U.S. patent application number 14/590892 was filed with the patent office on 2015-07-09 for methods and devices for analytical sensing of biogenic amines.
The applicant listed for this patent is South Carolina, University of. Invention is credited to John J. Lavigne, Mark Maynor, Toby Nelson.
Application Number | 20150192555 14/590892 |
Document ID | / |
Family ID | 40088742 |
Filed Date | 2015-07-09 |
United States Patent
Application |
20150192555 |
Kind Code |
A1 |
Lavigne; John J. ; et
al. |
July 9, 2015 |
Methods and Devices for Analytical Sensing of Biogenic Amines
Abstract
Disclosed herein are chromogenic response polymers and methods
and devices that utilize the disclosed polymers which are suitable
for use in detecting the presence of and identity of biogenic
amines.
Inventors: |
Lavigne; John J.; (Columbia,
SC) ; Nelson; Toby; (Columbia, SC) ; Maynor;
Mark; (Columbia, SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
South Carolina, University of |
Columbia |
SC |
US |
|
|
Family ID: |
40088742 |
Appl. No.: |
14/590892 |
Filed: |
January 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12079105 |
Mar 24, 2008 |
8927293 |
|
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14590892 |
|
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60919763 |
Mar 23, 2007 |
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Current U.S.
Class: |
436/98 ;
436/111 |
Current CPC
Class: |
G01N 33/12 20130101;
G01N 2021/7759 20130101; G01N 2021/7783 20130101; G01N 21/77
20130101; Y10T 436/173845 20150115; G01N 2021/7766 20130101; G01N
21/78 20130101; Y10T 436/147777 20150115; G01N 33/523 20130101;
Y10T 436/17 20150115 |
International
Class: |
G01N 33/12 20060101
G01N033/12; G01N 21/78 20060101 G01N021/78 |
Claims
1. A method for detecting the presence of a biogenic amine,
comprising: a) providing a chromogenic-responsive polymer; b)
contacting the polymer in step (a) with an analyte; and c)
detecting a chromogenic response.
2. A method according to claim 1, wherein the polymer comprises a
plurality of residues chosen from: ii) thiophenyl; iii) furanyl;
iv) pyrrolyl; v) fluorenyl; vi) 1,4-phenylene ethynylene; and vii)
1,4-phenylene.
3. A method according to claim 1, wherein the polymer is chosen
from: ##STR00035## wherein the index n has a value such that the
polymer has a molecular weight of from about 1,000 Da to about
1,000,000 Da; the indices a+b=n; X is chosen from S, N, or O; R and
R.sup.1 are each independently chosen from: i) --H; ii) --OM; iii)
--CO.sub.2M; iv) --SO.sub.3M; v) --PO.sub.3M.sub.2; and vi)
--OCH.sub.3; R.sup.2 is chosen from: i) --H; i) --OM; ii)
--CO.sub.2M; iii) --SO.sub.3M; iv) --PO.sub.3M.sub.2; and v)
--OCH.sub.3; R.sup.3 and R.sup.4 are each independently chosen
from: i) --H; ii) --OM; iii) --CO.sub.2M; iv) --SO.sub.3M; ii)
--PO.sub.3M.sub.2; and vi) --OCH.sub.3; or R.sup.3 and R.sup.4 can
be taken together to form a ring together with the two phenyl rings
to form a fused ring having from 5 to 7 carbon atoms; M is
hydrogen, an alkali metal, an alkali earth metal, a transition
metal, or a lanthanide metal; L, L.sup.1, L.sup.3, and L.sup.4 are
each independently chosen from: i) --(CR.sup.5aR.sup.5b).sub.m--;
or ii) --[O(CR.sup.5aR.sup.5b).sub.m].sub.k--; and L.sup.2 and
L.sup.5 are each chosen from: i) --(CR.sup.5aR.sup.5b).sub.m--; or
ii) --[O(CR.sup.5aR.sup.5b).sub.p].sub.k--; and iii)
--(CR.sup.5aR.sup.5b).sub.j(CH.dbd.CH)(CR.sup.5aR.sup.5b).sub.j--;
iv)
--(CR.sup.5aR.sup.5b).sub.j(C.ident.C)(CR.sup.5aR.sup.5b).sub.j--;
R.sup.5a and R.sup.5b are each independently hydrogen or methyl;
the index j is an integer from 0 to 10; the index k is an integer
from 1 to 20; the index m is an integer from 0 to 10; and the index
p is an integer from 2 to 20.
4. A method according to claim 1, wherein the polymer has the
formula: ##STR00036## wherein R is chosen from: i) --CO.sub.2M; ii)
--SO.sub.3M; or iii) --PO.sub.3M.sub.2; M is chosen from Na.sup.+,
Co.sup.2+, Cu.sup.2+, Fe.sup.2+, Ni.sup.2+, Ln.sup.3+, Eu.sup.3+,
and Tb.sup.3+; L is chosen from: i) --CH.sub.2--; ii)
--CH.sub.2CH.sub.2--; iii) --CH.sub.2CH.sub.2CH.sub.2--; or iv)
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--; and the index n is an integer
such that the molecular weight of the polymer is from about 2,000
Da to about 12,000 Da.
5. A method according to claim 1, wherein the polymer has the
formula: ##STR00037## wherein M is chosen from Na.sup.+, Co.sup.2+,
Cu.sup.2+, and Ni.sup.2+; and the index n is an integer such that
the molecular weight of the polymer is from about 5,000 Da to about
10,000 Da.
6. A method according to claim 1, wherein the analyte comprises one
or more amines.
7. A method according to claim 1, wherein the analyte comprises one
or more amines having the formula:
R.sup.12--(CR.sup.10aR.sup.10b).sub.p--(Y).sub.r--(CR.sup.10cR.sup.10d).s-
ub.q--NH.sub.2 wherein R.sup.10a, R.sup.10b, R.sup.10c, and
R.sup.10d are each independently chosen from: i) hydrogen; ii)
methyl; and iii) --NH.sub.2; the index p is an integer from 0 to 6;
the index q is an integer from 0 to 6; Y is: i) --NH--; or ii)
--(CR.sup.11aR.sup.11b).sub.sNH.sub.2; R.sup.11a and R.sup.11b are
each independently: i) hydrogen; or ii) methyl; the index r is 0 or
1; the index s is an integer from 1 to 6; and R.sup.12 is chosen
from: ii) hydrogen; iii) --NH.sub.2; iv) heterocyclic; v) phenyl;
or vi) heteroaryl.
8. A method according to claim 1, wherein the analyte comprises one
or more amines chosen from: ##STR00038##
9. A method according to claim 1, wherein the analyte further
comprises a solvent.
10. A method according to claim 9, wherein the solvent is
water.
11. A method according to claim 9, wherein the solvent is chosen
from a C.sub.1-C.sub.10 alcohol, a C.sub.3-C.sub.6 ester, a
C.sub.3-C.sub.6 ketone, a C.sub.4-C.sub.8 ether, a C.sub.1-C.sub.5
nitrile, a C.sub.1-C.sub.4 di-alkylformamide, a C.sub.1-C.sub.4
di-alkylsulfoxide, and mixtures thereof.
12. A method according to claim 1, wherein the analyte further
comprises a solvent chosen from water, acetonitrile,
dimethylformamide, dimethylsulfoxide, methyl ethyl ketone, methyl
acetate, ethyl acetate, and methyl tert-butyl ether.
13. A method according to claim 1, wherein the analyte further
comprises a buffer.
14. A method according to claim 1, wherein the pH of the analyte is
from about 6 to about 8.
15. A method according to claim 1, wherein the chromogenic response
is ultra violet absorption or emission.
16. A method according to claim 1, wherein the chromogenic response
is visible light absorption or emission.
17. A method according to claim 1, wherein the analyte is taken
from a food source.
18-39. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/079,105, filed Mar. 24, 2008, which claims
the benefit of priority from prior U.S. Provisional Application
Ser. No. 60/919,763, filed Mar. 23, 2007, the entire contents of
which are incorporated herein by reference.
FIELD
[0002] Disclosed herein are chromogenic responsive polymers and
methods and devices that utilize the disclosed polymers which are
suitable for use in detecting the presence of and identity of
biogenic amines.
BACKGROUND
[0003] The ability to sense or measure a physical quantity or
chemical substance has been a desirable research endeavor for
decades. Increasingly, there exists a need for rapid, accurate,
reproducible, and economical sensors. Of particular interest to
many are sensors capable of detecting biologically relevant
analytes. A method for the accurate detection, quantification, and
discrimination of biological molecules would be of great benefit to
many technical fields including biochemistry, food science, and
medicine.
[0004] Technology capable of sensing biologically relevant analytes
exists. Noteworthy are electronic nose and tongue technologies that
are finding increasing utility in the food science industry. While
these technologies and technologies alike are able to identify
different classes of analytes, their ability to discriminate
between analytes of the same class is limited. One class of
analytes that can often be difficult to detect and discriminate
between as well as from among other analytes is biogenic amines.
Biogenic amines have been associated with a variety of problems in
the food science and medical industries. An increased production of
biogenic amine by an organism, for example, can result from rapid
cell proliferation. Consequently, biogenic amine levels can serve
as indicators of health complications, including cancer, bacterial
infection, and food poisoning, to name a few. If a human, for
example, is exposed to elevated biogenic amine levels present in
food, this exposure can trigger a wide range of symptoms ranging
from headaches to life-threatening episodes of blood pressure
spikes.
[0005] Biogenic amines can also serve as indicators of food
spoilage caused by bacteria (i.e. to indirectly detect the presence
of bacteria). Food spoilage (e.g., meat and fish spoilage) occurs
as bacteria begin to grow shortly after the time of slaughter.
During the initial stages of food spoilage, free amino acids are
decarboxylated by enzymes released by invading spoilage
microorganisms. The product of decarboxylation includes biogenic
amines, namely putrescine and cadaverine. These two amines are
particularly distinctive in odor and correlate well with surface
bacterial counts. Another product, histamine, is of interest due to
its alleged ability to induce histamine intoxication, a form of
food poisoning associated with the consumption of spoiled fish.
[0006] Several methods exist for detecting biogenic amines.
Classical methods for detecting biogenic amines include
chromatographic techniques, such as gas chromatography, thin layer
chromatography, reversed phase liquid chromatography, and liquid
chromatography. However, these techniques often require sample
pre-treatment and relatively long analysis time, which can increase
costs and thereby make many of these methods not suitable for
routine use. Other more advanced methods for detecting biogenic
amines include the use of molecular imprinted polymers (MIPs),
enzymes, antibodies, single molecule, and array based sensors.
Although current amine sensors (e.g., biogenic amine sensors) show
promise, there is still a need to identify and develop new and
improved sensors. Particularly, a need exists for technology
capable of discriminating analytes within the same or similar class
of analytes.
SUMMARY
[0007] In accordance with the purposes of the disclosed materials,
compositions, articles, devices, and methods, as embodied and
broadly described herein, the disclosed subject matter, in one
aspect, relates to compounds and compositions (e.g.,
poly(thiophene) and poly(thiophene) derivatives) and methods for
providing and using such compounds and compositions. In another
aspect, the disclosed subject matter relates to devices comprising
the disclosed compounds and compositions.
[0008] Additional advantages will be set forth in part in the
description that follows, and in part will be obvious from the
description, or may be learned by practice of the aspects described
below. The advantages described below will be realized and attained
by means of the elements and combinations particularly pointed out
in the appended claims. 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.
BRIEF DESCRIPTION OF THE FIGURES
[0009] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several aspects
described below.
[0010] The application file contains at least one photograph
executed in color. Copies of this patent application publication
with color photographs will be providing by the Office upon request
and payment of the necessary fee.
[0011] FIG. 1 depicts the absorbance spectrum of a series of
solution comprising the chromogenic polymer of Example 1. The curve
indicated with a solid circle is the absorbance spectrum obtained
of the polymer alone (control), the curve indicated with a solid
square is the absorbance spectrum obtained from a solution of the
polymer in the presence of 1,2-ethylenediamine, the curve indicated
with a clear circle is the absorbance spectrum obtained from a
solution of the polymer in the presence of 1,4-butylenediamine, and
the curve indicated with a clear square is the absorbance spectrum
obtained from a solution of the polymer in the presence of
1,6-hexylenediamine.
[0012] FIG. 2 depicts a representation of the aggregative
interactions between a disclosed poly(thiophene) and three diamines
having differing lengths.
[0013] FIG. 3 depicts the change in the absorption spectrum of
polymer 1 with increasing concentrations of histamine in a tuna
fish matrix.
[0014] FIG. 4 depicts a linear ratiometric response
(A.sub.530/A.sub.420) with increasing histamine concentration in a
tuna fish matrix.
[0015] FIG. 5 depicts a photograph of solutions of polymer 2 with
three different substrate recognition elements and the
corresponding chromogenic response to a variety of diamines. Series
No. 1 comprises Co.sup.2+, series No. 2 comprises Cu.sup.2+ and
series No. 3 comprises Ni.sup.2+. Column A: polymer 2 in solution;
Column B: polymer 2 with metals as indicated above; Column C:
addition of 1,2-ethylenediamine; Column D: addition of
1,3-propylenediamine; Column E: addition of putrescine; Column F:
addition of cadaverine; Column G: addition of histamine; Column H:
addition of spermidine; Column I: addition of spermine.
[0016] FIG. 6 is a photograph depicting the color patterns observed
for various amines upon interaction with polymer 2 with different
recognition elements. Sample A is 1,2-ethylenediamine, sample B is
1,3-propylenediamine, sample C is putrescine, sample D is
cadaverine, sample E is histamine, sample F is spermidine, and
sample G is spermine.
[0017] FIG. 7 depicts a device schematic that can be used by either
the consumer or the inspector of food stuffs to determine the
degree of spoilage.
[0018] FIG. 8 depicts an exemplary dip-stick device, displaying
multiple polymer composites that would generate a pattern for
sensing biogenic amines.
[0019] FIG. 9 depicts the projection of the LDA results in three
dimensions for Polymer 1 differentiating structurally similar
diamines. Each point in the plot contains information from the nine
wavelengths taken from the spectrum for the specific diamine.
[0020] FIG. 10 depicts a plot of LDA results in two dimensions for
detecting amines in highly competitive aqueous media using Polymer
2. Each axis of the LDA plot represents weighted combinations of
the 36 dimensional data, where each point in the plot is an
individual replicate that contains information from the 36
wavelengths of the relevant spectrum. The circles around each
cluster represent 95% confidence limits.
[0021] FIG. 11 shows the absorbance spectra of 2 responding to the
addition of six different fish extracts from decaying tuna fish
flesh over time.
[0022] FIG. 12 depicts a linear ratiometric response
(A.sub.500/A.sub.420) from Polymer 2 as tuna fish flesh decays over
time.
[0023] FIG. 13 depicts a plot of the polymer 2 response in the red
channel from a lateral flow assay responding to putrescine at
various concentrations.
[0024] FIG. 14 depicts the absorbance spectra for combinations of
amines. The line with clear squares represents a 1:2 mixture of
putrescine and histamine, the line with solid squares represents a
1:2 mixture of putrescine and cadverine, and the line with solid
circles represents a 1:2 mixture of cadaverine and histamine
[0025] FIG. 15 depicts the absorbance spectra for combinations of
amines. The line with clear squares represents a 2:1 mixture of
putrescine and histamine, the line with solid squares represents a
2:1 mixture of putrescine and cadverine, and the line with circles
represents a 2:1 mixture of cadaverine and histamine
[0026] FIG. 16 depicts the absorbance spectra for combinations of
amines. The line with the solid squares represents a 1:1 mixture of
putrescine and histamine, the line with solid circles represents a
1:1 mixture of putrescine and cadverine, the line with clear
squares represents a 1:1 mixture of cadaverine and histamine, and
the line with clear circles represents a 1:1:1 mixture of
putrescine, cadaverine and histamine.
DETAILED DESCRIPTION
[0027] The materials, compounds, compositions, articles, and
methods described herein may be understood more readily by
reference to the following detailed description of specific aspects
of the disclosed subject matter and the Examples included therein
and to the Figures.
[0028] Before the present materials, compounds, compositions,
articles, and methods are disclosed and described, it is to be
understood that the aspects described below are not limited to
specific synthetic methods or specific reagents, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular aspects only and
is not intended to be limiting.
[0029] Also, throughout this specification, various publications
are referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which the disclosed matter pertains. The references disclosed are
also individually and specifically incorporated by reference herein
for the material contained in them that is discussed in the
sentence in which the reference is relied upon.
[0030] In this specification and in the claims that follow,
reference will be made to a number of terms, which shall be defined
to have the following meanings:
[0031] Throughout this specification, unless the context requires
otherwise, the word "comprise," or variations such as "comprises"
or "comprising," will be understood to imply the inclusion of a
stated integer or step or group of integers or steps but not the
exclusion of any other integer or step or group of integers or
steps.
[0032] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a pharmaceutical carrier" includes
mixtures of two or more such carriers, and the like.
[0033] "Optional" or "optionally" means that the subsequently
described event or circumstance can or cannot occur, and that the
description includes instances where the event or circumstance
occurs and instances where it does not.
[0034] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another aspect includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another aspect. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint.
[0035] A weight percent of a component, unless specifically stated
to the contrary, is based on the total weight of the formulation or
composition in which the component is included.
[0036] By "contacting" is meant the physical contact of at least
one substance to another substance.
[0037] By "sufficient amount" and "sufficient time" means an amount
and time needed to achieve the desired result or results, e.g.,
dissolve a portion of the polymer.
[0038] "Admixture" or "blend" as generally used herein means a
physical combination of two or more different components. In the
case of polymers, an admixture, or blend, of polymers is a physical
blend or combination of two or more different polymers as opposed
to a copolymer which is single polymeric material that is comprised
of two or more different monomers.
[0039] "Absorbable" as used herein means the complete degradation
of a material in vivo, and elimination of its metabolites from an
animal or human subject.
[0040] "Molecular weight" as used herein, unless otherwise
specified, refers generally to the relative average molecular
weight of the bulk polymer. In practice, molecular weight can be
estimated or characterized in various ways including gel permeation
chromatography (GPC) or capillary viscometry. GPC molecular weights
are reported as the weight-average molecular weight (Mw) or as the
number-average molecular weight (Mn). Capillary viscometry provides
estimates of molecular weight as the Inherent Viscosity (IV)
determined from a dilute polymer solution using a particular set of
concentration, temperature, and solvent conditions. Unless
otherwise specified, IV measurements are made at 30.degree. C. on
solutions prepared in chloroform at a polymer concentration of 0.5
g/dL.
[0041] "Analyte" as used herein, unless otherwise specified, refers
to a sample solution that contains one or more amines. The analyte
can be a sample directly taken from a source to be tested or the
analyte can be a solution, for example, a buffered solution into
which a sample has been placed prior to contacting the analyte with
the disclosed polymers or prior to being analyzed by the methods,
devices, and/or kits disclosed herein.
[0042] Substituted and unsubstituted acyclic units comprising from
1 to 24 carbon atoms encompass 3 categories of units: linear or
branched alkyl, non-limiting examples of which include, methyl
(C.sub.1), ethyl (C.sub.2), n-propyl (C.sub.3), iso-propyl
(C.sub.3), n-butyl (C.sub.4), sec-butyl (C.sub.4), iso-butyl
(C.sub.4), tert-butyl (C.sub.4), and the like; substituted linear
or branched alkyl, non-limiting examples of which includes,
hydroxymethyl (C.sub.1), chloromethyl (C.sub.1), trifluoromethyl
(C.sub.1), aminomethyl (C.sub.1), 1-chloroethyl (C.sub.2),
2-hydroxyethyl (C.sub.2), 1,2-difluoroethyl (C.sub.2),
3-carboxypropyl (C.sub.3), and the like; linear or branched
alkenyl, non-limiting examples of which include, ethenyl (C.sub.2),
3-propenyl (C.sub.3), 1-propenyl (also 2-methylethenyl) (C.sub.3),
isopropenyl (also 2-methylethen-2-yl) (C.sub.3), buten-4-yl
(C.sub.4), and the like; substituted linear or branched alkenyl,
non-limiting examples of which include, 2-chloroethenyl (also
2-chlorovinyl) (C.sub.2), 4-hydroxybuten-1-yl (C.sub.4),
7-hydroxy-7-methyloct-4-en-2-yl (C.sub.9),
7-hydroxy-7-methyloct-3,5-dien-2-yl (C.sub.9), and the like; and
linear or branched alkynyl, non-limiting examples of which include,
ethynyl (C.sub.2), prop-2-ynyl (also propargyl) (C.sub.3),
propyn-1-yl (C.sub.3), and 2-methyl-hex-4-yn-1-yl (C.sub.7);
substituted linear or branched alkynyl, non-limiting examples of
which include, 5-hydroxy-5-methylhex-3-ynyl (C.sub.7),
6-hydroxy-6-methylhept-3-yn-2-yl (C.sub.8),
5-hydroxy-5-ethylhept-3-ynyl (C.sub.9), and the like.
[0043] Substituted and unsubstituted cyclic units comprising from 3
to 24 carbon atoms encompass the following units: carbocyclic rings
having a single substituted or unsubstituted hydrocarbon ring,
non-limiting examples of which include, cyclopropyl (C.sub.3),
2-methyl-cyclopropyl (C.sub.3), cyclopropenyl (C.sub.3), cyclobutyl
(C.sub.4), 2,3-dihydroxycyclobutyl (C.sub.4), cyclobutenyl
(C.sub.4), cyclopentyl (C.sub.5), cyclopentenyl (C.sub.5),
cyclopentadienyl (C.sub.5), cyclohexyl (C.sub.6), cyclohexenyl
(C.sub.6), cycloheptyl (C.sub.7), cyclooctanyl (C.sub.8), decalinyl
(C.sub.10), 2,5-dimethylcyclopentyl (C.sub.5),
3,5-dichlorocyclohexyl (C.sub.6), 4-hydroxycyclohexyl (C.sub.6),
and 3,3,5-trimethylcyclohex-1-yl (C.sub.6); carbocyclic rings
having two or more substituted or unsubstituted fused hydrocarbon
rings, non-limiting examples of which include, octahydropentalenyl
(C.sub.8), octahydro-1H-indenyl (C.sub.9), 3a,
4,5,6,7,7a-hexahydro-3H-inden-4-yl (C.sub.9), decahydroazulenyl
(C.sub.10); and carbocyclic rings which are substituted or
unsubstituted bicyclic hydrocarbon rings, non-limiting examples of
which include, bicyclo-[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl,
bicyclo[3.1.1]heptanyl, 1,3-dimethyl[2.2.1]heptan-2-yl,
bicyclo[2.2.2]octanyl, and bicyclo[3.3.3]undecanyl.
[0044] Substituted and unsubstituted aryl units comprising from 6
to 24 carbon atoms encompass the following units: C.sub.6,
C.sub.10, or C.sub.14 substituted or unsubstituted aryl rings;
phenyl, naphthyl, anthracenyl, phenanthryl, and the like whether
substituted or unsubstituted, non-limiting examples of which
include, phenyl (C.sub.6), naphthylen-1-yl (C.sub.10),
naphthylen-2-yl (C.sub.10), 4-fluorophenyl (C.sub.6),
2-hydroxyphenyl (C.sub.6), 3-methylphenyl (C.sub.6),
2-amino-4-fluorophenyl (C.sub.6), 2-(N,N-diethylamino)phenyl
(C.sub.6), 2-cyanophenyl (C.sub.6), 2,6-di-tert-butylphenyl
(C.sub.6), 3-methoxyphenyl (C.sub.6), 8-hydroxynaphthylen-2-yl
(C.sub.10), 4,5-dimethoxynaphthylen-1-yl (C.sub.10), and
6-cyano-naphthylen-1-yl (C.sub.10); C.sub.6, C.sub.10, or C.sub.14
aryl rings fused with 1 or 2 saturated rings non-limiting examples
of which include, bicyclo[4.2.0]octa-1,3,5-trienyl (C.sub.8), and
indanyl (C.sub.9).
[0045] Substituted and unsubstituted heterocyclic or heteroaryl
units comprising from 1 to 24 carbon atoms encompasses the
following units all of which contain at least one heteroatom in at
least one ring chosen from nitrogen (N), oxygen (O), sulfur (S),
phosphorous (P) or mixtures of N, O, S, and P: heterocyclic units
having a single ring containing one or more heteroatoms chosen from
nitrogen (N), oxygen (O), or sulfur (S), or mixtures of N, O, and
S, non-limiting examples of which include, diazirinyl (C.sub.1),
aziridinyl (C.sub.2), urazolyl (C.sub.2), azetidinyl (C.sub.3),
pyrazolidinyl (C.sub.3), imidazolidinyl (C.sub.3), oxazolidinyl
(C.sub.3), isoxazolinyl (C.sub.3), isoxazolyl (C.sub.3),
thiazolidinyl (C.sub.3), isothiazolyl (C.sub.3), isothiazolinyl
(C.sub.3), oxathiazolidinonyl (C.sub.3), oxazolidinonyl (C.sub.3),
hydantoinyl (C.sub.3), tetrahydropyranyl (C.sub.4), pyrrolidinyl
(C.sub.4), morpholinyl (C.sub.4), piperazinyl (C.sub.4),
piperidinyl (C.sub.4), dihydropyranyl (C.sub.5), tetrahydropyranyl
(C.sub.5), piperidin-2-onyl (valerolactam) (C.sub.5),
2,3,4,5-tetrahydro-1H-azepinyl (C.sub.6), 2,3-dihydro-1H-indole
(C.sub.8), and 1,2,3,4-tetrahydro-quinoline (C.sub.9); heterocyclic
units having 2 or more rings one of which is a heterocyclic ring,
non-limiting examples of which include hexahydro-1H-pyrrolizinyl
(C.sub.7), 3a, 4,5,6,7,7a-hexahydro-1H-benzo[d]imidazolyl
(C.sub.7), 3a, 4,5,6,7,7a-hexahydro-1H-indolyl (C.sub.8),
1,2,3,4-tetrahydroquinolinyl (C.sub.9), and
decahydro-1H-cycloocta[b]pyrrolyl (C.sub.10); heteroaryl rings
containing a single ring, non-limiting examples of which include,
1,2,3,4-tetrazolyl (C.sub.1), [1,2,3]triazolyl (C.sub.2),
[1,2,4]triazolyl (C.sub.2), triazinyl (C.sub.3), thiazolyl
(C.sub.3), 1H-imidazolyl (C.sub.3), oxazolyl (C.sub.3), furanyl
(C.sub.4), thiopheneyl (C.sub.4), pyrimidinyl (C.sub.4),
2-phenylpyrimidinyl (C.sub.4), pyridinyl (C.sub.5),
3-methylpyridinyl (C.sub.5), and 4-dimethylaminopyridinyl
(C.sub.5); heteroaryl rings containing 2 or more fused rings one of
which is a heteroaryl ring, non-limiting examples of which include:
7H-purinyl (C.sub.5), 9H-purinyl (C.sub.5), 6-amino-9H-purinyl
(C.sub.5), 5H-pyrrolo[3,2-d]pyrimidinyl (C.sub.6),
7H-pyrrolo[2,3-d]pyrimidinyl (C.sub.6), pyrido[2,3-d]pyrimidinyl
(C.sub.7), 2-phenylbenzo[d]thiazolyl (C.sub.7), 1H-indolyl
(C.sub.8), 4,5,6,7-tetrahydro-1-H-indolyl (C.sub.8), quinoxalinyl
(C.sub.8), 5-methylquinoxalinyl (C.sub.8), quinazolinyl (C.sub.8),
quinolinyl (C.sub.9), 8-hydroxy-quinolinyl (C.sub.9), and
isoquinolinyl (C.sub.9).
[0046] The term "arylalkylene" is used throughout the specification
to refer to substituted or unsubstituted C.sub.6, C.sub.10, or
C.sub.14 aryl rings tethered to another unit through a substituted
or unsubstituted C.sub.1-C.sub.12 alkylene unit. These units can be
referred to by indicating the number of carbons contained in the
alkylene unit followed by the number of carbon atoms in the aryl
unit, or by their chemical name. A non-limiting example of tethered
cyclic hydrocarbyl units includes a substituted or unsubstituted
benzyl. A substituted or unsubstituted benzyl unit contains a
tether containing one carbon atom (methylene) and a substituted or
unsubstituted aryl ring containing six carbon atoms, or a
C.sub.1-(C.sub.6) unit, having the formula:
##STR00001##
[0047] wherein R.sup.a is optionally one or more independently
chosen substitutions for hydrogen. Further examples include other
aryl units, inter alia, (2-hydroxyphenyl)hexyl C.sub.6-(C.sub.6);
naphthalen-2-ylmethyl C.sub.1-(C.sub.10), 4-fluorobenzyl
C.sub.1-(C.sub.6), 2-(3-hydroxy-phenyl)ethyl C.sub.2-(C.sub.6), as
well as substituted and unsubstituted C.sub.3-C.sub.10
alkylenecarbocyclic units, for example, cyclopropylmethyl
C.sub.1-(C.sub.3), cyclopentylethyl C.sub.2-(C.sub.5),
cyclohexylmethyl C.sub.1-(C.sub.6).
[0048] The terms "heteroarylalkylene" and "heterocyclicalkylene"
are used throughout the specification to refer to substituted or
unsubstituted heteroaryl and heterocyclic rings as defined herein
above containing from 1 to 24 carbon atoms that are tethered to
another unit through a substituted or unsubstituted
C.sub.1-C.sub.12 alkylene unit. These units can be referred to by
indicating the number of carbons contained in the alkylene unit
followed by the number of carbon atoms in the heteroaryl and
heterocyclic unit, or by their chemical name. A non-limiting
example includes substituted and unsubstituted C.sub.1-C.sub.10
alkylene-heteroaryl units, for example a 2-picolyl
C.sub.1-(C.sub.6) unit having the formula:
##STR00002##
[0049] wherein R.sup.a is the same as defined above. In addition,
C.sub.1-C.sub.12 tethered cyclic hydrocarbyl units include
C.sub.1-C.sub.10 alkyleneheterocyclic units and alkylene-heteroaryl
units, non-limiting examples of which include, aziridinylmethyl
C.sub.1-(C.sub.2) and oxazol-2-ylmethyl C.sub.1-(C.sub.3).
[0050] Reference will now be made in detail to specific aspects of
the disclosed materials, compounds, compositions, articles, and
methods, examples of which are illustrated in the accompanying
Examples.
[0051] Disclosed herein are materials, compounds, compositions, and
components that can be used for, can be used in conjunction with,
can be used in preparation for, or are products of the disclosed
methods, devices, and compositions. These and other materials are
disclosed herein, and it is understood that when combinations,
subsets, interactions, groups, etc. of these materials are
disclosed that while specific reference of each various individual
and collective combinations and permutation of these compounds may
not be explicitly disclosed, each is specifically contemplated and
described herein. For example, if a composition is disclosed and a
number of modifications that can be made to a number of components
or residues of the composition are discussed, each and every
combination and permutation that are possible are specifically
contemplated unless specifically indicated to the contrary. Thus,
if a class of polymers A, B, and C are disclosed as well as a class
of polymers D, E, and F, and an example of a copolymer A-D is
disclosed, then even if each is not individually recited, each is
individually and collectively contemplated. Thus, in this example,
each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F
are specifically contemplated and should be considered disclosed
from disclosure of A, B, and C; D, E, and F; and the example
combination A-D. Likewise, any subset or combination of these is
also specifically contemplated and disclosed. Thus, for example,
the sub-group of A-E, B-F, and C-E are specifically contemplated
and should be considered disclosed from disclosure of A, B, and C;
D, E, and F; and the example combination A-D. This concept applies
to all aspects of this disclosure including, but not limited to,
steps in methods of making and using the disclosed compositions.
Thus, if there are a variety of additional steps that can be
performed it is understood that each of these additional steps can
be performed with any specific aspect or combination of aspects of
the disclosed methods, and that each such combination is
specifically contemplated and should be considered disclosed.
[0052] Certain materials, compounds, compositions, and components
disclosed herein can be obtained commercially or readily
synthesized using techniques generally known to those of skill in
the art. For example, the starting materials and reagents used in
preparing the disclosed compounds and compositions are either
available from commercial suppliers such as Aldrich Chemical Co.,
(Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher
Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are
prepared by methods known to those skilled in the art following
procedures set forth in references such as Fieser and Fieser's
Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,
1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and
Supplementals (Elsevier Science Publishers, 1989); Organic
Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's
Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and
Larock's Comprehensive Organic Transformations (VCH Publishers
Inc., 1989).
[0053] The disclosed methods and articles comprise one or more
chromogenic polymers. Without wishing to be limited by theory,
amines or polyamines interact with the chromogenic polymers in
solution to cause a distinct color change that can be equated to
the presence of a particular amine. Of particular importance are
diamines and polyamines, inter alia, the degradation products of
amino acids; putrescine, cadaverine, histamine, spermine and
spermidine. The diamines interact with the chromogenic polymers to
affect one or more bulk properties and or molecular level events of
the polymer, for example, the length of polymer conjugation, change
in polymer aggregation, and therefore changing the absorption
spectrum of a solution of the disclosed chromogenic polymers. The
change in bulk properties also results in the scattering of visible
light by the solutions containing an amine.
[0054] Of particular interest in the food industry and to the
consumer is the ability to detect the presence of amines that can
signal either spoilage of beef, fish, chicken, pork, and the like,
but whether spoilage has begun and to what degree spoilage, if any,
has occurred. In addition, the disclosed methods can be used to
identify the presence of biologically important amines.
Non-limiting examples of amines include 1,2-ethylenediamine,
putrescine, cadaverine, agmatine, spermine, spermidine, tryptamine,
histamine, phenylethylamine, tyramine, serotonin, and dopamine.
[0055] One aspect of the disclosed methods and articles relates to
identifying the presence of one or more amines chosen from:
##STR00003##
Chromogenic Polymers
[0056] The disclosed methods and articles utilize chromogenic
polymers. The chromogenic polymers as further described herein are
polymers comprising a conjugated backbone. Conjugated polymers have
been used as chemical and biological sensors because, at least in
part, of their distinctive optical and electronic properties. For
example, conjugated polymers have been used for the detection of
inorganic ions, small organic compounds, DNA, proteins, and for the
determination of stereochemistry. A principal signal transduction
mechanism for these materials has relied on the
planarization/deplanarization (i.e., a single structural change) of
the polymer backbone upon exposure of the side-chain functional
group to an analyte. As a result, methods relying on a signal
transduction mechanism (i.e. a single perturbation) can be limited
to a single-dimensional response that is often expressed as a
function of the change of one or two wavelengths (if the signal is
optical, for example) in a relevant polymer spectrum.
[0057] Disclosed herein is an alternative approach based on a
multistate, multidimensional response derived from numerous dynamic
polymer-analyte interactions. While not wishing to be bound by
theory, it is believed that such dynamic polymer-analyte
interactions cause main-chain conformational changes, as well as
.pi.-.pi. stacking between polymer chains and scattering of visible
light caused by the solution stable polymer-analyte aggregates. It
should be appreciated that given such a dynamic equilibrium between
a polymer and an analyte exists, multiple assemblies can, at least
in theory, form in solution, each of which can have their own
distinctive spectral properties. Thus, disclosed herein are methods
aimed at harnessing a collective response from multiple
interactions that characterize the overall shape of a spectrum,
thereby enabling the discrimination between specific compounds that
are found in the analytes or the concentration of a compound or
compounds in the analyte solution.
[0058] The disclosed chromogenic polymers comprise a backbone
having a plurality of one or more conjugated ring systems and a
substrate recognition element. Non-limiting examples of backbone
elements include thiophenyl, furanyl, pyrrolyl, fluorenyl,
1,4-phenylene ethynylene, and 1,4-phenylene. Non-limiting examples
of chromogenic polymer backbones include:
##STR00004##
wherein the index n has a value such that the polymer has a
molecular weight of from about 1,000 Da to about 2,000,000 Da. The
disclosed chromogenic response polymers have an average molecular
weight of from about 1000 Da to about 2,000,000 Da. In one
embodiment, the disclosed chromogenic response polymers have an
average molecular weight of from about 1000 Da to about 1,000,000
Da. In another embodiment, the disclosed chromogenic response
polymers have an average molecular weight of from about 1000 Da to
about 20,000 Da. In a further embodiment, the disclosed chromogenic
response polymers have an average molecular weight of from about
1000 Da to about 10,000 Da. In a yet another embodiment, the
disclosed chromogenic response polymers have an average molecular
weight of from about 5,000 Da to about 10,000 Da. In a still
further embodiment, the disclosed chromogenic response polymers
have an average molecular weight of from about 3,000 Da to about
8,000 Da. In a yet further embodiment, the disclosed chromogenic
response polymers have an average molecular weight of from about
5,000 Da to about 20,000 Da. In a still yet further embodiment, the
disclosed chromogenic response polymers have an average molecular
weight of from about 10,000 Da to about 20,000 Da. In one further
embodiment, the disclosed chromogenic response polymers have an
average molecular weight of from about 2,000 Da to about 5,000 Da.
However, the disclosed polymers can have any average molecular
weight, for example, 1,000 Da, 1,500 Da, 2,000 Da, 2,500 Da, 3,000
Da, 3,500 Da, 4,000 Da, 4,500 Da, 5,000 Da, 5,500 Da, 6,000 Da,
6,500 Da, 7,000 Da, 7,500 Da, 8,000 Da, 8,500 Da, 9,000 Da, 9,500
Da, or 10,000 Da rounded to the nearest 500 Da. However, the
polymers can have any discrete average molecular weight, for
example, 8,020 Da, 8,033 Da, 8,115 Da, 9,456 Da, and the like.
Therefore, the ranges of the various embodiments include all values
of average molecular weight within the range.
[0059] The average molecular weights of the disclosed polymers are
determined using only the backbone units prior to addition of the
substrate recognition elements, inter alia, transition metals. The
backbone unit having the formula:
##STR00005##
has an approximate molecular weight of 154 g/mol, therefore, a
chromogenic polymer having an average molecular weight of
approximately 1,500 Da will comprise about 10 of these units in the
backbone of the chromogeneic polymer.
X Units
[0060] X can be S, N, or O. When X is a sulfur atom, the
chromogenic polymers are poly(thiophene)s. When X is a nitrogen
atom, the chromogenic polymers are poly(pyrrole)s. When X is an
oxygen atom, the chromogenic polymers are poly(furan)s. However,
the disclosed polymers can have an admixture of different units,
for example, a mixture of thiophene units and furan units.
R Units
[0061] R units are moieties that interact with the disclosed
biogenic amines. R units are independently chosen from one another.
R units serve as a moiety that complexes with the one or more
substrate recognition elements and therefore serve to differentiate
between different biogenic amines. Non-limiting examples of R units
include:
[0062] i) --H;
[0063] ii) --OM;
[0064] iii) --CO.sub.2M;
[0065] iv) --SO.sub.3M;
[0066] v) --PO.sub.3M.sub.2; and
[0067] vi) --OCH.sub.3;
wherein M is hydrogen, ammonium, alkyl ammonium, an alkali metal,
an alkali earth metal, a transition metal, a lanthanide metal, or
mixtures thereof.
[0068] In one aspect of the disclosed chromogenic polymers, R units
comprise a carboxylate unit. Non-limiting examples of this aspect
of R units include:
##STR00006##
[0069] In another aspect of the disclosed chromogenic polymers, R
units comprise a sulfonate unit. Non-limiting examples of this
aspect of R units include:
##STR00007##
[0070] In a further aspect of the disclosed chromogenic polymers, R
units comprise a phosphonate unit. Non-limiting examples of this
aspect of R units include:
##STR00008##
[0071] In a yet further aspect of the disclosed chromogenic
polymers, R units are an admixture of charged units and non-charged
units. In one embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 90% of the R units
are carboxylate units and 10% of the R units are hydrogen. In
another embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 80% of the R units
are carboxylate units and 20% of the R units are hydrogen. In a
further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 70% of the R units
are carboxylate units and 30% of the R units are hydrogen. In a yet
further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 60% of the R units
are carboxylate units and 40% of the R units are hydrogen. In a
still further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 50% of the R units
are carboxylate units and 50% of the R units are hydrogen. However,
I one aspect of the disclosed polymers, all of the R units are
protonated.
[0072] In a further embodiment of this aspect, the chromogenic
polymers comprise a mixture of R units wherein at least 90% of the
R units are sulfonate units and 10% of the R units are hydrogen. In
another embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 80% of the R units
are sulfonate units and 20% of the R units are hydrogen. In a
further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 70% of the R units
are sulfonate units and 30% of the R units are hydrogen. In a yet
further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 60% of the R units
are sulfonate units and 40% of the R units are hydrogen. In a still
further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 50% of the R units
are sulfonate units and 50% of the R units are hydrogen.
[0073] In a yet further embodiment of this aspect, the chromogenic
polymers comprise a mixture of R units wherein at least 90% of the
R units are phosphonate units and 10% of the R units are hydrogen.
In another embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 80% of the R units
are phosphonate units and 20% of the R units are hydrogen. In a
further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 70% of the R units
are phosphonate units and 30% of the R units are hydrogen. In a yet
further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 60% of the R units
are phosphonate units and 40% of the R units are hydrogen. In a
still further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R units wherein at least 50% of the R units
are phosphonate units and 50% of the R units are hydrogen.
[0074] Another aspect relates to chromogenic polymers that comprise
a mixture of carboxylate and sulfonate units. In one embodiment of
this aspect, the chromogenic polymers comprise a mixture of R units
wherein at least 90% of the R units are carboxylate units and 10%
of the R units are sulfonate. In another embodiment of this aspect,
the chromogenic polymers comprise a mixture of R units wherein at
least 80% of the R units are carboxylate units and 20% of the R
units are sulfonate. In a further embodiment of this aspect, the
chromogenic polymers comprise a mixture of R units wherein at least
70% of the R units are carboxylate units and 30% of the R units are
sulfonate. In a yet further embodiment of this aspect, the
chromogenic polymers comprise a mixture of R units wherein at least
60% of the R units are carboxylate units and 40% of the R units are
sulfonate. In a still further embodiment of this aspect, the
chromogenic polymers comprise a mixture of R units wherein at least
50% of the R units are carboxylate units and 50% of the R units are
sulfonate.
R.sup.1 Units
[0075] R.sup.1 units are moieties that can interact with the
disclosed biogenic amines or R.sup.1 units can provide improved
organic phase or water phase solubility for the chromogenic
polymers. R.sup.1 units are independently chosen from one another.
R.sup.1 units when taken together with L.sup.1 linking units can
modify the hydrophilic or hydrophobic character of the chromogenic
polymers. In one aspect, R.sup.1 units can complex with the one or
more substrate recognition elements and thereby further enhance the
ability of the chromogenic polymers to differentiate between
different biogenic amines. When R.sup.1 units are the same or a
different anionic moiety, for example, a carboxylate moiety, the
L.sup.1 linking group is different than the L linking group present
for R units.
[0076] One aspect of R.sup.1 units relates to units that modify the
hydrophilic character of the polymers. In one embodiment, R.sup.1
is hydrogen and R.sup.1 is taken together with an L.sup.1 unit
comprising a polyalkyleneoxy unit as further described herein. In
another embodiment, R.sup.1 is hydroxyl and R.sup.1 is taken
together with an L.sup.1 unit comprising a polyalkyleneoxy unit as
further described herein. In a further embodiment, R.sup.1 is
methoxy and R.sup.1 is taken together with an L.sup.1 unit
comprising a polyalkyleneoxy unit as further described herein.
[0077] Another aspect of R.sup.1 units relates to units that modify
the hydrophobic character of the polymers. In one embodiment,
R.sup.1 is hydrogen and R.sup.1 is taken together with an L.sup.1
unit comprising a polyalkylene unit as further described
herein.
R.sup.2 Units
[0078] R.sup.2 units are moieties that interact with the disclosed
biogenic amines. R.sup.2 units are independently chosen from one
another. R.sup.2 units serve as a moiety that complexes with the
one or more substrate recognition elements and therefore serve to
differentiate between different biogenic amines. Non-limiting
examples of R.sup.2 units include:
[0079] i) --H;
[0080] ii) --OM;
[0081] iii) --CO.sub.2M;
[0082] iv) --SO.sub.3M;
[0083] v) --PO.sub.3M.sub.2; and
[0084] vi) --OCH.sub.3;
wherein M is hydrogen, an alkali metal, an alkali earth metal, a
transition metal, or mixtures thereof as defined herein above.
[0085] In one aspect of the disclosed chromogenic polymers, R.sup.2
units comprise a carboxylate unit. Non-limiting examples of this
aspect of R.sup.2 units include:
##STR00009##
[0086] In another aspect of the disclosed chromogenic polymers,
R.sup.2 units comprise a sulfonate unit. Non-limiting examples of
this aspect of R.sup.2 units include:
##STR00010##
[0087] In a further aspect of the disclosed chromogenic polymers,
R.sup.2 units comprise a phosphonate unit. Non-limiting examples of
this aspect of R.sup.2 units include:
##STR00011##
[0088] In a yet further aspect of the disclosed chromogenic
polymers, R.sup.2 units are an admixture of charged units and
non-charged units. In one embodiment of this aspect, the
chromogenic polymers comprise a mixture of R.sup.2 units wherein at
least 90% of the R.sup.2 units are carboxylate units and 10% of the
R.sup.2 units are hydrogen. In another embodiment of this aspect,
the chromogenic polymers comprise a mixture of R.sup.2 units
wherein at least 80% of the R.sup.2 units are carboxylate units and
20% of the R.sup.2 units are hydrogen. In a further embodiment of
this aspect, the chromogenic polymers comprise a mixture of R.sup.2
units wherein at least 70% of the R.sup.2 units are carboxylate
units and 30% of the R.sup.2 units are hydrogen. In a yet further
embodiment of this aspect, the chromogenic polymers comprise a
mixture of R.sup.2 units wherein at least 60% of the R.sup.2 units
are carboxylate units and 40% of the R.sup.2 units are hydrogen. In
a still further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R.sup.2 units wherein at least 50% of the
R.sup.2 units are carboxylate units and 50% of the R.sup.2 units
are hydrogen.
[0089] In a further embodiment of this aspect, the chromogenic
polymers comprise a mixture of R.sup.2 units wherein at least 90%
of the R.sup.2 units are sulfonate units and 10% of the R.sup.2
units are hydrogen. In another embodiment of this aspect, the
chromogenic polymers comprise a mixture of R.sup.2 units wherein at
least 80% of the R.sup.2 units are sulfonate units and 20% of the
R.sup.2 units are hydrogen. In a further embodiment of this aspect,
the chromogenic polymers comprise a mixture of R.sup.2 units
wherein at least 70% of the R.sup.2 units are sulfonate units and
30% of the R.sup.2 units are hydrogen. In a yet further embodiment
of this aspect, the chromogenic polymers comprise a mixture of
R.sup.2 units wherein at least 60% of the R.sup.2 units are
sulfonate units and 40% of the R.sup.2 units are hydrogen. In a
still further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R.sup.2 units wherein at least 50% of the
R.sup.2 units are sulfonate units and 50% of the R.sup.2 units are
hydrogen.
[0090] In a yet further embodiment of this aspect, the chromogenic
polymers comprise a mixture of R.sup.2 units wherein at least 90%
of the R.sup.2 units are phosphonate units and 10% of the R.sup.2
units are hydrogen. In another embodiment of this aspect, the
chromogenic polymers comprise a mixture of R.sup.2 units wherein at
least 80% of the R.sup.2 units are phosphonate units and 20% of the
R.sup.2 units are hydrogen. In a further embodiment of this aspect,
the chromogenic polymers comprise a mixture of R.sup.2 units
wherein at least 70% of the R.sup.2 units are phosphonate units and
30% of the R.sup.2 units are hydrogen. In a yet further embodiment
of this aspect, the chromogenic polymers comprise a mixture of
R.sup.2 units wherein at least 60% of the R.sup.2 units are
phosphonate units and 40% of the R.sup.2 units are hydrogen. In a
still further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R.sup.2 units wherein at least 50% of the
R.sup.2 units are phosphonate units and 50% of the R.sup.2 units
are hydrogen.
[0091] Another aspect relates to chromogenic polymers that comprise
a mixture of carboxylate and sulfonate units. In one embodiment of
this aspect, the chromogenic polymers comprise a mixture of R.sup.2
units wherein at least 90% of the R.sup.2 units are carboxylate
units and 10% of the R.sup.2 units are sulfonate. In another
embodiment of this aspect, the chromogenic polymers comprise a
mixture of R.sup.2 units wherein at least 80% of the R.sup.2 units
are carboxylate units and 20% of the R.sup.2 units are sulfonate.
In a further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R.sup.2 units wherein at least 70% of the
R.sup.2 units are carboxylate units and 30% of the R.sup.2 units
are sulfonate. In a yet further embodiment of this aspect, the
chromogenic polymers comprise a mixture of R.sup.2 units wherein at
least 60% of the R.sup.2 units are carboxylate units and 40% of the
R.sup.2 units are sulfonate. In a still further embodiment of this
aspect, the chromogenic polymers comprise a mixture of R.sup.2
units wherein at least 50% of the R.sup.2 units are carboxylate
units and 50% of the R.sup.2 units are sulfonate.
R.sup.3 and R.sup.4 Units
[0092] R.sup.3 and R.sup.4 units are moieties that interact with
the disclosed biogenic amines. R.sup.3 and R.sup.4 units are
independently chosen from one another. R.sup.3 and R.sup.4 units
serve as a moiety that complexes with the one or more substrate
recognition elements and therefore serve to differentiate between
different biogenic amines. Non-limiting examples of R.sup.3 and
R.sup.4 units include:
[0093] i) --H;
[0094] ii) --OM;
[0095] iii) --CO.sub.2M;
[0096] iv) --SO.sub.3M;
[0097] v) --PO.sub.3M.sub.2; and
[0098] vi) --OCH.sub.3;
wherein M is hydrogen, an alkali metal, an alkali earth metal, a
transition metal, or mixtures thereof as defined herein above.
[0099] In one aspect of the disclosed chromogenic polymers, R.sup.3
and R.sup.4 units comprise a carboxylate unit. Non-limiting
examples of this aspect of R.sup.3 and R.sup.4 units include:
##STR00012##
[0100] In another aspect of the disclosed chromogenic polymers,
R.sup.3 and R.sup.4 units comprise a sulfonate unit. Non-limiting
examples of this aspect of R.sup.3 and R.sup.4 units include:
##STR00013##
[0101] In a further aspect of the disclosed chromogenic polymers,
R.sup.3 and R.sup.4 units comprise a phosphonate unit. Non-limiting
examples of this aspect of R.sup.3 and R.sup.4 units include:
##STR00014##
[0102] In a yet further aspect of the disclosed chromogenic
polymers, R.sup.3 and R.sup.4 units are an admixture of charged and
non-charged units. In one embodiment of this aspect, the
chromogenic polymers comprise a mixture of R.sup.3 and R.sup.4
units wherein at least 90% of the R.sup.3 and R.sup.4 units are
carboxylate and 10% of the R.sup.3 and R.sup.4 units are hydrogen.
In another embodiment of this aspect, the chromogenic polymers
comprise a mixture of R.sup.3 and R.sup.4 units wherein at least
80% of the R.sup.3 and R.sup.4 units are carboxylate and 20% of the
R.sup.3 and R.sup.4 units are hydrogen. In a further embodiment of
this aspect, the chromogenic polymers comprise a mixture of R.sup.3
and R.sup.4 units wherein at least 70% of the R.sup.3 and R.sup.4
units are carboxylate and 30% of the R.sup.3 and R.sup.4 units are
hydrogen. In a yet further embodiment of this aspect, the
chromogenic polymers comprise a mixture of R.sup.3 and R.sup.4
units wherein at least 60% of the R.sup.3 and R.sup.4 units are
carboxylate and 40% of the R.sup.3 and R.sup.4 units are hydrogen.
In a still further embodiment of this aspect, the chromogenic
polymers comprise a mixture of R.sup.3 and R.sup.4 units wherein at
least 50% of the R.sup.3 and R.sup.4 units are carboxylate and 50%
of the R.sup.3 and R.sup.4 units are hydrogen.
[0103] In a further embodiment of this aspect, the chromogenic
polymers comprise a mixture of R.sup.3 and R.sup.4 units wherein at
least 90% of the R.sup.3 and R.sup.4 units are sulfonate and 10% of
the R.sup.3 and R.sup.4 units are hydrogen. In another embodiment
of this aspect, the chromogenic polymers comprise a mixture of
R.sup.3 and R.sup.4 units wherein at least 80% of the R.sup.3 and
R.sup.4 units are sulfonate and 20% of the R.sup.3 and R.sup.4
units are hydrogen. In a further embodiment of this aspect, the
chromogenic polymers comprise a mixture of R.sup.3 and R.sup.4
units wherein at least 70% of the R.sup.3 and R.sup.4 units are
sulfonate and 30% of the R.sup.3 and R.sup.4 units are hydrogen. In
a yet further embodiment of this aspect, the chromogenic polymers
comprise a mixture of R.sup.3 and R.sup.4 units wherein at least
60% of the R.sup.3 and R.sup.4 units are sulfonate and 40% of the
R.sup.3 and R.sup.4 units are hydrogen. In a still further
embodiment of this aspect, the chromogenic polymers comprise a
mixture of R.sup.3 and R.sup.4 units wherein at least 50% of the
R.sup.3 and R.sup.4 units are sulfonate and 50% of the R.sup.3 and
R.sup.4 units are hydrogen.
[0104] In a yet further embodiment of this aspect, the chromogenic
polymers comprise a mixture of R.sup.3 and R.sup.4 units wherein at
least 90% of the R.sup.3 and R.sup.4 units are phosphonate and 10%
of the R.sup.3 and R.sup.4 units are hydrogen. In another
embodiment of this aspect, the chromogenic polymers comprise a
mixture of R.sup.3 and R.sup.4 units wherein at least 80% of the
R.sup.3 and R.sup.4 units are phosphonate and 20% of the R.sup.3
and R.sup.4 units are hydrogen. In a further embodiment of this
aspect, the chromogenic polymers comprise a mixture of R.sup.3 and
R.sup.4 units wherein at least 70% of the R.sup.3 and R.sup.4 units
are phosphonate and 30% of the R.sup.3 and R.sup.4 units are
hydrogen. In a yet further embodiment of this aspect, the
chromogenic polymers comprise a mixture of R.sup.3 and R.sup.4
units wherein at least 60% of the R.sup.3 and R.sup.4 units are
phosphonate and 40% of the R.sup.3 and R.sup.4 units are hydrogen.
In a still further embodiment of this aspect, the chromogenic
polymers comprise a mixture of R.sup.3 and R.sup.4 units wherein at
least 50% of the R.sup.3 and R.sup.4 units are phosphonate and 50%
of the R.sup.3 and R.sup.4 units are hydrogen.
[0105] Another aspect relates to chromogenic polymers wherein
R.sup.3 and R.sup.4 units are taken together with the two phenyl
rings to form a fused ring having from 5 to 7 carbon atoms that
comprise a mixture of carboxylate and sulfonate units. A
non-limiting embodiment of this aspect relates to R.sup.3 and
R.sup.4 units taken together when the index z is equal to 0 and
therefore L.sup.5 is absent, to form a 5-member ring, thereby
forming a fluorenyl group having the formula:
##STR00015##
Linking Units L, L.sup.1, L.sup.3, and L.sup.4
[0106] Linking units L, L.sup.1, L.sup.3, and L.sup.4 are each
independently chosen from:
[0107] i) --(CR.sup.5aR.sup.5b).sub.m--; or
[0108] ii)
--(CR.sup.5aR.sup.5b).sub.m[O(CR.sup.5aR.sup.5b).sub.m].sub.k---
;
wherein R.sup.5a and R.sup.5b are each independently hydrogen or
methyl; the index k is an integer from 1 to 20; and the index m is
an integer from 0 to 10.
[0109] A first category of L, L.sup.1, L.sup.3, and L.sup.4 linking
units relates to alkylene units having the formula:
--(CH.sub.2).sub.m--
wherein R.sup.5a and R.sup.5b are each hydrogen; and the index m is
from 1 to 10. Non-limiting examples of this category include:
[0110] i) --CH.sub.2--;
[0111] ii) --CH.sub.2CH.sub.2--;
[0112] iii) --CH.sub.2CH.sub.2CH.sub.2--;
[0113] iv) --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--;
[0114] v) --CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--;
[0115] vi)
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--;
[0116] vii)
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--;
[0117] viii)
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--;
[0118] ix)
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.su-
b.2CH.sub.2--; and
[0119] x)
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub-
.2CH.sub.2CH.sub.2--.
[0120] One embodiment of this category relates to chromogenic
polymers comprising an L unit having the formula:
[0121] i) --CH.sub.2CH.sub.2--;
[0122] ii) --CH.sub.2CH.sub.2CH.sub.2--; or
[0123] iii) --CH.sub.2CH.sub.2CH.sub.2CH.sub.2--.
[0124] Another category of L, L.sup.1, L.sup.3, and L.sup.4 linking
units relates to alkylene units having the formula:
--[CH.sub.2CH(CH.sub.3)].sub.m-- or
--[CH(CH.sub.3)CH.sub.2].sub.m--
wherein R.sup.5a and R.sup.5b are each hydrogen or methyl; and the
index m is from 1 to 10. Non-limiting examples of this category
include:
[0125] i) --CH.sub.2CH(CH.sub.3)--;
[0126] ii) --CH(CH.sub.3)CH.sub.2--;
[0127] iii) --CH(CH.sub.3)CH.sub.2CH.sub.2CH(CH.sub.3)--;
[0128] iv) --CH.sub.2CH(CH.sub.3)CH.sub.2CH(CH.sub.3)--;
[0129] iii) --CH(CH.sub.3)CH.sub.2CH(CH.sub.3)CH.sub.2--;
[0130] iv) --CH.sub.2CH(CH.sub.3)CH(CH.sub.3)CH.sub.2--;
[0131] iii)
--CH(CH.sub.3)CH.sub.2CH.sub.2CH(CH.sub.3)CH.sub.2CH(CH.sub.3)--;
[0132] iv)
--CH.sub.2CH(CH.sub.3)CH.sub.2CH(CH.sub.3)CH.sub.2CH(CH.sub.3)--
-;
[0133] iii)
--CH(CH.sub.3)CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.2CH(CH.sub.3)--;
[0134] iv)
--CH.sub.2CH(CH.sub.3)CH(CH.sub.3)CH.sub.2CH.sub.2CH(CH.sub.3)--
-;
[0135] iii)
--CH(CH.sub.3)CH.sub.2CH.sub.2CH(CH.sub.3)CH(CH.sub.3)CH.sub.2--;
[0136] iv)
--CH.sub.2CH(CH.sub.3)CH.sub.2CH(CH.sub.3)CH(CH.sub.3)CH.sub.2--
-;
[0137] iii)
--CH(CH.sub.3)CH.sub.2CH(CH.sub.3)CH.sub.2CH(CH.sub.3)CH.sub.2--;
and
[0138] iv)
--CH.sub.2CH(CH.sub.3)CH(CH.sub.3)CH.sub.2CH(CH.sub.3)CH.sub.2--
-.
[0139] A further category of L, L.sup.1, L.sup.3, and L.sup.4
linking units relates to alkyleneoxy units having the formula:
--(CR.sup.5aR.sup.5b).sub.m[O(CR.sup.5aR.sup.5b).sub.m].sub.k--
wherein the index k is an integer from 1 to 20 and the index m is
an integer from 1 to 10. Non-limiting examples of this category of
L, L.sup.1, L.sup.3, and L.sup.4 linking units includes units
chosen from:
[0140] i) --(CH.sub.2CH.sub.2)[O(CH.sub.2CH.sub.2)]O--;
[0141] ii) --(CH.sub.2CH.sub.2)[O(CH.sub.2CH.sub.2)].sub.2O--;
[0142] iii) --(CH.sub.2CH.sub.2)[O(CH.sub.2CH.sub.2)].sub.3O--;
[0143] iv) --(CH.sub.2CH.sub.2)[O(CH.sub.2CH.sub.2)].sub.4O--;
[0144] v) --(CH.sub.2CH.sub.2)[O(CH.sub.2CH.sub.2)].sub.5O--;
[0145] vi) --(CH.sub.2CH.sub.2)[O(CH.sub.2CH.sub.2)].sub.6O--;
[0146] vii) --(CH.sub.2CH.sub.2)[O(CH.sub.2CH.sub.2)].sub.7O--;
[0147] viii) --(CH.sub.2CH.sub.2)[O(CH.sub.2CH.sub.2)].sub.8O--;
and
[0148] ix) --(CH.sub.2CH.sub.2)[O(CH.sub.2CH.sub.2)].sub.9O--.
L.sup.2 and L.sup.5 Linking Units
[0149] L.sup.2 and L.sup.5 units are backbone linking units that
can be present when the index z is equal to 1 or absent when the
index z is equal to 0. L.sup.5 linking units are chosen from:
[0150] i)
--(CR.sup.5aR.sup.5b).sub.j(CH.dbd.CH)(CR.sup.5aR.sup.5b).sub.j--
-;
[0151] ii) --(CR.sup.5aR.sup.5b).sub.j
(C.ident.C)(CR.sup.5aR.sup.5b).sub.j--;
R.sup.5a and R.sup.5b are each independently hydrogen or methyl;
the index j is an integer from 0 to 10.
[0152] One category of L.sup.2 and L.sup.5 units relates to
chromogenic polymers having an alkynyl linking unit. Non-limiting
examples of polymer units comprising an alkynyl linking group
include:
##STR00016##
Another category of L.sup.5 units relates to chromogenic polymers
having an alkenyl linking unit. Non-limiting examples of polymer
units comprising an alkenyl linking group include:
##STR00017##
[0153] One category of chromogenic polymers relates to
poly(thiophene)s having the formula:
##STR00018##
wherein R is independently chosen from:
[0154] i) --CO.sub.2M;
[0155] ii) --SO.sub.3M; and
[0156] iii) --PO.sub.3M.sub.2;
M is a substrate recognition element, and L is linking unit having
from 1 to 10 methylene units.
[0157] One aspect of this category relates to chromogenic polymers
comprising a poly(thiophene) backbone having the formula:
##STR00019##
wherein the index n has a value such that the chromogenic polymer
has an average molecular weight of from about 1,000 Da to about
20,000 Da, and M is chosen from copper, cobalt, or nickel.
[0158] One embodiment of this aspect relates to chromogenic
polymers having an average molecular weight of from about 1,000 Da
to about 20,000 Da. and wherein M is copper. An iteration of this
embodiment is a chromogenic polymer having an average molecular
weight of from about 1,000 Da to about 5,000 Da. Another iteration
of this embodiment is a chromogenic polymer having an average
molecular weight of from about 2,000 Da to about 6,000 Da. A
further iteration of this embodiment is a chromogenic polymer
having an average molecular weight of from about 3,000 Da to about
7,000 Da. A yet further iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 4,000 Da to about 8,000 Da. A still further iteration of this
embodiment is a chromogenic polymer having an average molecular
weight of from about 5,000 Da to about 9,000 Da.
[0159] Another aspect relates to chromogenic polymers having a
backbone comprising units having the formula:
##STR00020##
wherein the chromogenic polymer has an average molecular weight of
from about 1,000 Da to about 20,000 Da. One embodiment of this
aspect relates to chromogenic polymers having an average molecular
weight of from about 1,000 Da to about 20,000 Da. and wherein the
recognition element associated therewith can be copper, cobalt,
nickel, and the like. An iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 1,000 Da to about 5,000 Da. Another iteration of this
embodiment is a chromogenic polymer having an average molecular
weight of from about 2,000 Da to about 6,000 Da. A further
iteration of this embodiment is a chromogenic polymer having an
average molecular weight of from about 3,000 Da to about 7,000 Da.
A yet further iteration of this embodiment is a chromogenic polymer
having an average molecular weight of from about 4,000 Da to about
8,000 Da. A still further iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 5,000 Da to about 9,000 Da.
[0160] Another aspect relates to chromogenic polymers having a
backbone comprising units having the formula:
##STR00021##
wherein the chromogenic polymer has an average molecular weight of
from about 1,000 Da to about 20,000 Da. One embodiment of this
aspect relates to chromogenic polymers having an average molecular
weight of from about 1,000 Da to about 20,000 Da. and wherein the
recognition element associated therewith can be copper, cobalt,
nickel, and the like. An iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 1,000 Da to about 5,000 Da. Another iteration of this
embodiment is a chromogenic polymer having an average molecular
weight of from about 2,000 Da to about 6,000 Da. A further
iteration of this embodiment is a chromogenic polymer having an
average molecular weight of from about 3,000 Da to about 7,000 Da.
A yet further iteration of this embodiment is a chromogenic polymer
having an average molecular weight of from about 4,000 Da to about
8,000 Da. A still further iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 5,000 Da to about 9,000 Da.
[0161] A yet another aspect of this category relates to chromogenic
polymers comprising a poly(thiophene) backbone having the
formula:
##STR00022##
wherein the index n has a value such that the chromogenic polymer
has an average molecular weight of from about 1,000 Da to about
20,000 Da, and M is chosen from copper, cobalt, or nickel.
[0162] One embodiment of this aspect relates to chromogenic
polymers having an average molecular weight of from about 1,000 Da
to about 20,000 Da. and wherein M is copper. An iteration of this
embodiment is a chromogenic polymer having an average molecular
weight of from about 1,000 Da to about 5,000 Da. Another iteration
of this embodiment is a chromogenic polymer having an average
molecular weight of from about 2,000 Da to about 6,000 Da. A
further iteration of this embodiment is a chromogenic polymer
having an average molecular weight of from about 3,000 Da to about
7,000 Da. A yet further iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 4,000 Da to about 8,000 Da. A still further iteration of this
embodiment is a chromogenic polymer having an average molecular
weight of from about 5,000 Da to about 9,000 Da.
[0163] Another category of chromogenic polymers relates to
poly(thiophene)s having the formula:
##STR00023##
wherein R is independently chosen from:
[0164] i) --CO.sub.2M;
[0165] ii) --SO.sub.3M; and
[0166] iii) --PO.sub.3M.sub.2;
R.sup.1 is hydrogen or methoxy; L has the formula
--(CH.sub.2).sub.m--, the index m is an integer from 1 to 10;
L.sup.1 has the formula --(CH.sub.2).sub.m-- or
--(CH.sub.2).sub.m[O(CH.sub.2).sub.m].sub.k--; the index k is an
integer from 1 to 20, the index m is an integer from 1 to 10; M is
a substrate recognition element.
[0167] One aspect of this category relates to chromogenic polymers
wherein L.sup.1 and R.sup.1 together provide a unit that enhances
the solubility of the polymers in hydrophobic systems, inter alia,
organic solvents. One non-limiting example of a backbone according
to this aspect has the formula:
##STR00024##
[0168] Another aspect of this category relates to chromogenic
polymers wherein L.sup.1 and R.sup.1 together provide a unit that
enhances the solubility of the polymers in hydrophilic systems,
inter alia, aqueous systems. One non-limiting example of a backbone
according to this aspect has the formula:
##STR00025##
[0169] Another category of chromogenic polymers relates to random
poly(thiophene) copolymer having, for example, the formula:
##STR00026##
wherein the indices a+b=n.
[0170] A yet further category of chromogenic polymers relates to
poly(furan)s having the formula:
##STR00027##
wherein R is independently chosen from:
[0171] i) --CO.sub.2M;
[0172] ii) --SO.sub.3M; and
[0173] iii) --PO.sub.3M.sub.2;
M is a substrate recognition element, and L is linking unit having
from 1 to 10 methylene units.
[0174] One aspect of this category relates to chromogenic polymers
comprising a poly(thiophene) backbone having the formula:
##STR00028##
wherein the index n has a value such that the chromogenic polymer
has an average molecular weight of from about 1,000 Da to about
20,000 Da, and M is chosen from copper, cobalt, or nickel.
[0175] One embodiment of this aspect relates to chromogenic
polymers having an average molecular weight of from about 1,000 Da
to about 20,000 Da. and wherein the recognition element associated
therewith can be copper, cobalt, nickel, and the like. An iteration
of this embodiment is a chromogenic polymer having an average
molecular weight of from about 1,000 Da to about 5,000 Da. Another
iteration of this embodiment is a chromogenic polymer having an
average molecular weight of from about 2,000 Da to about 6,000 Da.
A further iteration of this embodiment is a chromogenic polymer
having an average molecular weight of from about 3,000 Da to about
7,000 Da. A yet further iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 4,000 Da to about 8,000 Da. A still further iteration of this
embodiment is a chromogenic polymer having an average molecular
weight of from about 5,000 Da to about 9,000 Da.
[0176] Another aspect relates to chromogenic polymers having a
backbone comprising units having the formula:
##STR00029##
wherein the chromogenic polymer has an average molecular weight of
from about 1,000 Da to about 20,000 Da. One embodiment of this
aspect relates to chromogenic polymers having an average molecular
weight of from about 1,000 Da to about 20,000 Da. and wherein the
recognition element associated therewith can be copper, cobalt,
nickel, and the like. An iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 1,000 Da to about 5,000 Da. Another iteration of this
embodiment is a chromogenic polymer having an average molecular
weight of from about 2,000 Da to about 6,000 Da. A further
iteration of this embodiment is a chromogenic polymer having an
average molecular weight of from about 3,000 Da to about 7,000 Da.
A yet further iteration of this embodiment is a chromogenic polymer
having an average molecular weight of from about 4,000 Da to about
8,000 Da. A still further iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 5,000 Da to about 9,000 Da.
[0177] A still further category of chromogenic polymers relates to
poly(pyrrole)s having the formula:
##STR00030##
wherein R is independently chosen from:
[0178] i) --CO.sub.2M;
[0179] ii) --SO.sub.3M; and
[0180] iii) --PO.sub.3M.sub.2;
M is a substrate recognition element, and L is linking unit having
from 1 to 10 methylene units.
[0181] One aspect of this category relates to chromogenic polymers
comprising a poly(thiophene) backbone having the formula:
##STR00031##
wherein the index n has a value such that the chromogenic polymer
has an average molecular weight of from about 1,000 Da to about
20,000 Da, and M is chosen from copper, cobalt, or nickel.
[0182] One embodiment of this aspect relates to chromogenic
polymers having an average molecular weight of from about 1,000 Da
to about 20,000 Da. and wherein the recognition element associated
therewith can be copper, cobalt, nickel, and the like. An iteration
of this embodiment is a chromogenic polymer having an average
molecular weight of from about 1,000 Da to about 5,000 Da. Another
iteration of this embodiment is a chromogenic polymer having an
average molecular weight of from about 2,000 Da to about 6,000 Da.
A further iteration of this embodiment is a chromogenic polymer
having an average molecular weight of from about 3,000 Da to about
7,000 Da. A yet further iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 4,000 Da to about 8,000 Da. A still further iteration of this
embodiment is a chromogenic polymer having an average molecular
weight of from about 5,000 Da to about 9,000 Da.
[0183] Another aspect relates to chromogenic polymers having a
backbone comprising units having the formula:
##STR00032##
wherein the chromogenic polymer has an average molecular weight of
from about 1,000 Da to about 20,000 Da. One embodiment of this
aspect relates to chromogenic polymers having an average molecular
weight of from about 1,000 Da to about 20,000 Da. and wherein the
recognition element associated therewith can be copper, cobalt,
nickel, and the like. An iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 1,000 Da to about 5,000 Da. Another iteration of this
embodiment is a chromogenic polymer having an average molecular
weight of from about 2,000 Da to about 6,000 Da. A further
iteration of this embodiment is a chromogenic polymer having an
average molecular weight of from about 3,000 Da to about 7,000 Da.
A yet further iteration of this embodiment is a chromogenic polymer
having an average molecular weight of from about 4,000 Da to about
8,000 Da. A still further iteration of this embodiment is a
chromogenic polymer having an average molecular weight of from
about 5,000 Da to about 9,000 Da.
[0184] The preparation of the disclosed chromogenic polymer can be
carried out using methods known in the art or other methods
disclosed herein. As one of skill in the art will readily
recognize, synthetic polymer procedures can be readily modified to
produce varying polymer chain lengths without undue experimentation
(e.g., shortening or lengthening a polymerization reaction time).
HT-poly(3-(2-(4,5-dihydro-4,4-dimethyl-2-oxazolyl)ethyl)thiophene
was synthesized using methods disclosed by McCullough as described
in the examples herein, for example, using Gronowitz conditions for
the formation of dimers, as described by Ewbank et al. (Ewbank, P.
C et al., Tetrahedron 2004, 60, 11269-11275).
Example 1
HT-2,5-poly(3-(2-(4,5-dihydro-4,4
dimethyl-2-oxazolyl)ethyl)thiophene
[0185]
2-(2-(2-bromo-5-(trimethylstannyl)thiophen-3-yl)ethyl)-4,5-dihydro--
4,4dimethyloxazole (7.0 g, 15.5 mmol) was weighed in 100 mL Schlenk
flask followed by the addition of 30 mL dry DMF then purged with
nitrogen for 15 min. To this mixture, copper (II) oxide (1.24 g,
15.5 mmol, 1 eq.), triphenylphosphine (814 mg, 0.031 mmol, 0.20
eq.) and bis(dibenzylidene acetone)palladium(0) (446 mg, 0.776
mmol, 0.05 eq) was added all at once. The greenish-brown suspension
solution was purged with nitrogen for 15 min then heated to
100.degree. C. and stirred for 18 hrs under argon atmosphere. The
reaction mixture was filtered thru celite and the purple
precipitate was extracted with chloroform. The chloroform extract
was concentrated and the resulting solid was soxhlet-extracted with
hexanes then extracted with chloroform. The solvent was removed to
afford a purple solid (2.1 g, 66% S5 yield). .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta.=7.06 (s, 1H), 3.94 (s, 2H), 3.14 (d, 2H), 2.66
(d, 2H), 1.28 (s, 6H); .sup.13C NMR (75 MHz, CDCl.sub.3):
.delta.=166.7, 138.4, 134.4, 132.4, 129.8, 80.0, 68.0, 29.6, 29.2,
26.4. CDCl.sub.3
##STR00033##
[0186] HT-poly(thiophene-3-propionoic acid) (1), and
HT-poly(thiophene-3-propionate) (2) have been reported by
McCullough. For example, polymer 1 can be prepared by Stille/CuO
polymerization of an oxazoline protected monomer followed by acid
hydrolysis according to the method of McCullough et al. Polymer 1
can be deprotonated to form 2, a salt, by exposing 1 to an
appropriate base (McCullough, R. D.; Ewbank, P. C.; Loewe, R. S. J.
Am. Chem. Soc. 1997, 119, 633-634). A representative, exemplary
procedure for the synthesis of polymer 1 is given herein below.
[0187] Polymer (1). HT-2,5-poly(3-(2-(4,5-dihydro-4,4
dimethyl-2-oxazolyl)ethyl)thiophene (1.9 g, 10.1 mmol) was
dissolved in 50 mL chloroform then 50 mL 3 N HCl was added to the
mixture. The reaction was refluxed overnight. The resulting dark
purple polymer was filtered, washed with water and chloroform and
dried to give the product (1.05 g, 67% yield). .sup.1H NMR (300
MHz, CD3OD) (characterized as the cesium salt): .delta.=7.16 (s,
1H), 3.11 (t, 2H), 2.54 (t, 2H). Molecular weights and distribution
of polymer 1 were determined as the butyl ester using MALDI-TOF MS.
The ester was obtained by acid-catalyzed esterification of polymer
5 with sulfuric acid in refluxing n-butanol. .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta.=7.02 (s, 1H), 4.11 (t, 2H), 3.14 (t, 2H), 2.70
(t, 2H), 1.62 (m, 2H), 1.35 (m, 2H), 0.92 (t, 3H); MALDITOF MS:
M.sub.n=3170, M.sub.w=3230, PDI=1.02.
##STR00034##
[0188] The tetrabutylammonium polymer 3 can be prepared by adding
polymer 1 to deionized water followed by the addition of an aliquot
of 40% (w/v) tetrabutylamonium hydroxide solution. The pH of a
polymer solution can be adjusted to an appropriate pH using acid or
base (e.g., hydrochloric acid or sodium hydroxide).
Methods
[0189] Disclosed herein are methods for detecting the presence of a
biogenic amine, comprising:
[0190] a) providing a chromogenic-responsive polymer;
[0191] b) contacting the polymer in step (a) with an analyte;
and
[0192] c) detecting a chromogenic response.
It is understood that the methods disclosed herein can be used in
combination with the various chromogenic polymers and compositions
disclosed herein, devices, and articles of manufacture, and
iterations of both visual and mathematic analysis.
Linear Discriminate Analysis
[0193] In various aspects, the present invention can use pattern
recognition protocols, such as linear discriminate analysis (LDA).
LDA of a data set can be carried out using methods known in the art
with commercially available software (e.g., Systat, Systat
Software, Inc., 2004, Version 11.00.01)
Devices
[0194] One aspect of the disclosed devices relates to an assay
device for determining the presence of one or more biogenic amines,
comprising one or more substrates having deposited thereon a
chromogenic responsive polymer, as such, disclosed herein are
devices suitable for use in sensing applications. The devices
disclosed herein can be useful for sensing one or more amines in an
analyte. The devices can also be useful for differentiating between
different amines that comprise analytes. Methods of using the
disclosed devices comprise, in one aspect, identifying an amine
from reference data.
[0195] In one aspect, devices can comprise a polymeric substrate
having a first side and a second side wherein further the
chromogenic responsive polymer is deposited onto the first side.
The polymeric substrate can comprise one of more suitable polymers,
for example, polyethylene, polypropylene,
poly(propylene-b-terephthalate), and the like. In another aspect,
the devices can comprise a paper having the second side (the side
opposite the first side wherein the chromogenic response polymer is
applied) comprise a thin metallic film, for example, aluminum.
Further aspects and devices are described herein below.
[0196] In a further aspect, devices disclosed herein can at least
partially detect and/or discriminate between amines. In a specific
aspect, the disclosed devices can detect and/or discriminate
between diamines. Amines (e.g., diamines) contemplated for use with
the disclosed devices can be any amine. Amines can typically be
associated with biological and/or bacterial spoilage. As such, the
devices disclosed herein can be useful for detecting and/or
discriminating between biogenic amines that are released during
biological spoilage.
[0197] In another aspect, the disclosed devise can be used as a
diagnostic tool to detect the quality of protein containing
samples. Exemplary protein samples comprise, inter alia, food
samples, medical samples, and forensic samples. In a specific
aspect, the disclosed devices can be used to detect and/or
discriminate between biogenic amines present in, for example, food
samples, medical samples, and forensic samples.
[0198] Any polymer and/or polymer composition (or combination of
more than one polymer or polymer composition) disclosed herein can
be used in combination with the herein disclosed devices.
[0199] FIG. 5 is a photograph of solutions of polymer 2 with three
different substrate recognition elements and the corresponding
chromogenic response to a variety of diamines. Series No. 1
comprises Co.sup.2+, series No. 2 comprises Cu.sup.2+ and series
No. 3 comprises Ni.sup.2+ Vials A-I contain the following:
TABLE-US-00001 Series No. 1 Series No. 2 Series No. 3 A polymer 2 A
polymer 2 A polymer 2 B Polymer 2 + CoCl.sub.2 B Polymer 2 +
CuCl.sub.2 B Polymer 2 + NiCl.sub.2 C Polymer 2 + CoCl.sub.2 + C
Polymer 2 + CuCl.sub.2 + C Polymer 2 + NiCl.sub.2 + 1,2-ethylene
diamine 1,2-ethylene diamine 1,2-ethylene diamine D Polymer 2 +
CoCl.sub.2 + D Polymer 2 + CuCl.sub.2 + D Polymer 2 + NiCl.sub.2 +
1,3-propylenediamine 1,3-propylenediamine 1,3-propylenediamine E
Polymer 2 + CoCl.sub.2 + E Polymer 2 + CuCl.sub.2 + E Polymer 2 +
NiCl.sub.2 + putrescine putrescine putrescine F Polymer 2 +
CoCl.sub.2 + F Polymer 2 + CuCl.sub.2 + F Polymer 2 + NiCl.sub.2 +
cadaverine cadaverine cadaverine G Polymer 2 + CoCl.sub.2 + G
Polymer 2 + CuCl.sub.2 + G Polymer 2 + NiCl.sub.2 + histamine
histamine histamine H Polymer 2 + CoCl.sub.2 + H Polymer 2 +
CuCl.sub.2 + H Polymer 2 + NiCl.sub.2 + spermidine spermidine
spermidine I Polymer 2 + CoCl.sub.2 + I Polymer 2 + CuCl.sub.2 + I
Polymer 2 + NiCl.sub.2 + spermine spermine spermine
As depicted in FIG. 5, the chromogenic response for each sample is
dependent on both the substrate recognition element and biogenic
amine.
[0200] FIG. 6 is a photograph depicting the color changes observed
for various amines in polymer 1 with different recognition
elements. Sample A is ethylenediamine, sample B is
propylenediamine, sample C is putrescine, sample D is cadaverine,
sample E is histamine, sample F is spermidine, and sample F is
spermine. The consumer or the inspector can use a color reference
guide to aid in determining and differentiating the presence of
various biogenic amines.
Lateral Flow Device
[0201] One or more polymers or polymer compositions disclosed
herein can be coated onto or into a solid or liquid component (e.g.
a solid substrate) to form a device article. A polymer can adhere
or be in communication with, for example, a substrate, through any
appropriate means. If, for example, a substrate is used in a
lateral flow device as described herein, it can be preferable that
a polymer have some physical or chemical interaction with the
substrate. For one contemplated lateral flow device, the extent of
the interaction can change in the presence of different analyte
species and can thereby produce a differential response to
different analyte species.
[0202] In one aspect, a silica coated plate can be used as a
substrate and a polymer can be introduced onto the substrate
through any appropriate means (e.g., spotting the substrate with a
capillary tube filled with polymer solution). In this example, if a
stimulatory analyte (e.g., biogenic amine) is present in a sample
(e.g. a water sample) that is developed with the silica substrate,
the polymer can move laterally across the substrate. The distance
that the polymer moves can be directly proportional to the amount
(and type) of analyte present. The polymer can also become
fluorescent under a stimulus (e.g., UV light) in the presence of an
analyte. If the analyte is a diamine, for example, the polymer can
change color from blue-purple to red-orange upon exposure to the
diamine in the analyte solution. In addition the matrix can be
nitrocellulose or mixtures of materials, for example,
nitrocellulose and silica.
[0203] In one aspect, a lateral flow device as described herein can
be a hand-held device. The device can be any appropriate size
(e.g., for example, small enough to transport). One example is an
assay device for determining the presence of one or more biogenic
amines within an analyte, comprising a substrate having one or more
detection zones wherein each zone contains a chromogenic responsive
polymer that undergoes a chromogenic response upon reaction with a
biogenic amine and wherein the response is dependent upon the
biogenic amine present.
[0204] In a further aspect, an assay device for determining the
presence of one or more biogenic amines within an analyte,
comprising a substrate having a first end, a second end, and a
plurality of channels wherein a different chromogenic responsive
polymer is deposited onto each channel, the first end having a
means for receiving an analyte and directing the analyte into each
of the plurality of channels, wherein further each channel provides
a chromogenic response that is specific to a particular biogenic
amine.
[0205] FIG. 6 depicts a device that can be used by either the
consumer or the inspector of food stuffs to determine the degree of
spoilage. Such a device can function using a mechanism similar to
that which ejects the ink stylus from an ink pen housing. A small
needle can be ejected from the device that can be used to pierce a
sample (liquid or solid). This piercing action can withdraw an
amount of liquid from the sample via capillary action and wet a
polymer-bound support, thereby initiating the development process.
The distance which the polymer moves can be correlated to a
particular type and/or amount of diamine present, or can be
correlated to a particular type of diamine forming food product.
FIG. 6 uses the example of analyzing for the level of a diamine or
amine in a fish sample. The amount of amine present is correlated
to the distance a sample will move on a substrate coated with one
or more of the disclosed chromogenic responsive polymers.
[0206] While FIG. 6 displays one lane for a polymer, a plurality of
lanes could be used, for example, to create a pattern.
Alternatively, it can be possible for each lane to have specific
sensitivity to a particular desired analyte. For example, if three
lanes, lanes 1, 2, and 3, and three analytes, analyte 1, 2, and 3
were present, lane 1 could sense analyte 1, lane 2 could sense
analyte 2, and lane 3 could sense analyte 3. For example, lane 1
could sense an analyte in tuna, lane 2 could sense an analyte in
salmon, lane 3 could sense an analyte in beef, etc. As such, one
embodiment of the device depicted in FIG. 6 relates to an assay
device for determining the presence of one or more biogenic amines
within an analyte, comprising a substrate having one or more
detection zones wherein each zone contains a chromogenic responsive
polymer that undergoes a chromogenic response upon reaction with a
biogenic amine and wherein the response is dependent upon the
biogenic amine present. Furthermore, the presence of this or these
biogenic amines relates to the extent of spoilage of the food.
Dip-Stick Device
[0207] A device suitable for use with the disclosed compounds,
compositions, and methods can be a dip-stick device. In one aspect,
a dip-stick device can operate functionally similar to a pH
dip-stick device. For example, different indicators (e.g., in the
form of strips or pads) can be exposed to an analytical sample, and
the result (e.g., a visible change) can be compared to a reference
pattern (e.g., a pattern present on a device package).
Such a device can comprise a polymer or polymer composition, such
as a polymer or polymer compositions disclosed herein. If a strip
or pad is used in a device, a strip or pad can be impregnated with
a sensing polymer. Such polymers can, for example, have different
main-chain structures and/or different side-chain functionalities
and/or different side-chain modifications of the same polymer, or
the polymers can be in different matrices (e.g., polar and apolar
polymer composites or immobilized solutions). These polymers can
turn different colors in the presence of different types and
amounts of analytes (e.g., a biogenic amine). A permeable,
semi-permeable, or partially permeable membrane can be used that
can allow sample into the device while at least partially keeping
the polymer from leaching out of the device.
[0208] FIG. 7, for example, displays an exemplary device
composition that could be used with a dip-stick device. Zones A-C
of the dip-stick in one embodiment can be correlated to the results
depicted in FIG. 5. For example, zone A can comprise the polymer
from Series No. 1, zone B the polymer from Series 2, and zone C the
polymer from Series No. 3. An analyte applied to the dip-stick that
registers the chromogenic response seen in the vials marked E would
indicate the presence of putrescine. The identity of the amine in
the analyte can be determined by comparing the color pattern along
the vertical columns of vials depicted in FIG. 5.
Lateral Flow Assay Device
[0209] A lateral flow device suitable for use with the disclosed
methods and compositions can be based on polymer aggregation. Such
a device can be based on multiple interactions between an analyte
and a receptor. For example, polymers in such a device can be used
for aggregate formation thereby producing a yes or no (or 1 or 0)
signal. Some polymers disclosed herein have multiple acidic
side-chains and thereby can be suitable for detecting the presence
of, for example, biogenic amines since most biogenic amines contain
multiple basic amines. Alternatively, metal nano-particles or
polymer microspheres coated with a polymer or polymer composition,
such as a polymer or polymer compositions as disclosed herein, or
coated, for example, with a carboxylic acid based compound or
polymer could be used to produce a platform from which aggregation
could stem.
Analytical Methods Using Chromogenic Responsive Polymers
[0210] The chromogenic responsive polymers can be used for
analytical methods in determining the presence and concentration of
one or more amines in an analyte. Using the measured absorbance of
reference samples containing single amines and combinations of
amines, the formulator can construct a reference library to
determine the composition of amines in an analyte.
[0211] The following are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices,
and/or methods described and claimed herein are made and evaluated,
and are intended to be purely exemplary and are not intended to
limit the scope of what the inventors regard as their invention.
Efforts have been made to ensure accuracy with respect to numbers
(e.g., amounts, temperature, etc.) but some errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, temperature is in .degree. C. or is at ambient
temperature, and pressure is at or near atmospheric. There are
numerous variations and combinations of conditions, e.g., component
concentrations, desired solvents, solvent mixtures, temperatures,
pressures and other reaction ranges and conditions that can be used
to optimize the methods described herein. Only reasonable and
routine experimentation will be required to optimize such process
conditions.
Organic Solution Based Sensing Using Polymer 1
[0212] For analyses in organic media, absorbance studies were
performed using a Beckman Coulter 640 DU spectrophotometer and
quartz cuvets from Starna. .sup.1H and .sup.13C spectra were
recorded on a Varian Mercury300 spectrometer operating at 300 and
75 MHz, respectively. MALDI-TOF MS was performed using a Bruker
Ultraflex TOF/TQF mass spectrometer. 2,2':5',2''-Terthiophene
(Aldrich) was used as the matrix.
[0213] An exemplary, representative assay experiment used in
combination with the methods disclosed herein. A polymer solution
was prepared by adding 16 mg of 1 to N,N-dimethylformamide (1 mL)
then sonicated for one hour. The resulting mixture was diluted with
1,4-dioxane (9 mL) and again sonicated for one hour. Subsequently,
the mixture was filtered thru a 0.45 micron teflon disk to remove
undissolved polymer. The orange filtrate (1 mL) was added to a
mixture of acetonitrile (5 mL) and deionized water (250 .mu.L).
This preparation produces 6.25 mL of the purple working polymer
solution. The 100 mM amine solutions (1,2-ethylenediamine,
1,3-propylenediamine, 1,4-butylenediamine, histamine,
1,5-pentylenediamine and 1,6-hexylenediamine) were prepared by
adding 1,4-dioxane to the appropriate amount of amine. For each
assay, the amine solution (50 .mu.L) was added to polymer solution
(1 mL) in a quartz cuvet and then shaken for 30 seconds before the
absorbance was measured. Each amine was measured at five different
concentrations: 1.5 and 3.5 mM, in triplicate and 0.5, 2.5, and 5.0
mM all with six replicates for a total of 24 measurements for each
amine and 144 measurements overall. Spectral data was obtained from
300-750 nm for each experiment at 25.degree. C.
[0214] In wet acetonitrile solution, the addition of each diamine
to a solution of polymer 1 resulted in an immediate change in the
solution color from purple to different shades of red. All spectral
data were normalized such that the area under each absorption
spectrum was equal to one. Analysis was performed on the response
of the polymer across the entire spectrum using nine different
wavelengths between 420 and 740 nm every 40 nm (Absorbance values
above 420 nm were used to eliminate potential bias in the analysis
due to absorption from histamine).
[0215] Absorbance data can be analyzed using pattern recognition
protocols. Linear discriminant analysis (LDA), through commercially
available software, was used to minimize variation within each
diamine group while maximizing differences between each different
diamine. FIG. 9 depicts the projection of the LDA results in three
dimensions. Each point in the plot contains information from the
nine wavelengths taken from the spectrum for the specific diamine.
Using the leave-one-out cross-validation method, for this example,
conjugated polymer 1 correctly classified 143 out of 144 measured
samples (>99% accuracy). In addition, the polymer response can
be dependant on the concentration of amine in solution. In the
example depicted in FIG. 8, the absorbance maximum for the
polymer-analyte complex did not follow a linear trend when
correlated with amine concentration. The formulator can perform
multiple linear regression on unnormalized absorbance data thereby
predicting the expected value. Additional analysis using principal
component analysis (PCA) can also further enhance the accuracy.
Utilizing the disclosed methods, diamines can be detected and
quantified in the nanomolar range (.about.100 nM).
Aqueous Based Sensing Using Polymer 2
[0216] For aqueous based sensing, absorbance studies were performed
using a Beckman Coulter 640 DU spectrophotometer or a SPECTRAmax
Plus plate reader from Molecular Devices in plastic (polypropylene)
cuvets or plastic microtiter plates, respectively. All measurements
were carried out in a Peltier thermostated sample holder maintained
at 25.degree. C. Canned tuna fish was purchased from area grocery
stores.
[0217] The following example describes methods of sensing,
identifying, and classifying structurally similar diamines using
polymer 2 in the presence of a buffered aqueous media (i.e. a
competitive solvent). The analysis carried out relied on the
optical signature of a 1 mM aqueous solution of a polymer 2
responding to 1 eq. of amine in a solution of 40 mM HEPES buffer
(pH 7.4) at a constant temperature.
[0218] For assay experiments carried out in water, deionized water
can be used. HEPES buffer solution was prepared to the
concentration of 50 mM as a stock solution and the pH was adjusted
to 7.4 using a Thermo Orion pH reader. Solutions of the amines were
prepared to the concentration of 10 mM in 50 mM HEPES buffer
solution (pH=7.4). The polymer solution was prepared by adding 61.6
mg of 1 to 80 mL of deionized water and the pH was adjusted to 7.4
using 1 M NaOH and 1M HCl and then sonicated for thirty minutes.
This preparation produces 80 mL of a 5 mM reddish/purple working
polymer solution.
[0219] For spectroscopic assay analysis in water, the following is
given as a representative procedure. A 5 mM stock solution of
polymer 2 (pH=7.4) was loaded at 60 .mu.l per well into a
multi-well plate. To each well 30 .mu.l of a 10 mM solution of
amine was added. Finally 210 .mu.l of 50 mM HEPES buffer solution
(pH 7.4) was added for a total volume of 300 .mu.l. Final
concentrations in each well are: buffer=40 mM, polymer=1 mM, and
amine=1 mM. The absorbance was measured from 300-700 nm with 47
replicates each. The entire spectrum between 350 and 700 nm every
10 nm (36 wavelengths) was used for the analysis. Absorbance values
below 350 nm were excluded so that the assay would not be
influenced by absorption from aromatic amines.
[0220] The formulator can reduce systematic errors and other
sources of error by using techniques familiar to the artisan.
Absorption spectra were recorded using a microtiter plate reader
with the samples randomized on the plates to avoid systematic
errors. For example, one method of reducing or minimizing
systematic error, is to conduct replicate analyses. For example, 47
replicate analyses (282 total samples) were carried out on
different days, using different solutions. The spectrum between
350-700 nm (at 10 nm intervals) was used for the analysis; 36 total
wavelengths were analyzed, thereby providing a 36 dimensional data
set. Absorbance values below about 350 nm were excluded in order to
minimize the influence of aromatic analytes. The pH was controlled
to ensure that discrimination between amines was based upon polymer
response to the amine rather than pH changes.
[0221] FIG. 10 is a plot of LDA results in two dimensions for
detecting amines in highly competitive aqueous media. Each axis of
the LDA plot represents weighted combinations of the 36 dimensional
data, where each point in the plot is an individual replicate that
contains information from the 36 wavelengths of the relevant
spectrum. The circles around each cluster represent 95% confidence
limits. Leave-one-out cross-validation was used to estimate the
predictive ability of the LDA model; this method showed excellent
discrimination between amines with the analyte being accurately
identifying the 99% of the time (i.e., 278/282 samples).
Aqueous Based Sensing Using Polymer 2 in a Fish Matrix
[0222] The following example is directed towards demonstrating the
utility of the described design to detect differing amounts of
biogenic amine in a food matrix, namely tuna fish extract. In this
particular example, the exact nature of the biogenic amine present
is histamine, the principle biogenic amine formed as tuna fish
decomposes, i.e. spoils.
[0223] For fish assay analysis, the following is provided herein as
a representative procedure. Generation of the "fish matrix"
followed methods known in the art. Fresh canned tuna fish was
drained and only solid meat was used. The meat (113.85 g) was
blended with 10% (v/v) trichloroacetic acid (TCA, 200 mL) to
extract biogenic amines. A 40 mL aliquot was centrifuged at 3000
rpm at 4.degree. C. for 10 minutes to remove precipitated protein
and other particulate. The liquid was decanted and washed with
hexanes to remove lipids and other oils. The resulting aqueous
solution was used as the fish matrix which was spiked with varying
amounts of histamine. The 100 mM histamine stock solution was made
by dissolving 184 mg histamine in 10 mL of 10% TCA. Twelve
different concentrations of histamine in the fish matrix were
generated from 0.0225 mM (2.5 ppm) to 4.5 mM (500 ppm). Each
histamine spiked fish matrix was loaded at 20 .mu.l per well into a
multi-well plate (8 replicates). 280 .mu.l of a 0.536 mM solution
of polymer 2 in 50 mM HEPES (pH=7.4) was added to each well. The
final polymer concentration was 0.5 mM. The entire spectrum between
350 and 700 nm every 10 nm (36 wavelengths) was used for the
analysis.
[0224] Polymer 2 was used to assess the amount of biogenic amine
present in a fish sample. The most prevalent biogenic amine found
in tuna is histamine. A tuna sample was obtained and spiked with
histamine. The fish sample was processed by extracting the biogenic
amines with trichloroacetic acid, while simultaneously
precipitating undesired proteins. FIG. 3 depicts the change in the
absorption spectrum of polymer 2 with increasing concentrations of
histamine and therefore the sensitivity of the disclosed methods to
indicate the degree of food spoilage. FIG. 4 depicts a linear
ratiometric response (A.sub.530/A.sub.420) with increasing
histamine concentration over the range useful for detecting food
spoilage associated with food poisoning. The sensitivity of the
described assay is better than the typical mammalian sense of
smell. Specifically, this method could allow for the detection of
the non-volatile biogenic amines at hazardous levels before the
fish begins to smell bad (e.g., in the early stages of
spoilage).
Aqueous Based Sensing Using Polymer 2 to Assess Fish Spoilage
[0225] The following example is directed toward the detection of
biogenic amines in a sample of fish with the goal of determine the
spoilage degree. In this particular example, the exact nature of
the biogenic amines present was unknown. The general quality of the
fish sample was measured as a function of the amount of biogenic
amine present.
[0226] Specifically, the canned tuna used in this analysis was
Bumblebee white albacore in water, purchased from a local
supermarket. A solution of polymer 2 in aqueous sodium hydroxide
(2.5 mM) was prepared by adding 1.1 mg of polymer 1 to 2.5 mL of
deionized water followed by 0.05 mL of 2 N sodium hydroxide
solution. The fish solutions were prepared by homogenizing canned
tuna. About 5 grams of the fish was placed in unsealed vials and
was set to spoil at 0, 4, 8, 24, and 48 hours. For each experiment,
at the designated time, 10% (w/v) trichloroacetic acid (3 mL) was
added to the fish flesh and minced using a mortar and pestle until
a slush-like consistency was obtained. The fish slush was
centrifuged at 3000 rpm (4.degree. C.) for about 30 minutes. The
fish extract (supernatant liquid) was removed with a pipette and
allowed to equilibrate to 30.degree. C. in a temperature bath. For
each assay, 900 .mu.L, of the 50 mM HEPES solution (pH 7.4) was
added to 50 .mu.L, of the polymer solution followed by 50 .mu.L, of
the fish extract. The absorbance was obtained for each time for
both polymer solutions.
[0227] FIG. 11 shows the absorbance spectra of 2 responding to the
addition of six different fish extracts from decaying tuna fish
flesh over time. With increasing concentration of biogenic amines
in the fish extract, i.e. as the fish spoils over time, the polymer
produces a unique chromogenic response. FIG. 12 shows the linear
ratiometric response from this assay correlated to spoilage
time.
Aqueous Based Sensing Using Polymer 2 Composites
[0228] The following example describes experiments directed at
detecting biogenic amines with the polymer compositions of the
present disclosure. In situ ion exchange was used to form
polymer-metal composites based on 2+ cations. The polymer chosen to
detect these amines was HT-poly(thiophene-3-propionate) (2), used
in combination with three transition metal salts including Cobalt,
Copper and Nickel.
[0229] A selected metal was first added to the polymer, and amines
were subsequently detected and identified using the metallic
polymer composition. HEPES buffer solution (50 mM) used in this
Example was prepared using methods known in the art. The pH of the
buffer solution was adjusted to 7.4. Amine solutions were prepared
to concentrations of 100 mM (of amine) in the 50 mM HEPES buffer
solution. A stock solution of 2 was prepared in HEPES buffer
solution in a concentration of 5 mM polymer 2.
[0230] A 5 mM solution of the polymer 2 was loaded at 12 .mu.l per
well into a multi-well plate. Fifteen .mu.l of a 100 mM solution of
amine mixture was added to each well. Twelve .mu.l of a 5 mM
solution of transition metal salt was added to each well. The wells
were then filled to a volume of 300 .mu.l with 50 mM HEPES buffer
solution, and the absorbance was subsequently measured.
Twenty-eight replicates were completed for each mixture in each
assay.
[0231] The addition of the transition metals to the polymer led to
precipitation (FIG. 5, vial B). While not wishing to be bound by
theory, it was believed that the subsequent addition of the amines
competes with the polymer for metal binding and breaks up the
aggregated polymer through coordination between the metal and the
amine. This produces a "turn-on" sensor since the colorless
solution becomes highly colored upon addition of amine, thereby
enhancing the sensitivity and utility of the assay. A composite
color signature or fingerprint of each polymer composite in the
array responding differently to each analyte can be obtained from
the chromic responses of the polymer array (FIG. 5, vials C-I).
Advanced pattern recognition protocols are not required to
differentiate the identity of the biogenic amine present. Rather,
the unique color pattern produced clearly indicates the identity of
the analyte by simple visual analysis (i.e. by eye).
Aqueous Based Sensing Using Polymer 2 Composites to Assess Biogenic
Amine Mixtures
[0232] These same polymer-metal composites described above can be
used to analyze mixtures of biogenic amines. Three of the most
common biogenic amines include putrescine, cadverine and histamine.
Therefore, mixtures of these three targets were investigated based
on the chromogenic response from the aggregate
polymer-composites.
[0233] The assay conditions were as described above. The amine
mixtures were produced according to ratios that can be seen in the
legend of FIGS. 14-16. The absorbance spectra for 0.4 mM of the
polythiophene/metal composite sensor upon addition of 5 mM of the
amine mixture were collected and normalized. FIGS. 14-16 depict the
averaged absorbance spectra for polymer 2 on its own, and with
Co.sup.2+, Cu.sup.2+ and Ni.sup.2+ in response to combinations of
three relevant biogenic amines: putrescine, caraverine and
histamine. In FIG. 14, the line with clear squares represents a 1:2
mixture of putrescine and histamine, the line with solid squares
represents a 1:2 mixture of putrescine and cadverine, and the line
with solid circles represents a 1:2 mixture of cadaverine and
histamine. In FIG. 15, the line with clear squares represents a 2:1
mixture of putrescine and histamine, the line with solid squares
represents a 2:1 mixture of putrescine and cadverine, and the line
with circles represents a 2:1 mixture of cadaverine and histamine.
In FIG. 16, the line with the solid squares represents a 1:1
mixture of putrescine and histamine, the line with solid circles
represents a 1:1 mixture of putrescine and cadverine, the line with
clear squares represents a 1:1 mixture of cadaverine and histamine,
and the line with clear circles represents a 1:1:1 mixture of
putrescine, cadaverine and histamine.
[0234] LDA was performed on the spectral data to differentiate and
classify the differences of the spectra from 420-700 nm (at 30 nm
intervals) for each sensor element of the array. Twenty-eight
replicates of each mixture were analyzed to ensure reproducibility
of this approach. Table 1 lists the response of each sensor element
of the array and the combined total response. Using the
polymer/metal complex array, a 99% classification rate was
obtained. The leave-one-out cross-validation number accuracy was
found to be 97%. It will be apparent to those skilled in the art
that various modifications and variations can be made in the
present invention without departing from the scope or spirit of the
invention. Other embodiments of the invention will be apparent to
those skilled in the art from consideration of the specification
and practice of the invention disclosed herein. It is intended that
the specification and examples be considered as exemplary only,
with a true scope and spirit of the invention being indicated by
the following claims.
[0235] The differences in polymer responses provide a
distinguishable optical signature for the analytes tested. These
signatures can be further tuned to generate maximum responses that
can be used to improve the detection of biogenic amines.
Alternatively, these signals can be combined to provide an array
based response, where the diagnostic value of the analysis can be
enhanced when the response from multiple functional polymers or
polymer composites are evaluated simultaneously. The optical
signature can be used to determine relative concentrations of
biogenic amines in foods by establishing trends between the
spectra. The techniques used in these experiments can be used to
detect the quality of other fish, meats, and other foods as a
function of spoilage and bacterial contamination.
[0236] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
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