variety of fluorescent particles ranging in size,
spectral characteristic of fluorescence, fluorescence lifetime,
fluorescence intensity, and/or surface functional group are
available. Classes of particles that differ in their spectral
characteristic of fluorescence emission can be chosen to identify
specific enzymatic activity, enzyme-reactive specific latent
fluorophores which fluoresce in the FITC or PE channel that is
associated with that class.
[0088] Depending on the application, the available excitation
sources, and the emission filter(s) used, a variety of combinations
can be selected. For example, available Internet tools such as the
interactive spectrum viewer provided by BD Biosciences, Franklin
Lakes, N.J., or the interactive spectrum viewer provided by Life
Technologies Corporation, Carlsbad, Calif. can be used for dye
selection. In at least certain embodiments, combinations may be
chosen so that the emission characteristics (e.g., emission
spectra) are sufficiently different so that a first emission
spectrum (for example, from an enzyme-reactive latent fluorophore)
does not significantly overlap with second and subsequent emission
spectrum(a) (for example, fluorescent microspheres of type 1, type
2, and type 3).
[0089] By way of yet further non-limiting example, microspheres
having different scattering characteristics may be chosen. In at
least on exemplary embodiment, SPHERO.TM. Polystyrene Particles,
Spherotech, Lake Forest, Ill., may be used in multiplex assays.
[0090] A wide variety of particle sizes with surface functional
groups are available. For example, by linking a specific
enzyme-reactive latent fluorophore (enzyme 1) to a bead of a
specific size (microsphere type 1) and a second specific
enzyme-reactive latent fluorophore (enzyme 2) to a bead of a
different size (microsphere type 2), the specific enzyme activity
(fluorescence from the latent fluorophore) can be associated with
the scattering properties of the microsphere. The size of the beads
may be selected so that there is a sufficient difference in the
scattering properties of the various types of microspheres so that
each can be uniquely identified.
[0091] In yet further exemplary embodiments, supports may be chosen
that are coated. By way of example only, streptavidin-coated
microspheres may be used in various embodiments of the disclosure.
In yet further, non-limiting exemplary embodiments, other binding
proteins, or either component of any conjugative linking pair, may
be coated on the support.
Magnetic Support
[0092] In at least certain exemplary embodiments according to the
disclosure, magnetic microspheres, microbeads, and beads may be
chosen as the support for the latent fluorophore. As used here, the
terms "magnetic microsphere," "magnetic microbead," and "magnetic
bead," which may be used interchangeably, as well as variations
thereof, mean a particle exhibiting magnetism. For the manipulation
of beads, paramagnetism or superparamagnetism in the presence of an
externally applied magnetic field is often used. Paramagnetic
materials exhibit a positive change in magnetic moment in the
presence of an externally applied magnetic field. Superparamagnetic
materials exhibit high levels of paramagnetism. The high levels of
paramagnetism exhibited in superparamagnetic materials are due to
microscale or nanoscale ordering of spin orientations, which may be
lacking in other paramagnetic materials. In certain exemplary
embodiments, the support comprises a superparamagnetic
material.
[0093] Sizes of commercially available magnetic beads typically
range from about 0.1 .mu.m to about 120 .mu.m. They often contain a
paramagnetic material, for example iron oxide, resulting in the
beads' paramagnetic properties, and a biocompatible shell that can
be functionalized. The paramagnetic material may constitute the
core of the particle or may be a shell-like coating on a core, for
example made of polystyrene. However, it should be noted that
magnetic supports useful according to the disclosure are not
required to have any particular size, functionality, and the like,
such as found in commercially available products.
[0094] Because of their embedded paramagnetic materials, such beads
can be manipulated by an external magnetic field that induces a
non-negligible magnetic field in the bead. In particular, the beads
can be moved through a solution, collected, dispersed, mixed and
extracted in a controlled manner by means of an applied external
magnetic field. Paramagnetic beads have the advantage of a magnetic
field that vanishes once the external field vanishes, so that the
beads do not influence each other in undesirable ways, for example
cluster together. Methods for controlled sample preparation
involving such magnetic beads are known in the art.
[0095] Additionally, fluorophores can be embedded in the bead for
detection or identification (for example UMC3F COMPEL.TM. Magnetic
COOH modified, fluorescent particles from Bangs Laboratories Inc.).
Connecting a fluorophore to a magnetic microsphere may allow
concentration of the fluorescence signal after performing an
enzymatic reaction in a larger volume. The technique can allow the
enzyme assay to be performed in complex solutions, for example
whole blood. It also enables efficient rinsing of interfering
compounds, components, and the like, prior to detecting the
fluorescence signal. In addition, fluorophores connected to
magnetic microspheres can, in at least certain exemplary
embodiments, enable automated sample preparation and analysis.
Methods of Preparing Latent Fluorophores Linked to a Support
[0096] Any known method for preparing latent fluorophores
comprising at least one enzyme-reactive group and at least one
conjugative group may be used. It is well within the skill of those
in the art to design appropriate chemical reactions in order to
prepare a latent fluorophore having at least one enzyme-reactive
group and at least one conjugative group.
[0097] An exemplary and non-limiting method of synthesizing a
latent fluorophore represented by Chemical Formula 1, which method
is presented solely for purposes of illustrating one optional
embodiment, is shown in Reaction Scheme 1.
##STR00002## ##STR00003## ##STR00004##
[0098] Any known connection between a support and latent
fluorophores comprising at least one enzyme-reactive group and at
least one conjugative group is also contemplated to be within the
scope of the disclosure. By way of non-limiting example only, a
latent fluorophore such as that of Chemical Formula 1 prepared
according to Reaction Scheme 1 may be linked to the microsphere
with a PEG linker. For example, a heterobifunctional PEG may be
chosen, such as thiol-PEG-Amine (e.g. HS-PEG-NH.sub.2; Creative
PEGworks, Winston Salem, N.C.). The thiol group reacts with the
maleimide group on Chemical Formula 1, the amine group is reacted
with a microsphere containing a carboxylic acid functionality (for
example, Dynabeads.RTM. M-270 Carboxylic Acid (magnetic,
nonfluorescent) or FluoSpheres.RTM. Carboxylate-Modified
Microspheres (fluorescent, nonmagnetic), both Life Technologies
Corporation, Carlsbad, Calif.; Polybead.RTM. Carboxylate
Microspheres (nonfluorescent, nonmagnetic) or Fluoresbrite.RTM. BB
Carboxylate Microspheres (fluorescent, nonmagnetic); both
Polysciences, Inc., Warrington, Pa.)) following activation with
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)
to yield the desired latent fluorophore linked to a microsphere.
Optionally, the amine group at the end of the PEG linker on the
latent fluorophore can be linked to a Tosylactivated microsphere
(for example, Dynabeads.RTM. M-280 Tosylactivated; Life
Technologies Corporation, Carlsbad, Calif.).
[0099] In yet a further non-limiting exemplary embodiment where the
latent fluorophore is chosen from those of Chemical Formula 2,
thiol-PEG-acid may be chosen (e.g. HS-PEG-COON; Creative PEGworks,
Winston Salem, N.C.) to link to the support. The thiol group is
reacted with the maleimide group on Chemical Formula 2, the
carboxylic acid group is reacted with
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)
and crosslinked to a microsphere containing a carboxylic acid
functionality (for example, Dynabeads.RTM. M-270 Carboxylic Acid;
Life Technologies Corporation, Carlsbad, Calif., or Polybead.RTM.
Carboxylate Microspheres; Polysciences, Inc., Warrington, Pa.).
[0100] In yet a further exemplary embodiment for preparing latent
fluorophores and linking to a support, the latent fluorophores
comprising at least one enzyme-reactive group and at least one
conjugative group may be synthesized with a biotin group as at
least one conjugative group. The latent fluorophore-biotin compound
may then be reacted with streptavidin-coated microspheres, to yield
a latent fluorophore linked to a microsphere.
[0101] Further, any known method of linking latent fluorophores
comprising at least one enzyme-reactive group and at least one
conjugative group to a support is also contemplated to be within
the scope of the disclosure. In certain exemplary embodiments, the
latent fluorophores may spontaneously connect to the support when
both are placed in solution. In further exemplary embodiments, the
solution may be stirred, heated, and/or cooled to facilitate the
linkage between the fluorophore and the support. In yet further
exemplary embodiments, additives, for example surfactants,
promoting agents, enzymes and the like, may be added to the
solution to facilitate the linkage between the fluorophore and the
support.
Methods of Using Latent Fluorophores Linked to a Support
[0102] As used herein, the term "measuring enzyme activity" and
variations thereof refers to a measure of the quantity of active
enzyme, and/or of the quantity of analyte acted upon by the enzyme.
As used herein, the terms "an enzyme reacting with the analyte,"
"an enzyme acting upon the analyte," and variations thereof mean a
chemical reaction catalyzed by an enzyme, wherein an analyte is
chemically altered. In certain embodiments, the chemical reaction
may be a dehydrogenation reaction.
[0103] The fluorophore compositions described according to various
embodiments of the disclosure may be useful in enzyme assays. For
example, the fluorophore compositions may be useful for methods of
measuring activities of enzymes. In various embodiments, the
fluorescence signal of the unlocked fluorophore may be proportional
to the enzyme activity to be measured due to the quenching group
being altered and/or released by the enzyme in question, and/or by
a downstream enzyme in a series of enzyme reactions, or enzyme
cascade, including the enzyme in question. In certain embodiments,
the enzyme activities of two or more enzymes may be compared.
[0104] Further, the fluorophore compositions may be useful for
detecting and/or measuring the concentration of analytes in a
sample, for example when the enzyme activity of a known
concentration of enzyme in the presence of an unknown amount of
analyte is compared with the enzyme activity of a known
concentration of enzyme in the presence of a known amount of
analyte.
[0105] As used herein, a sample may be any substance, in particular
a sample of liquid substance, and in at least certain embodiments,
may be a sample of bodily fluids such as urine, saliva, or blood.
The samples used according to various methods described herein may
be obtained or collected by any technique known. A "test" sample
and a "reference" sample may, in at least certain embodiments, be
two samples from the same source, e.g. two samples of blood taken
from one blood collection, where the test sample and reference
sample may be treated with the compositions described herein in
different manners.
[0106] Methods of Measuring the Activities of Enzymes
[0107] Enzymatic activity dependent on the presence of a particular
substrate is known to be correlatable with the concentration of
that substrate. Compositions prepared according to various
embodiments of the disclosure may permit detection of such
enzymatic activity via enzymatic assay. Exemplary enzyme assays
useful according to various embodiments include, but are not
limited to, enzyme cascade assays, coupled assays and direct
assays.
[0108] As used herein, the terms "contact," "contacting," and
variations thereof mean bringing components into close enough
proximity to allow for a chemical or enzymatic reaction to occur
between or among the components. The contacting may be between or
among any combination of components in any manner. In various
non-limiting exemplary embodiments, the contacting between a sample
and a fluorophore is performed by including or mixing a fluorophore
in a liquid sample. In certain embodiments, the liquid sample may
be stirred, sonicated, heated and/or cooled, and/or additives may
be added to the liquid sample to facilitate the contacting between
the sample and the fluorophore.
[0109] As used herein, the terms "compare," "comparing," and
variations thereof mean determining the relative quantitative
and/or qualitative fluorescence signals of two or more samples, by
any method, and including either human or machine/computer
determination. The fluorescence signals of the samples may be
compared by any known methods. In one non-limiting exemplary
embodiment, the fluorescence signals of two samples may be compared
by subtracting the quantitative signal of one sample from the
quantitative signal of the other sample.
[0110] Methods for measuring the activity and/or presence of an
enzyme in a test sample including an analyte include one or more
steps chosen from:
[0111] a. preparing a fluorophore composition comprising: [0112] i.
at least one enzyme-reactive latent fluorophore comprising at least
one enzyme-reactive quenching group and at least one conjugative
group, and [0113] ii. a support connectable to the latent
fluorophore by at least one conjugative group;
[0114] b. providing a test sample to be analyzed and a reference
sample to be analyzed, wherein the reference sample contains a
known quantity of the analyte;
[0115] c. contacting the test sample with the latent fluorophore
composition, at least one first unquenching enzyme capable of
releasing the enzyme-reactive quenching group from the latent
fluorophore, and at least one second enzyme capable of reacting
with the analyte;
[0116] d. contacting the reference sample with the latent
fluorophore composition and the at least one first unquenching
enzyme;
[0117] e. measuring the fluorescence signal of the test sample and
the fluorescence signal of the reference sample; and
[0118] f. comparing the fluorescence signal of the test sample with
the fluorescence signal of the reference sample.
[0119] In various embodiments of the method of measuring the
activity of an enzyme, the first unquenching enzyme that unlocks
the latent fluorophore added to the test sample may be the same as
or different from the second enzyme capable of reacting with the
analyte. When the first unquenching enzyme is the same as the
second enzyme, this may be referred to as a direct assay. When the
first unquenching enzyme is different from the second enzyme, this
may be referred to as an enzyme cascade or coupled assay, and in
various embodiments the first enzyme may be considered downstream
of the second enzyme because the first enzyme uses a by-product of
the reaction between the second enzyme and the analyte in order to
unquench the quenching group.
[0120] By way of non-limiting example, in one embodiment the first
unquenching enzyme that unlocks the latent fluorophore added to the
test sample is the same as the first unquenching enzyme added to
the reference sample. When the same enzyme is added to both
samples, the resulting difference in fluorescence may be attributed
to the activity of the enzyme on the analyte, when the only
difference between the test sample and the reference sample is the
quantity of analyte. In certain exemplary embodiments, the
reference sample is free or substantially free of the analyte. In
contrast, in a non-limiting embodiment where the test sample
includes a different unquenching enzyme than the unquenching enzyme
in the reference sample, a difference in fluorescence signal
between the two samples may be at least partially attributable to
the difference in unquenching activity and/or rate between
different unquenching enzymes.
[0121] Methods for measuring activities of two or more enzymes in a
sample, for example in a multiplex assay, include one or more steps
chosen from:
[0122] a. providing a first fluorophore composition comprising:
[0123] i. at least one first enzyme-reactive latent fluorophore
comprising at least one first enzyme-reactive quenching group and
at least one conjugative group, and [0124] ii. at least one support
connectable to the at least one first latent fluorophore by at
least one conjugative group;
[0125] b. providing a second fluorophore composition comprising:
[0126] i. at least one second enzyme-reactive latent fluorophore
comprising at least one second enzyme-reactive quenching group and
at least one conjugative group, wherein the at least one second
enzyme-reactive latent fluorophore is different from said first
enzyme-reactive latent fluorophore in said first fluorophore
composition, and [0127] ii. at least one support connectable to the
at least one first latent fluorophore by at least one conjugative
group;
[0128] c. providing a test sample to be analyzed and a reference
sample to be analyzed;
[0129] d. contacting the test sample with the first and second
latent fluorophore compositions, at least one first unquenching
enzyme capable of releasing the enzyme-reactive quenching group
from the first latent fluorophore, and at least one second
unquenching enzyme capable of releasing the enzyme-reactive
quenching group from the second latent fluorophore;
[0130] e. contacting the reference sample with the first and second
latent fluorophore compositions;
[0131] f. measuring the fluorescence signals of the test sample and
the fluorescence signals of the reference sample; and
[0132] g. comparing the fluorescence signals of the test sample
with the fluorescence signals of the reference sample.
[0133] In various embodiments of the method of the multiplex assay
above, the first unquenching enzyme that unlocks the latent
fluorophore added to the test sample may be the same as or
different from the first enzyme added to the reference sample. When
the first unquenching enzyme is the same as the first enzyme, this
may be referred to as a multiplex direct assay. When the first
unquenching enzyme is different from the first enzyme, this may be
referred to as a first enzyme cascade, at in various embodiments
the first unquenching enzyme may be considered downstream of the
first enzyme because the first unquenching enzyme uses a by-product
of the reaction between the first enzyme and the analyte in order
to unquench the first quenching group. An enzyme cascade including
two enzymes, one that reacts with the analyte and the other that
unquenches the latent fluorophore, may be referred to as a coupled
assay.
[0134] By way of example, in one non-limiting embodiment, the first
quenching group is different from the second quenching group. For a
multiplex direct assay, wherein the analyte-active enzyme is the
same as the unquenching enzyme, the first and second quenching
groups are unlocked by different unquenching enzymes; otherwise the
difference in fluorescence signal could not be correlated with the
activity of a particular enzyme on a particular analyte. For a
multiplex enzyme cascade assay, wherein an enzyme having activity
in the presence of an analyte is part of an enzyme cascade that
includes a downstream unquenching enzyme, the first and second
quenching groups are unlocked by different unquenching enzymes;
otherwise the difference in fluorescence signal could not be
correlated with the activity of a particular enzyme cascade on a
particular analyte.
[0135] In various embodiments, the first fluorophore has a first
emission spectrum that may be the same as or different from a
second emission spectrum of the second fluorophore. When the first
and second emission spectra have at least one different property,
for example peak emission wavelength, peak absorption wavelength,
and/or fluorescence lifetime, the first and second fluorescence
signals from the first and second fluorophores, respectively, may
be distinguished.
[0136] Alternatively, when the first and second emission spectra
are the same, the first fluorophore may be distinguished from the
second fluorophore by employing other techniques. In various
embodiments, one such technique includes physically separating the
first fluorophore from the second fluorophore, for example by
linking at least one of the fluorophores to a support and
physically separating the support, for example by spatial
concentration using an external magnetic, electric or acoustic
field, or filtering; and/or linking the first fluorophore to a
first support and the second fluorophore to the second support, and
subsequently distinguishing the first support and the second
support. In certain embodiments, the first support and the second
support may be distinguished by the differential fluorescence of
the first and second supports, and/or by differential scattering by
first and second supports having different sizes.
[0137] In certain embodiments of the multiplex assay method above,
the conjugative group linking the first fluorophore to the support
may be the same as or different from the conjugative group linking
the second fluorophore to the support. By way of non-limiting
example, in one embodiment the conjugative group may be different
in order to selectively link one of the fluorophores with a
support, while the other fluorophore is linked to a different
support or left freely suspended in the test or reference sample.
In other non-limiting exemplary embodiments, the conjugative group
is the same for both the first and second fluorophores, for example
when the fluorophores are linked to the same or different support
temporally and/or spatially independently of one another, when the
fluorophores are linked to the same support, and/or when the
fluorophores have sufficiently distinct fluorescence signals that
separation of the fluorophores during the fluorescence measurement
is not necessary.
[0138] In various embodiments of the multiplex assay method above,
the first fluorophore and the second fluorophore may be linked to
the same support, the same type of support, a different support, or
a different type of support.
[0139] By way of example, a coupled assay may include at least one
first enzyme that generates a first reaction product from a first
enzymatic reaction on a first substrate, and at least one second
enzyme that uses the first reaction product generated from the
first enzymatic reaction to produce a fluorescence signal by
reacting with and releasing the enzyme-reactive quenching group on
the latent fluorophore. In certain embodiments, the fluorescence
signal may be approximately proportional to the concentration of
the first substrate.
[0140] According to various exemplary and non-limiting embodiments
of the disclosure, the at least one first enzyme may be chosen from
protease, phosphatase, and dehydrogenase. In at least one exemplary
embodiment, the first enzyme is a dehydrogenase, and the first
enzymatic reaction is a dehydrogenation reaction. In certain
embodiments, the first substrate is phenylalanine, the first enzyme
is phenylalanine dehydrogenase, and the first reaction products are
NADH (or NADPH)+phenylpyruvate +NH.sub.3+H. The first substrate,
first enzyme, and first reaction products for other exemplary
embodiments are shown in Table 1 below.
TABLE-US-00001 TABLE 1 First Substrate First Enzyme First Reaction
Product Cholesterol Cholesterol NADH + cholest-4-en-3-
dehydrogenase one + H.sup.+ Lactate Lactate dehydrogenase NADH +
pyruvate + H.sup.+ Leucine Leucine dehydrogenase NADH + 4-methyl-2-
oxopentanoate + NH.sub.3 + H.sup.+ D-glucose Glucose dehydrogenase
NADH + D-glucono-1,5- lactone + H.sup.+ Ethanol Alcohol
dehydrogenase NADH + acetaldehyde
[0141] Other non-limiting examples of enzymes contemplated
according to various embodiments of the disclosure may use
modification of an enzyme-reactive group to achieve specificity for
the enzyme of interest, for example alkaline phosphatase using a
rhodamine fluorophore (Levine, M. N. et al, Analytical Biochemistry
418 (2011), 247-252); esterase (Levine, M. N. et al, Molecules
(2008), 13, 204-211); and serine protease using a
bis(N-benzyloxycarbonyl-L-argininamido)Rhodamine substrate (Leytus,
S. P. et al, Biochem. J. (1983), 209, 299-307).
[0142] According to various exemplary embodiments, the at least one
second enzyme may be a diaphorase. As used here, the term
"diaphorase" is intended to encompass several different enzyme
classes, some of which have different substrate specificities for
NADH or NADPH. By way of example only, the diaphorase may be chosen
from the enzyme classes EC 1.8.1.4-dihydrolipoyl dehydrogenase, EC
1.6.5.2-NAD(P)H dehydrogenase (quinone or DT-diaphorase), EC
1.6.99.1-NADPH dehydrogenase, EC 1.6.99.3-NADH dehydrogenase, and
mixtures thereof, all of which may catalyze oxidation/reduction
reactions. In at least one exemplary embodiment, the diaphorase may
be chosen from dihydrolipoyl dehydrogenase and NAD(P)H
dehydrogenase.
[0143] As shown in FIG. 1, an exemplary coupled assay for measuring
the amount of an analyte in a sample may include a dehydrogenase
and a diaphorase. In this exemplary embodiment, the dehydrogenase
reacts with the analyte in the sample to produce an oxidized
analyte and NADH in a dehydrogenase reaction. The diaphorase uses
the NADH created by the dehydrogenase reaction to react with and
release the enzyme-reactive quenching group from the latent
fluorophore, thus revealing a fluorescent compound.
[0144] In various embodiments, the concentrations of dehydrogenase
and diaphorase may be adjusted such that the oxidation of the
analyte is the rate-limiting reaction.
[0145] As shown in FIG. 2, an exemplary coupled assay for measuring
the amount of phenylalanine in a sample may include a phenylalanine
dehydrogenase (PheDH) and a diaphorase. In this embodiment, the
PheDH reacts with phenylalanine in the sample to produce
phenylpyruvate, NH.sub.3 and NADH in a dehydrogenase reaction. The
diaphorase uses the NADH created by the dehydrogenase reaction to
react with and release the enzyme-reactive quenching group from the
latent fluorophore, thus revealing a fluorescent compound.
[0146] In various embodiments, the concentrations of PheDH and
diaphorase may be adjusted such that the oxidative deamination of
phenylalanine is the rate-limiting reaction.
[0147] In various exemplary embodiments, a direct assay may include
at least one enzyme that acts upon a substrate to produce a
fluorescence signal by reacting with and releasing the
enzyme-reactive quenching group on the latent fluorophore. In at
least one exemplary embodiment, the enzyme may be pyruvate oxidase
and the substrate may be pyruvate.
[0148] As shown in FIG. 3, an exemplary direct assay for measuring
the amount of pyruvate in a sample includes a pyruvate oxidase. In
this exemplary embodiment, the pyruvate oxidase reacts with the
pyruvate in the sample to produce acetate and CO.sub.2, and also
reacts with and releases a quinone enzyme-reactive quenching group
from the latent fluorophore, thus revealing a fluorescent
compound.
[0149] Methods of Detecting and Measuring Analytes in a Sample
[0150] Enzyme-reactive latent fluorophores linked to microspheres
prepared according to various embodiments described herein may, in
exemplary embodiments, be used to detect the presence and/or
concentration of certain analytes in a sample. The analyte may, for
example, be detected by utilizing a direct or coupled assay.
[0151] Exemplary methods for detecting and measuring an analyte in
a sample include one or more steps chosen from:
[0152] a. preparing a fluorophore composition comprising: [0153] i.
at least one enzyme-reactive latent fluorophore comprising at least
one enzyme-reactive quenching group and at least one conjugative
group, and [0154] ii. a support connectable to the latent
fluorophore by at least one conjugative group;
[0155] b. providing a test sample to be analyzed and a reference
sample to be analyzed;
[0156] c. contacting the test sample with the latent fluorophore
composition, at least one first unquenching enzyme capable of
releasing the enzyme-reactive quenching group from the latent
fluorophore, and at least one second enzyme capable of reacting
with the analyte;
[0157] d. contacting the reference sample with the latent
fluorophore composition and the at least one first unquenching
enzyme;
[0158] e. measuring the fluorescence signal of the test sample and
the fluorescence signal of the reference sample; and
[0159] f. comparing the fluorescence signal of the test sample with
the fluorescence signal of the reference sample.
[0160] In various embodiments of the method of measuring the
activity of an enzyme above, the first unquenching enzyme that
unlocks the latent fluorophore added to the test sample is the same
as or different from the second enzyme capable of reacting with the
analyte. When the first unquenching enzyme is the same as the
second enzyme, this may be referred to as a direct assay. When the
first unquenching enzyme is different from the second enzyme, this
may be referred to as an enzyme cascade or coupled assay, wherein
the first enzyme is considered downstream of the second enzyme
because the first enzyme requires a by-product of the reaction
between the second enzyme and the analyte in order to unquench the
quenching group.
[0161] By way of non-limiting example, an assay, e.g. a coupled
assay, may be used to measure the presence and/or concentration of
phenylalanine in whole blood. In at least certain exemplary
embodiments, an assay, e.g. a coupled assay, can be used to measure
the presence and/or concentration of any analyte in any solution,
e.g. those that have a specific dehydrogenase that generates NADH
or NADPH in a solution, for example whole blood. In various
embodiments, the assay can be used to measure the presence and/or
concentration of a metabolite, for example a human metabolite or an
animal metabolite, in a bodily fluid, for example blood, urine,
saliva, bile, spinal fluid, gastric juices, mucus, tears, amniotic
fluid, semen, sweat, lymph fluids, and the like.
[0162] FIG. 4a is an exemplary reaction schematic showing a latent
fluorescent compound revealed by a trimethyl lock reaction. In this
exemplary embodiment, the latent fluorophore having an
enzyme-reactive quenching group is connected to a microsphere with
a linking group including a pyrrole-2,5-dione bioconjugate and a
PEG linker. In the presence of a suitable enzyme, for example
diaphorase, and any cofactors, for example NADH, the
enzyme-reactive quenching group is released from the latent
fluorophore via a trimethyl lock reaction. The fluorophore
connected to the microsphere is now fluorescent.
[0163] FIG. 4b is an exemplary reaction schematic showing a latent
fluorescent compound revealed by a trimethyl lock reaction and
connected to a microsphere with a biotin-streptavidin linkage. In
this exemplary embodiment, the latent fluorophore having an
enzyme-reactive quenching group and a biotin group is connected to
a microsphere coated with streptavidin. In the presence of a
suitable enzyme, for example diaphorase, and any cofactors, for
example NADH, the enzyme-reactive quenching group is released from
the latent fluorophore via a trimethyl lock reaction. The
fluorophore connected to the microsphere is now fluorescent.
[0164] FIG. 5 is an exemplary reaction schematic showing a latent
fluorescent compound revealed by a double trimethyl lock reaction.
In this exemplary embodiment, the latent fluorophore has two
enzyme-reactive quenching groups, and is connected to a microsphere
with a linking group including a pyrrole-2,5-dione bioconjugate and
a PEG linker. In the presence of an enzyme, for example diaphorase,
and any necessary cofactor, for example NADH, the enzyme releases
one or both of the enzyme-reactive groups. In certain embodiments,
having two enzyme-reactive groups can help increase or maximize the
difference in fluorescence intensity upon reaction with the
enzyme.
[0165] In various embodiments, the microspheres may be used to
isolate the fluorophores from the sample solution prior to the
fluorescence measurement. In at least one embodiment, fluorophores
connected to magnetic microspheres may be collected using a magnet,
and the other reactants, compounds, and materials in the sample may
be washed away. In certain embodiments, the isolated magnetic
microspheres may be suspended in a suitable buffer, for example
phosphate-buffered saline. The fluorescence of the fluorophores
linked to the magnetic microspheres can then be then measured.
[0166] The fluorescence of the active fluorophore may be measured
by any known process, including but not limited to bulk
fluorescence, flow cytometry, or spatially modulated fluorescence
detection technology.
[0167] In certain exemplary and non-limiting embodiments according
to the disclosure, the fluorescence signal may be measured using
bulk fluorescence measurements. In certain embodiments,
fluorescence in the sample may be induced by directing light of an
appropriate wavelength region into the sample that contains the
fluorophores. Fluorescent light of longer wavelengths than the
excitation light may be collected and detected from the same sample
region. To block out unwanted excitation light from the detection
path, the optical axis of excitation and detection path may be
arranged perpendicularly or in opposing direction (so called
epi-detection). Additionally, excitation and background light can
be separated and filtered with wavelength selective (dichroic)
mirrors and filters, for example bandpass filters.
[0168] In yet further exemplary embodiments, the fluorescence
signal may be measured using flow cytometry, the principles of
which are well known, and thus one of skill in the art would be
able to determine the appropriate procedures and/or parameters for
use according to various embodiments of the disclosure.
[0169] In flow cytometry, the same measurement principles as in
bulk fluorescence detection are applied. However, rather than
measuring bulk fluorescence, the fluorescence intensity of
particles is detected in a flow cytometer. The excitation and
detection region of a flow cytometer are placed in the path of
particles that are transported through a fluidic system. The
excitation/detection region covers usually the lateral width of the
flow channel and expands some tens of micrometers along the flow
direction. Typical particle sizes detected in a flow cytometer
generally range from 100 s nm to 10 s of .mu.m. The size of the
excitation/detection region is limited in flow direction to ensure
that ideally only a single particle is present in this region at
any given time. Commonly, flow cytometers can excite and detect in
several wavelength regions, including scattered light at the
excitation wavelength. The subsequent (fluorescence) intensity
measurement of hundreds, thousands or even ten thousands of
particles contains the statistical information of the basic
population.
[0170] In flow cytometry it is common practice to measure particle
sizes by forward scattering signals. This measurement requires a
precise alignment of the excitation laser and the detector by a
light block. This block prevents laser light from entering the
detector when no particle is present. A particle with a refractive
index ("RI") different from the suspension solution may scatter
light within the excitation spot of the laser. This scattered light
is detected by the scatter detector, because it passes by the sides
of the light block. The intensity of the scattered light depends on
a variety of particle properties, including RI, size, and scatter
center distribution. In a forward scattering direction, the amount
of scattered light is usually a measure of particle size, as the RI
of bioparticles does not differ from bioparticle type to
bioparticle type enough to provide relevant bioparticle information
while size (shape, orientation) dominates the scatter properties of
the particle.
[0171] In yet further exemplary embodiments, the fluorescence
signal may be measured using spatially modulated fluorescence
detection technology, which uses fluorescence detection comparable
to a flow cytometer. However, the spatial modulation technique uses
a large excitation/detection area (ca. 0.1.times.1 mm) to increase
the total flux of fluorescence light that originates from a
particle. This configuration generates a time-dependent signal as a
continuously fluorescing particle traverses a predefined pattern
for optical transmission. Correlating the detected signal with the
known pattern achieves high discrimination of the particle signal
from background noise and simplifies optical system alignment. At
the same time, multiple particles that simultaneously occur in the
detection region can be differentiated computationally by the
correlation of the signal sequences and the known transmission mask
pattern.
[0172] FIG. 6 is a flowchart showing the steps for an exemplary
dehydrogenase differential assay of a sample, for example blood.
The sample is divided between a test sample and a reference sample.
The test sample contains the analyte-specific enzyme, for example
dehydrogenase, the diaphorase, the latent fluorophore, plus any
necessary cofactors, for example NAD+. The reference sample
contains all of the components in the test sample except for the
analyte-specific enzyme. The fluorescence signals from the test
sample and the reference sample are measured, and the fluorescence
signal of the reference sample is subtracted from that of the test
sample to yield a differential measurement.
[0173] In certain embodiments, differential measurements may be
desired, e.g. to increase the reliability of the assay. For
example, some individuals may have varying levels of other analytes
in their blood, which can result in different background signals
that are independent of enzymatic action on the desired
analyte.
[0174] FIG. 7 is a flowchart showing the steps for an exemplary
phenylalanine dehydrogenase differential assay of blood. The sample
is divided between a test sample and a reference sample. The test
sample contains phenylalanine dehydrogenase (PheDH), the
diaphorase, the latent fluorophore, plus any necessary cofactors,
for example NAD+. The reference sample contains all of the
components in the test sample except for the PheDH. The
fluorescence signals from the test sample and the reference sample
are measured, and the fluorescence signal of the reference sample
is subtracted from that of the test sample to yield a differential
measurement.
[0175] In certain embodiments, the latent fluorophore including a
suitable linking group, for example biotin, can be used in solution
and bound to the microsphere coated with a suitable functional
group, for example streptavidin, after the enzyme assay is
performed. FIG. 8a is a reaction schematic showing an exemplary
embodiment of a latent fluorescent compound revealed by a trimethyl
lock reaction and subsequently linked to a streptavidin-coated
microsphere. The enzyme assay is performed as described above using
latent fluorophores including a biotin group. Streptavidin-coated
microspheres are subsequently introduced into the test sample and
reference sample. The biotin binds to the streptavidin, which
connects the fluorophore to the microsphere. The microsphere can
then be used to isolate the fluorophore from the samples as
described above.
[0176] In further exemplary embodiments according to the
disclosure, a multiplex array can be used to examine the activity
of multiple enzymes at the same time by using different
enzyme-reactive quenching groups and a different fluorophore for
each enzymatic reaction. The fluorescence of the different
fluorophores connected to different microspheres can be analyzed
using flow cytometry or spatially modulated fluorescence detection
technology. Suitable enzymes include, but are not limited to,
proteases, phosphatases, and dehydrogenases.
[0177] FIG. 9a is a flowchart showing the steps for a general
enzymatic multiplexed differential assay of a sample using
fluorophores having different emission properties, according to an
exemplary embodiment of the disclosure. Although three different
enzymes are used in the exemplary embodiment shown in FIG. 9a, a
greater or fewer number of enzymes may be used. The sample is
divided between a test sample and a reference sample. The test
sample contains three different enzymes, three different latent
fluorophores, plus any necessary cofactors and coupling enzymes,
for example diaphorase. The reference sample contains all of the
components in the test sample except for the three different
enzymes. The three different fluorophores have different
enzyme-reactive quenching groups and different emission properties,
for example maxima. The fluorescence signals from the test sample
and the reference sample are measured, and the fluorescence signals
of the reference sample are subtracted from those of the test
sample to yield differential measurements. In certain embodiments,
the microspheres are magnetic.
[0178] FIG. 9b is a flowchart showing the steps for a general
enzymatic multiplexed differential assay of a sample using
fluorescent microspheres having different emission properties
according to an exemplary embodiment of the disclosure. Although
three different enzymes are used in the exemplary embodiment shown
in FIG. 9b, a greater or fewer number of enzymes may be used. The
sample is divided between a test sample and a reference sample. The
test sample contains three different enzymes, three different
latent fluorophores, plus any necessary cofactors and coupling
enzymes, for example diaphorase. The reference sample contains all
of the components in the test sample except for the three different
enzymes. The three different fluorophores have different
enzyme-reactive quenching groups and the same emission properties.
Each of the three different latent fluorophores is coupled to a
microsphere having a specific fluorescent property, i.e., latent
fluorophore 1 is coupled to microsphere type 1, latent fluorophore
2 is coupled to microsphere type 2, and latent fluorophore 3 is
coupled to microsphere type 3, where microsphere types 1-3 have
different fluorescent properties, for example emission maxima.
Although the three latent fluorophores have the same fluorescence
properties, the activity of the three different enzymes may be
monitored by separating the microbeads having specific fluorescent
properties, for example by flow cytometry. The fluorescence signals
from the fluorophores and/or microspheres of the test sample and
the reference sample are measured, and the fluorescence signals of
the reference sample are subtracted from those of the test sample
to yield differential measurements. In another embodiment, the
three latent fluorophores have different emission properties. In
certain embodiments, all or some of the microspheres are
magnetic.
[0179] FIG. 9c is a flowchart showing the steps for a general
enzymatic multiplexed differential assay of a sample using
fluorescent microspheres having different sizes, according to an
exemplary embodiment of the disclosure. Although three different
enzymes are used in the exemplary embodiment shown in FIG. 9c, a
greater or fewer number of enzymes may be used. The sample is
divided between a test sample and a reference sample. The test
sample contains three different enzymes, three different latent
fluorophores, plus any necessary cofactors and coupling enzymes,
for example diaphorase. The reference sample contains all of the
components in the test sample except for the three different
enzymes. The three different fluorophores have different
enzyme-reactive quenching groups and the same emission properties.
Each of the three different latent fluorophores is coupled to a
microsphere having a specific particle size, i.e., latent
fluorophore 1 is coupled to microsphere type 1, latent fluorophore
2 is coupled to microsphere type 2, and latent fluorophore 3 is
coupled to microsphere type 3, where microsphere types 1-3 have
different sizes. The microspheres may be magnetic and/or
fluorescent, with the same or different fluorescent properties. The
difference in particle sizes between the different microspheres
causes different scattering signals from each size of microsphere.
Although the three latent fluorophores have the same fluorescence
properties, the activity of the three different enzymes may be
monitored by distinguishing the microspheres by size based on the
different scattering properties, for example using flow cytometry.
The fluorescence signals and scattering signals from the
fluorophores and/or microspheres of the test sample and the
reference sample are measured, and the fluorescence signals of the
reference sample are subtracted from those of the test sample to
yield differential measurements. In another embodiment, the three
latent fluorophores have different emission properties.
[0180] In at least certain exemplary embodiments, the enzymatic
multiplexed differential assays shown in FIGS. 9a-9c may be applied
to simultaneously screen compounds on multiple enzyme targets in a
multiplexed differential screening assay. For example, a
multiplexed differential screening assay may be used to determine
which, if any, of a number of enzymes shows activity in the
presence of a test compound.
[0181] FIG. 10a is a flowchart showing the steps for a general
enzymatic multiplexed differential screening assay of a sample
using fluorophores having different emission properties, according
to an exemplary embodiment of the disclosure. Although three
different enzymes are used in the exemplary embodiment shown in
FIG. 10a, a greater or fewer number of enzymes may be used. The
sample is divided between a test sample and a reference sample. The
test sample contains three different enzymes, three different
latent fluorophores, a test compound, plus any necessary cofactors
and coupling enzymes, for example diaphorase. The reference sample
contains all of the components in the test sample except for the
test compound. The three different fluorophores have different
enzyme-reactive quenching groups and different emission properties,
for example maxima. Although the three latent fluorophores have the
same fluorescence properties, the activity of the three different
enzymes may be monitored by separating the microbeads having
specific fluorescent properties, for example by flow cytometry. The
fluorescence signals from the test sample and the reference sample
are measured, and the fluorescence signals of the reference sample
are subtracted from those of the test sample to yield differential
measurements. In certain embodiments, the microspheres are
magnetic
[0182] FIG. 10b is a flowchart showing the steps for a general
enzymatic multiplexed differential screening assay of a sample
using fluorescent microspheres having different emission
properties, according to an exemplary embodiment of the disclosure.
Although three different enzymes are used in the exemplary
embodiment shown in FIG. 10b, a greater or fewer number of enzymes
may be used. The sample is divided between a test sample and a
reference sample. The test sample contains three different enzymes,
three different latent fluorophores, a test compound, plus any
necessary cofactors and coupling enzymes, for example diaphorase.
The reference sample contains all of the components in the test
sample except for the test compound. The three different
fluorophores have different enzyme-reactive quenching groups and
the same emission properties. Each of the three different latent
fluorophores is coupled to a microsphere having a specific
fluorescent property, i.e., latent fluorophore 1 is coupled to
microsphere type 1, latent fluorophore 2 is coupled to microsphere
type 2, and latent fluorophore 3 is coupled to microsphere type 3,
where microsphere types 1-3 have different fluorescent properties,
for example emission maxima. Although the three latent fluorophores
have the same fluorescence properties, the activity of the three
different enzymes may be monitored by separating the microbeads
having specific fluorescent properties, for example by flow
cytometry. The fluorescence signals from the fluorophores and/or
microspheres of the test sample and the reference sample are
measured, and the fluorescence signals of the reference sample are
subtracted from those of the test sample to yield differential
measurements. In another embodiment, the three latent fluorophores
have different emission properties. In certain embodiments, all or
some of the microspheres are magnetic.
[0183] FIG. 10c is a flowchart showing the steps for a general
enzymatic multiplexed differential screening assay of a sample
using fluorescent microspheres having different particle sizes,
according to an exemplary embodiment of the disclosure. Although
three different enzymes are used in the exemplary embodiment shown
in FIG. 10c, a greater or fewer number of enzymes may be used. The
sample is divided between a test sample and a reference sample. The
test sample contains three different enzymes, three different
latent fluorophores, a test compound, plus any necessary cofactors
and coupling enzymes, for example diaphorase. The reference sample
contains all of the components in the test sample except for the
test compound. The three different fluorophores have different
enzyme-reactive quenching groups and the same emission properties.
Each of the three different latent fluorophores is coupled to a
microsphere having a specific particle size, i.e., latent
fluorophore 1 is coupled to microsphere type 1, latent fluorophore
2 is coupled to microsphere type 2, and latent fluorophore 3 is
coupled to microsphere type 3, where microsphere types 1-3 have
different sizes. The microspheres may be magnetic and/or
fluorescent, with the same or different fluorescent properties. The
difference in particle sizes between the different microspheres
causes different scattering signals from each size of microsphere.
Although the three latent fluorophores have the same fluorescence
properties, the activity of the three different enzymes may be
monitored by distinguishing the microspheres by size based on the
different scattering properties, for example during flow cytometry.
The fluorescence signals from the fluorophores and/or microspheres
of the test sample and the reference sample are measured, and the
fluorescence signals of the reference sample are subtracted from
those of the test sample to yield differential measurements. In
another embodiment, the three latent fluorophores have different
emission properties. In certain embodiments, all or some of the
microspheres are magnetic.
[0184] Further exemplary embodiments of the disclosure relates to
kits comprising a composition, where the composition comprises at
least one latent fluorophore comprising at least one
enzyme-reactive quenching group and at least one conjugative group,
and at least one support connectible to the at least one latent
fluorophore via at least one conjugative group, as described
herein. These kits may include, for example, at-home testing kits
for analyzing phenylalanine levels in blood.
[0185] As used herein, the terms "a", "an", and "the" are intended
to encompass the plural as well as the singular. In other words,
for ease of reference only, the terms "a" or "an" or "the" may be
used herein, such as "a support", "an enzyme", "the microsphere",
etc., but are intended, unless explicitly indicated to the
contrary, to mean "at least one," such as "at least one support",
"at least one enzyme", "the at least one microsphere", etc. This is
true even if the term "at least one" is used in one instance, and
"a" or "an" or "the" is used in another instance, e.g. in the same
paragraph or section. Furthermore, as used herein, the phrase "at
least one" means one or more, and thus includes individual
components as well as mixtures/combinations.
[0186] The term "comprising" (and its grammatical variations) as
used herein is used in the inclusive sense of "having" or
"including," with which it may be used interchangeably. These terms
are not to be construed as being used in the exclusive sense of
"consisting only of" unless explicitly so stated.
[0187] Other than where expressly indicated, all numbers expressing
quantities of ingredients and/or reaction conditions are to be
understood as being modified in all instances by the term "about."
This includes terms such as "all" or "none" and variants thereof.
As used herein, the modifier "about" means within the limits that
one of skill in the art would expect with regard to the particular
quantity defined; this may be, for example, in various embodiments,
.+-.10% of the indicated number, .+-.5% of the indicated number,
.+-.2% of the indicated number, .+-.1% of the indicated number,
.+-.0.5% of the indicated number, or .+-.0.1% of the indicated
number.
[0188] Additionally, where ranges are given, it is understood that
the endpoints of the range define additional embodiments, and that
subranges including those not expressly recited are also intended
to include additional embodiments.
[0189] As used herein, "formed from," "generated by," and
variations thereof, mean obtained from chemical reaction of,
wherein "chemical reaction," includes spontaneous chemical
reactions and induced chemical reactions. As used herein, the
phrases "formed from" and "generated by" are open ended and do not
limit the components of the composition to those listed.
[0190] The compositions and methods according to the present
disclosure can comprise, consist of, or consist essentially of the
elements and limitations described herein, as well as any
additional or optional ingredients, components, or limitations
described herein or otherwise known in the art.
[0191] It should be understood that, unless explicitly stated
otherwise, the steps of various methods described herein may be
performed in any order, and not all steps must be performed, yet
the methods are still intended to be within the scope of the
disclosure.
[0192] It will be appreciated that variants of the above-disclosed
and other features and functions, or alternatives thereof, may be
combined into many other different systems or applications. Various
presently unforeseen or unanticipated alternatives, modifications,
variations, or improvements therein may be subsequently made by
those skilled in the art which are also intended to be encompassed
by the following claims.
* * * * *