U.S. patent application number 11/820882 was filed with the patent office on 2010-02-04 for cross-reactive sensors.
Invention is credited to Caroline L. Schauer, Frank J. Steemers, David R. Walt.
Application Number | 20100028923 11/820882 |
Document ID | / |
Family ID | 22696352 |
Filed Date | 2010-02-04 |
United States Patent
Application |
20100028923 |
Kind Code |
A1 |
Walt; David R. ; et
al. |
February 4, 2010 |
Cross-reactive sensors
Abstract
The present invention provides a novel cross-reactive sensor
system utilizing cross-reactive recognition elements. In the
inventive system, each of said one or more cross-reactive
recognition elements is capable of interacting with more than one
species of liquid analyte of interest, whereby each of said one or
more cross-reactive recognition elements reacts in a different
manner with each of said one or more species of liquid analytes of
interest to produce a detectable agent of each analyte of interest,
whereby said detectable agent is analyzed and the information is
processed for data acquisition and interpretation. In certain
preferred embodiments, the detectable agent and/or change is
detected directly, while in certain other preferred embodiments,
the detectable agent and/or change is detected with the help of a
transducing agent capable of relaying information about each
detectable agent generated for each of said species of liquid
analyte of interest, whereby said information is processed for data
acquisition and interpretation. The present invention also provides
method for the analysis of analytes comprising contacting one or
more analytes with the inventive system described above.
Inventors: |
Walt; David R.; (Lexington,
MA) ; Schauer; Caroline L.; (Silver Spring, MD)
; Steemers; Frank J.; (San Diego, CA) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Family ID: |
22696352 |
Appl. No.: |
11/820882 |
Filed: |
June 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10221815 |
Mar 5, 2003 |
7250267 |
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PCT/US01/08126 |
Mar 14, 2001 |
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11820882 |
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60189200 |
Mar 14, 2000 |
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Current U.S.
Class: |
435/19 ;
506/18 |
Current CPC
Class: |
C12Q 1/37 20130101; C12Q
1/44 20130101 |
Class at
Publication: |
435/19 ;
506/18 |
International
Class: |
C12Q 1/44 20060101
C12Q001/44; C40B 40/10 20060101 C40B040/10 |
Goverment Interests
GOVERNMENT SUPPORT
[0001] The work described herein was supported by Office of Naval
Research contract N00014-95-1-1340 and National Institutes of
Health grant GM 48142. Therefore, the government may have certain
rights in this invention.
Claims
1-114. (canceled)
115. A sensor system for liquid analytes comprising: one or more
cross-reactive recognition elements, wherein each of said
cross-reactive recognition elements is an enzyme or a protein
receptor and wherein each of said cross-reactive recognition
elements interacts directly with more than one species of liquid
analyte of interest, whereby each of said cross-reactive
recognition elements interacts in a different manner with each of
said more than one species of liquid analyte of interest to produce
a detectable event for each analyte of interest, wherein said
detectable event is a detectable agent, a detectable change or
both; wherein said detectable event is analyzed to obtain
information about the detectable event and said information is
processed for data acquisition and interpretation.
116. A sensor system for liquid analytes comprising: two or more
cross-reactive recognition elements, wherein each of said
cross-reactive recognition elements is an enzyme or a protein
receptor and wherein each of said cross-reactive recognition
elements interacts directly with more than one species of liquid
analyte of interest, whereby each of said cross-reactive
recognition elements interacts in a different manner with each of
said more than one species of liquid analyte of interest to produce
a detectable event for each analyte of interest, wherein said
detectable event is a detectable agent, a detectable change or
both; wherein said detectable event is analyzed to obtain
information about the detectable event and said information is
processed for data acquisition and interpretation.
117. A sensor system for liquid analytes comprising: two to five
cross-reactive recognition elements, wherein each of said
cross-reactive recognition elements is an enzyme or a protein
receptor and wherein each of said cross-reactive recognition
elements interacts directly with more than one species of liquid
analyte of interest, whereby each of said cross-reactive
recognition elements interacts in a different manner with each of
said more than one species of liquid analyte of interest to produce
a detectable event for each analyte of interest, wherein said
detectable event is a detectable agent, a detectable change, or
both; wherein said detectable event is analyzed to obtain
information about the detectable event and said information is
processed for data acquisition and interpretation.
118. A sensor system for liquid analytes comprising: two to ten
cross-reactive recognition elements, wherein each of said
cross-reactive recognition elements is an enzyme or a protein
receptor and wherein each of said cross-reactive recognition
elements interacts directly with more than one species of liquid
analyte of interest, whereby each of said cross-reactive
recognition elements interacts in a different manner with each of
said more than one species of liquid analyte of interest to produce
a detectable event for each analyte of interest, wherein said
detectable event is a detectable agent, a detectable change, or
both; wherein said detectable event is a detectable agent, a
detectable change, or both; wherein said detectable event is
analyzed to obtain information about the detectable event and said
information is processed for data acquisition and
interpretation.
119. The sensor system of any one of claims 115-118, further
comprising a processing unit.
120. The sensor system of any one of claims 115-118, wherein said
cross-reactive recognition elements are provided in array format
having a plurality of addresses, wherein each address in the array
contains one cross-reactive recognition element.
121. The sensor system of any one of claims 115-118, wherein said
cross-reactive recognition elements are provided in array format
having a plurality of addresses, wherein each address in the array
contains more than one cross-reactive recognition element.
122. The sensor system of claim 120, wherein the array comprises a
plurality of addresses, wherein two or more of the addresses
contain the same type of cross-reactive recognition element.
123. The sensor system of claim 120, wherein the array comprises a
plurality of addresses, wherein each address contains the same
cross-reactive recognition element.
124. The sensor system of claim 115-118, wherein at least one
cross-reactive recognition element is an enzyme selected from the
group consisting of esterases, proteases, hydrolases, isomerases,
lysases, transferases, oxido-reductases, and ligases.
125. The sensor system of claim 124, wherein the cross-reactive
recognition element is an esterase selected from the group
consisting of esterase from rabbit liver, esterase from porcine
liver, acetylcholine esterase from electrophorous electricus,
cholesterol esterase from hog pancrease, esterase from hog liver,
esterase from horse liver, esterase from mucor miehei, esterase
from bacillus sp., and esterase from bacillus
thermoglucosidasius.
126. The sensor system of claim 124, wherein the cross-reactive
recognition element is a protease selected from the group
consisting of proteinase K, chymotrypsin, papain, carboxypepsidase
A, substilisin, protease (staphylococcus), and protease VII.
127. The sensor system of any one of claims 115-118, wherein each
of said cross-reactive recognition elements is attached to a solid
support.
128. The sensor system of claim 127, wherein the solid support is a
bead, or a resin.
129. The sensor system of claim 127, wherein the solid support is a
bead, each bead is attached to one type of cross-reactive
recognition element, and wherein together the beads are provided in
array format having a plurality of addresses, wherein each address
in the array contains one bead.
130. The sensor system of claim 127, wherein the solid support is a
bead, each bead is attached to one type of cross-reactive
recognition element, and wherein together the beads are provided in
array format having a plurality of addresses, wherein each address
in the array contains more than one bead.
131. The sensor system of claim 129, wherein the array comprises a
plurality of addresses, wherein two or more of the addresses
contain beads having the same type of cross-reactive recognition
element.
132. The sensor system of claim 129, wherein the array comprises a
plurality of addresses, wherein each address contains beads having
the same cross-reactive recognition element.
133. A sensor system for liquid analytes comprising: one or more
cross-reactive recognition elements, wherein each of said
cross-reactive recognition elements is attached to a solid support,
wherein each of said cross-reactive recognition elements is an
enzyme or a protein receptor and wherein each of said
cross-reactive recognition elements interacts directly with more
than one species of liquid analyte of interest, whereby each of
said cross-reactive recognition elements interacts in a different
manner with each of said more than one species of liquid analyte of
interest to produce a detectable event for each analyte of
interest, wherein said detectable event is a detectable agent, a
detectable change or both; wherein said detectable event is
analyzed to obtain information about said detectable event and said
information is processed for data acquisition and
interpretation.
134. The sensor system of claim 133 further comprising a processing
unit.
135. The sensor system of claim 133, wherein said solid support is
a bead.
136. The sensor system of claim 135, wherein together the beads are
provided in array format having a plurality of addresses, wherein
each address in the array contains one bead.
137. The sensor system of claim 135, wherein together the beads are
provided in array format having a plurality of addresses, wherein
each address in the array contains more than one bead.
138. The sensor system of claim 136, wherein the array comprises a
plurality of addresses, wherein two or more of the addresses
contain a bead containing the same type of cross-reactive
recognition element.
139. The sensor system of claim 136, wherein the array comprises a
plurality of addresses, wherein each address contains a bead
containing the same cross-reactive recognition element.
140. The sensor system of claim 133, wherein at least one
cross-reactive recognition element is an enzyme selected from the
group consisting of esterases, proteases, hydrolases, isomerases,
lysases, transferases, oxido-reductases, and ligases.
141. The sensor system of claim 140, wherein the cross-reactive
recognition element is an esterase selected from the group
consisting of esterase from rabbit liver, esterase from porcine
liver, acetylcholine esterase from electrophorous electricus,
cholesterol esterase from mucor miehei, esterase from bacillus sp.,
and esterase from bacillus thermoglucosidasius.
142. The sensor system of claim 140, wherein the cross-reactive
recognition element is a protease selected from the group
consisting of proteinase K, chymotrypsin, papain, carboxypeptidase
A, subtilisin, protease (staphylococcus), and protease VII.
Description
BACKGROUND OF THE INVENTION
[0002] The ability to more efficiently detect and analyze specific
components (analytes) of a mixture or sample would greatly benefit
medicine, environmental analysis, and consumer industries (e.g.,
food analysis), to name a few. For example, the food industry
depends upon chemical analysis for quality control,
environmentalists depend upon chemical analysis for the detection
of harmful agents in natural resources, such as water, and the
medical community depends upon analysis for the detection of agents
such as metabolites, drugs, and glucose to name a few. Although
many methods suitable for sensing applications have been developed
(see, for example, Wolfbeis et al., Analytica Chim. Acta, 1991,
250, 181), there still remains a need to develop chemical sensors
that are capable of detecting analytes with specificity and
selectivity.
[0003] In general, a sensor device includes the following: 1) a
recognition element capable of identifying and interacting with the
analyte which usually is contained in low concentration in a
mixture of a variety of other components; 2) a transducer element
that can transform the recognition process into a measurable
signal; and 3) a processing unit, which, after amplification of the
primary signal, converts it into a familiar readout (e.g., pH, ppm,
etc.). One approach that has been utilized in the development of
more selective sensors is the use of bioorganic species (enzymes,
ion carriers, and natural or synthetic receptor/carriers) that are
believed to mimic the selectivity of nature and undergo specific
reactions with the entity to be recognized, resulting in specific
recognition and, consequently, sensing. The difficulty with this
approach, however, is that identification of agents that can
selectively interact with analytes of interest can be problematic.
For example, synthetic receptors often exhibit poor selectivity,
and have difficulties in transducing the recognition process.
Additionally, these approaches have generally focused on the
interaction of one specific agent with one analyte, creating a
cumbersome system if many analytes need to be detected. It would
thus be desirable to develop a system that would minimize the
number of recognition elements necessary, whereby the recognition
elements utilized would be cross-reactive, thus each interacting
with more than one analyte to generate a unique agent and/or change
that can be readily detected.
[0004] Towards this end, Walt et al. (see, for example, Dickinson
et al., Anal. Chem. 1999, 71, 2192; White et al., Anal. Chem. 1996,
68, 2191; Dickinson et al., Nature 1996, 382, 697) described a
novel approach, the "artificial nose", in which high-density
optical arrays that directly incorporate a number of structural and
operational features of the olfactory system were developed for the
cross-reactive analysis of vapors. Clearly, it would also be
desirable if an efficient and sensitive cross-reactive sensor
system could be developed for the analysis of liquid analytes,
preferably in an array format for high-throughput complex
analysis.
SUMMARY OF THE INVENTION
[0005] In recognition of the need for the development of novel and
efficient sensors, the present invention provides a sensor system
for liquid analytes comprising one or more cross-reactive
recognition elements, wherein each of said one or more
cross-reactive recognition element is capable of interacting with
more than one species of liquid analyte of interest, whereby each
of said one or more cross-reactive recognition elements reacts in a
different manner with each of said one or more species of liquid
analytes of interest to produce a detectable agent for each analyte
of interest, and whereby said detectable agent is analyzed and the
information is processed for data acquisition and interpretation.
In other preferred embodiments the sensor system employs at least
two or more cross-reactive recognition elements, for example, two
to five cross reactive recognition elements or two to ten
cross-reactive recognition elements. In yet other preferred
embodiments, the sensor system employs at least ten or more cross
reactive recognition elements. In a final embodiment, the present
invention employs at least fifty or more cross-reactive recognition
elements. For example, the "artificial nose" currently utilizes 39
equivalents of the cross-reactive recognition elements described
herein.
[0006] In certain embodiments, the detectable agent and/or change
can be analyzed directly, however in certain other embodiments, a
transducer agent is present, whereby said transducer is capable of
relaying information about each detectable agent generated for each
of said species of liquid analyte of interest, whereby said
information is processed for data acquisition and interpretation.
Thus, in another aspect, the present invention provides a system
for analysis comprising 1) a sensor system as described above,
wherein the sensor system optionally includes a transducer; and 2)
a processing unit, which, after amplification of the primary
signal, converts it into a familiar signal for subsequent data
analysis.
[0007] In yet another aspect, the present invention provides a
method for the analysis of analytes that involves contacting one or
more analytes of interest with a cross-reactive sensor system as
described above, and analyzing the agents and/or change associated
with the interaction. It will be appreciated that this agent and/or
change is either analyzed directly, or with the help of a
transducer. In certain embodiments, a processing unit (e.g.,
fluorescence detector) is utilized for the analysis of the agent
and/or change associated with the interaction of the cross-reactive
recognition element and the analytes of interest. It will also be
appreciated that the method of the present invention may further
include a chemoinformatic step, for example a step involving
computational analysis, to sort, analyze, or process the data
obtained.
[0008] In certain embodiments of the inventive sensor system, the
system for analysis, and the method of analysis, as described
above, the cross-reactive recognition elements are provided in
array format having a plurality of addresses, whereby each address
in the array contains one cross-reactive recognition element. In
certain other preferred embodiments, one or more cross-reactive
recognition elements are provided in array format having a
plurality of addresses, whereby each address in the array contains
more than one cross-reactive element. In still other embodiments,
two or more of the addresses contains the same type of
cross-reactive recognition element.
[0009] Alternatively, the inventive sensor system for analysis and
the method of analysis attaches the cross-reactive recognition
elements to a solid support, for example, beads or resin. The
cross-reactive recognition element on the solid support are
contacted with the analyte(s) of interest for capture and/or
reaction with, and identification of, the analyte of interest.
Alternatively, the solid support containing the cross-reactive
recognition element, e.g., a bead, is placed in array format having
a plurality of addresses, whereby each address in the array
contains one bead having an attached cross-reactive recognition
element. In certain other preferred embodiments, one or more beads
having attached cross-reactive recognition elements are provided in
array format having a plurality of addresses, whereby each address
in the array contains more than one bead having an attached
cross-reactive element. Finally, as with the array described above,
two or more of the addresses may further contain beads having the
same type of cross-reactive recognition element.
[0010] In certain embodiments of the present invention, each of
said one or more cross-reactive recognition elements is an enzyme
or a receptor. Exemplary enzymes for use in the present invention
include, but are not limited to those selected from the group
consisting of esterases, hydrolases, isomerases, lysases,
transferases, oxido-reductases, and ligases. In certain
embodiments, the enzyme is an esterase selected from the group
consisting of esterase from rabbit liver, esterase from porcine
liver, acetylcholine esterase from electrophorous electricus,
cholesterol esterase from hog pancrease, esterase from hog liver,
esterase from horse liver, esterase from mucor miehei, esterase
from bacillus sp., and esterase from bacillus
thermoglucosidasius.
[0011] Exemplary receptors include receptors wherein the binding
event is coupled to the
transduction scheme, e.g., antibody, protein, and small molecule
receptors. In certain embodiments of the present invention, the
cross-reactive recognition element is a receptor selected from the
group consisting of chemosensors, phosphorescent chemosensors,
cryptands, carcerands, hemicarcerands, hemicarceplexes,
carceplexes, spherands, hemispherands, cryptahemispherands,
coraplexes, velcraplexes, cyclophanes, cyclic oligonucleotides,
cyclic ureas, cyclic peptides, nanotubes, discrete aggregates,
clefts and polyaza clefts, macrolactams, macrobicyclics,
macrocyclics, macrotricyclics, calix[n]arenes, crown ethers,
cyclodextrins, hemispherands, cages, chlorophyls, cavitands,
cavitand dimers, catenanes, grids, polymers, double and triple
helicates, porphryns, viruses, self-assembling enzymes, DNA, RNA,
peptides, proteins, micelles, fibers and discs.
[0012] As mentioned above, in certain embodiments of the present
invention, a transducer is also present, wherein said transducer is
selected from the group consisting of electrochemical transducer,
optical transducer, thermal transducer, and acoustic transducer.
Exemplary electrochemical transducers include, but are not limited
to those having an energy transduction mode selected the group
consisting of amperometric, conductimetric, impedimetric,
potentiometric, and potentiometric stripping analysis. Exemplary
optical transducers include, but are not limited to, those having
an energy transduction mode selected from the group consisting of
absorbance, chemiluminescence, electrogenerated chemiluminescence,
fluorescence, fluorescence lifetime, fiber optic waveguides,
near-field microscopy, near-field spectroscopy, near-infared,
planar waveguides, surface enhanced raman, and surface plasmon
resonance. In certain preferred embodiments, the optical transducer
is a pH sensitive dye, including, but not limited to, those
selected from the group consisting of fluorescein,
carboxyfluorescein, SNAFL, SNARF, LysoSensor Green DND-189, Oregon
Green, NERF, LysoSensor Yellow/Blue DND-160, HPTS (pyranine),
BCECF, BCPCF, and Bodipy. In other preferred embodiments, the
optical transducer comprises an oxygen sensitive dye, including,
but not limited to Ru(4,7-diphenyl-1,10-phen).sub.3(Cl).sub.2,
Ru(bipy).sub.3Cl.sub.2 and trans-1(2'-methoxyvinyl)pyrene.
Exemplary acoustic transducers include, but are not limited to
those having an energy transduction mode selected from the group
consisting of acoustic plate mode, flexural plate mode, surface
acoustic wave, surface transverse wave, and thickness shear mode.
Exemplary thermal transducers include, but are not limited to those
having an energy transduction mode selected from the group
consisting of adiabatic and heat transduction.
DESCRIPTION OF THE DRAWING
[0013] The invention is described with reference to the several
figures of the drawing, in which,
[0014] FIG. 1 depicts a microtiter plate as a sensor array,
contrasting selective sensor arrays and cross-reactive sensor
arrays.
[0015] FIG. 2 depicts five initially tested esters that are
distinguishable by two esterases.
[0016] FIG. 3 depicts the separation of five esters using nine
esterases.
[0017] FIG. 4 depicts certain exemplary analytes (esters).
[0018] FIG. 5 depicts certain exemplary analytes (esters).
[0019] FIG. 6 depicts the relative fluorescence versus time for
methylcyclohexane carboxylate and for methyl 2-methyl butyrate.
[0020] FIG. 7 depicts the relative fluorescence versus time for
methyl 2-methyl glycidate and for methyl 6-methyl nicotinate.
[0021] FIG. 8 depicts the relative fluorescence versus time for
methyl nicotinate and for propyl butyrate.
[0022] FIG. 9 depicts the relative fluorescence versus time for
L-alanine methyl ester and for methyl butyrate.
[0023] FIG. 10 depicts the relative fluorescence versus time for
acetylcholine chloride and for napthyl acetate.
[0024] FIG. 11 depicts the relative fluorescence versus time for
isopropyl acetate and for isopropyl nicotinate.
[0025] FIG. 12 depicts the relative fluorescence versus time for
ethyl valerate and for hexyl acetate.
[0026] FIG. 13 depicts the relative fluorescence versus time for
D-alanine methyl ester and for ethyl propionate.
[0027] FIG. 14 depicts the relative fluorescence versus time for
t-butyl acetate and for ethyl acetate.
[0028] FIG. 15 depicts the relative fluorescence versus time for
phenyl acetate and for methyl benzoate.
[0029] FIG. 16 depicts the relative fluorescence versus time for
ethyl benzoate and for butyric acid ethyl ester (ethyl
butyrate).
[0030] FIG. 17 depicts the three-dimensional principal component
analysis for nine esterases and twelve esters.
[0031] FIG. 18 depicts the three-dimensional principal component
analysis for eleven esters and eight esterases.
[0032] FIG. 19 depicts the three-dimensional principal component
analysis for twenty-three substrates and eight enzymes.
[0033] FIG. 20 depicts a Lineweaver-Burk plot of various
concentrations of methyl 6-methyl nicotinate with bacteria esterase
1. Vmax and Km values are 4-10.sup.-7 Ms.sup.-1 and 2.6 mM,
respectively.
[0034] FIG. 21 depicts a Lineweaver-Burk plot of various
concentrations of ethyl valerate with bacteria esterase 1. Vmax and
Km values are 2.times.10.sup.-7 Ms.sup.-1 and 1 mM,
respectively.
[0035] FIG. 22 is a table that depicts the slope of the reaction of
nine different esterases with methyl 6-methyl nicotinate in
fluorescence units/time from data collected over a number of
days.
[0036] FIG. 23 depicts the reaction rate curves of esterase from
Mucor miehei (denoted fungi) with twenty-three different ester
analytes.
[0037] FIG. 24 depicts a principal component analysis (PCA)
confusion matrix that indicates the actual vs. the calculated
identity of twenty-three ester analytes based on reaction rates
with nine different esterases. Esterases are Rabbit Liver, Porcine
liver, Horse liver, Hog liver, Mucor miehei (fungi), Bacillus sp.
(bacteria-1), Bacillus th. bacteria-2), Acetylcholine Esterase from
Electrophorus electricus, and Cholesterol Esterase from hog
pancreas. Esters are ethyl propionate (EP), ethyl benzoate (EB),
ethyl valerate (EV), ethyl acetate (EA), ethyl butyrate (BA),
propyl butyrate (PB), isopropyl nicotinate (IN), isopropyl acetate
(IA), methyl 2-methyl butyrate (MMBU), methyl butyrate (MU), methyl
benzoate (MB), methyl 2-methyl glycidate (MMG), methyl nicotinate
(MNI), methyl 6-methyl nicotinate (MMNI), methyl cyclohexane
carboxylate (MC), L-alanine methyl ester (LM), D-alanine methyl
ester (DM), t-butyl acetate (TB), hexyl acetate (HA), 2-naphthyl
acetate (NA), acetylcholine chloride (AC), phenyl acetate (PA), and
propyl acetate (PRA).
[0038] FIG. 25 depicts the chemical structure of four esters, ethyl
acetate (EA), ethyl butyrate (BA), methyl 2-methyl butyrate (MMBU),
and methyl butyrate (MBU), and highlighted regions of the confusion
matrix of FIG. 24 showing the actual vs. the calculated identity of
each ester based on reaction rates with nine different
esterases.
[0039] FIG. 26 depicts the chemical structure of two esters,
L-alanine methyl ester (LM) and D-alanine methyl ester (DM), and
highlighted regions of the confusion matrix of FIG. 24 showing the
actual vs. the calculated identity of each ester of based on
reaction rates with nine different esterases.
[0040] FIG. 27 depicts the fluorescence ratio versus time for
rabbit esterase immobilized onto beads upon interaction with twenty
different ester analytes.
[0041] FIG. 28 depicts the fluorescence ratio versus time for
bacteria esterase 1 immobilized onto beads with six different ester
analytes.
[0042] FIG. 29 depicts the fluorescence ratio versus time for
porcine esterase immobilized onto beads with twelve different ester
analytes.
[0043] FIG. 30 depicts a PCA confusion matrix for protein analytes
digested with the seven cross-reactive proteases.
[0044] FIG. 31 depicts highlighted regions of the principal
component analysis (PCA) confusion matrix of FIG. 30 for albumin
protein analytes digested with the seven cross-reactive
proteases.
DETAILED DESCRIPTION OF THE INVENTION
[0045] As mentioned above, the development of more efficient
sensors has been a challenging problem in analytical chemistry and,
as a result, there has been continuous research and development in
this important area. In recognition of this need, the present
invention provides, for the first time, a sensor system for liquid
analytes utilizing cross-reactive recognition elements. In general,
the system of the present invention provides one or more
cross-reactive recognition elements, wherein each of the one or
more cross-reactive recognition elements is capable of interacting
with more than one species of liquid analyte of interest, whereby
each of the one or more cross-reactive recognition elements
interacts in a different manner with each of the one or more
species of liquid analytes of interest to produce a detectable
agent for each analyte of interest. By "cross-reactive" it is
meant, as used herein, that the recognition element utilized is
capable of interacting with more than one species of analyte of
interest, and additionally interacts with each of the more than one
species in a different and uniquely identifiable manner (e.g.,
different rate of reaction, different reaction product produced, to
name a few). In certain embodiments of the present invention, the
detectable agent and/or change can be monitored or identified
directly, wherein the information is processed for data acquisition
and interpretation. In certain other embodiments of the present
invention, a transducer agent is additionally provided, whereby the
transducer is capable of relaying information about each detectable
agent and/or change generated for each of the species of liquid
analyte of interest, whereby the information is processed for data
acquisition and interpretation.
[0046] Thus, in another aspect, the present invention provides a
system for analysis comprising: 1) a sensor system as described
above, wherein the sensor system optionally includes a transducer;
and 2) a processing unit, which, after amplification of the primary
signal, converts it into a familiar signal for subsequent data
analysis. As used herein, the terms "transducer" or "energy
transducer" are meant to include agents that are capable of
relaying information about each detectable agent and/or change
generated by the recognition event for each of the species of
liquid analyte of interest. It will be appreciated that, in certain
embodiments, two or more cross-reactive recognition elements will
be required to analyze a solution of one or more analytes. In
certain other preferred embodiments, however, only one
cross-reactive recognition element will be required to analyze a
solution of one or more analytes.
[0047] The present invention thus also provides a method for the
analysis of analytes comprising: 1) contacting one or more analytes
of interest with a cross-reactive sensor system as described above,
and 2) analyzing the agents and/or change associated with the
interaction. It will be appreciated that this agent and/or change
is either analyzed directly, or with the help of a transducer. In
certain embodiments, a processing unit (e.g., fluorescence
detector, which is capable of detecting a fluorescent transducer)
is utilized for the analysis of the agent and/or change associated
with the interaction of the cross-reactive recognition element and
the analytes of interest. It will also be appreciated that the
method of the present invention may further include a
chemoinformatic step, for example a step involving computational
analysis, to sort, analyze, or process the data obtained.
[0048] In preferred embodiments of the inventive sensor system, the
system for analysis, and the method of analysis, as described
above, the cross-reactive recognition elements are provided in
array format having a plurality of addresses, whereby the array
comprises a plurality of addresses wherein two or more of the
addresses contain the same type of cross-reactive recognition
element. As shown in FIG. 1, the traditional sensor array system
utilizing selective recognition agents is contrasted with the
inventive cross-reactive sensor arrays. In this fashion, a
"combinatorial sensor array" is generated, whereby a plurality of
analytes can be detected by relatively few cross-reactive
recognition agents.
[0049] It will be appreciated that the inventive sensors, in
addition to being attached to array supports, can also be attached
to solid supports, such as beads, and resins. As used herein, these
terms are intended to include: beads, columns, plates, vials,
tubes, slides, pellets, disks, strips, wafers, electrical leads,
electrodes, wires, fibers, gels, or particles such as cellulose
beads, controlled pore-glass beads, silica gels, polystyrene beads
optionally cross-linked with divinylbenzene and optionally grafted
with polyethylene glycol and optionally functionalized with amino,
hydroxy, carboxy, or halo groups, grafted co-poly beads,
polyacrylamide beads, latex beads, dimethylacrylamide beads
optionally cross-linked with N,N'-bis-acryloyl ethylene diamine, or
glass particles coated with hydrophobic polymer, to name a few. In
general, the solid supports are made of any of a variety of
materials, such as polymer, glass, silica, metal and the like. In
certain embodiments, amino-functionalized or hydroxy-terminating
beads are utilized to effect attachment of the sensors to the
support. Those skilled in the art will further appreciate that
attachment of any cross-reactive recognition element to any solid
support merely requires choosing the appropriate cross-linker.
Attachment of the cross-linker may occur during or after synthesis
of the solid substrate.
[0050] Once attached to the solid support, the cross-reactive
recognition elements are contacted with the analyte(s) of interest.
In certain embodiments, the cross-reactive recognition element on
the bead is contacted directly with the analyte. In other
embodiments, the solid support containing the cross-reactive
recognition element, e.g., the bead, is placed in array format
having a plurality of addresses, whereby each address in the array
contains one bead having an attached cross-reactive recognition
element. In yet other embodiments, one or more beads having
attached cross-reactive recognition elements are provided in array
format having a plurality of addresses, whereby each address in the
array contains more than one bead having an attached cross-reactive
element. For example, once synthesized, the beads containing the
cross-reactive recognition elements may be placed in complimentary
wells of an etched optical imaging fiber Illumina (San Diego,
Calif.).
[0051] Finally, as with the array described above, two or more of
the addresses may further contain beads having the same type of
cross-reactive recognition element. Alternatively, two or more of
the addresses may further contain beads having different types of
cross-reactive recognition elements. In yet another embodiment,
each address contains a bead having attached to it a different
cross-reactive recognition element. Placing the solid supports in
an array format allows analysis of analytes that utilize multiple
cross-reactive recognition elements simultaneously.
[0052] The present invention will be described in more detail below
with respect to certain exemplary embodiments. It will be
appreciated, however, that these embodiments are not intended to
limit the scope of the present invention.
Exemplary Cross-Reactive Recognition Agents
[0053] It will be appreciated that a variety of cross-reactive
recognition agents can be utilized in the present invention for the
analysis of a variety of desired analytes or species of analytes.
In particular, cross-reactive recognition agents are selected for
their ability to interact with one or more analytes or species of
analytes of interest, such that the cross-reactive recognition
agent interacts in a different manner with each individual analyte
of interest. As used herein, the term "interacts in a different
manner" means that a distinct agent and/or change is produced upon
interaction of the analyte and the cross-reactive agent, such that
each distinct agent and/or change can be uniquely identified.
Analytes and cross-reactive agents may interact in a different
manner by producing agents (e.g., reaction products) at different
rates, by producing agents having different chemical properties, or
by inducing a detectable conformational change, to name a few.
[0054] It will be appreciated that the system of the present
invention contemplates the use of any suitable cross-reactive
recognition agent for the analysis of desired analytes. As
discussed above, and as will become readily apparent below,
suitable cross-reactive recognition agents comprise those agents
that are capable of interacting in a different manner with each
individual analyte of interest to produce a distinct agent and/or
change that can be readily detected. It will be appreciated that,
in certain embodiments, two or more cross-reactive recognition
elements will be required to analyze a solution of analytes,
because certain cross-reactive recognition elements will interact
only with certain analytes in a solution and not others. In certain
other preferred embodiments, only one cross-reactive recognition
element will be required to analyze a solution of analytes, because
the cross-reactive recognition element will be able to interact
with each analyte to produce a unique agent and/or change that can
subsequently be analyzed. As mentioned above, the inventive system
is also preferably utilized in array format having a plurality of
addresses, wherein two or more of the addresses contain the same
cross-reactive recognition element for analysis of multiple
analytes. In preferred embodiments, one cross-reactive recognition
agent per address is utilized, however, it will also be appreciated
that, in other embodiments, more than one cross-reactive
recognition agent per address can be utilized for analysis. One
example of such a system utilized for selective recognition agents
includes a sequential microenzymatic assay of cholesterol,
triglycerides, and phospholipids in a single aliquot. See, Nanjee
et al., Clinical Chem, 1996, 42, 915. Additionally, in another
example, the sensing of acetylcholine by a tricomponent-enzyme
layered electrode using Faradaic Impedance Spectroscopy, cyclic
voltammetry and microgravimetric quartz crystal microbalance
transduction methods is described in Alfonta et al., Anal. Chem.,
2000, 72, 927.
[0055] Certain exemplary cross-reactive recognition agents that can
be utilized in the present invention include, but are not limited
to, cross-reactive enzymes, cross-reactive receptors,
cross-reactive transition metals, cross-reactive ligands for
transition metals, and cross-reactive synthetic catalysts, to name
a few. For example, certain cross-reactive enzymes that can be
utilized include, but are not limited to, esterases, hydrolases,
isomerases, lysases, transferases, oxido-reductases, and ligases.
Such enzymes can be utilized to detect and/or analyze a variety of
reagents including, but not limited to amino acids (using L-amino
acid oxidase, D-amino acid oxidase), alcohols (using alcohol
dehydrogenase, alcohol oxidase), sugars, esters (using esterases),
and proteins (using proteases).
[0056] In certain embodiments of the present invention,
cross-reactive esterases are utilized, whereby the esterases are
capable of hydrolyzing different esters (analytes) at different
reaction rates, thus producing desired products at different rates.
The hydrolysis and production of reaction products can then be
monitored over time to produce distinct patterns for different
analytes. As depicted below in Equation 1, esterases hydrolyze
esters to produce alcohols and carboxylic acids:
##STR00001##
As depicted, the enzyme reacts with the analyte (ester) causing a
change in pH (via production of the carboxylic acid). The change in
pH over time (due to differing reaction rates with different
analytes) can then be monitored (in one example, by using pH
sensitive fluorescent dyes) to produce distinct patterns for
specific analytes. As described in the examples below, certain
exemplary esterases for use in the present invention include, but
are not limited to, esterase from rabbit liver, esterase from
porcine liver, acetylcholine esterase from electrophorous
electricus, cholesterol esterase from hog pancrease, esterase from
hog liver, esterase from horse liver, esterase from mucor miehei,
esterase from bacillus sp., and esterase from bacillus
thermoglucosidasius.
[0057] In certain other embodiments, the cross-reactive recognition
element is an agent that is capable of undergoing a cross-reactive
biorecognition event. For example, the cross-reactive
biorecognition event may be based on a catalytic conversion with an
enzyme or organelle acting as a catalytic agent transforming an
agent or a substrate into a measurable product. Alternatively, the
analyte may only take part in a binding event based upon an
antibody or receptor. As discussed above, such agents are
cross-reactive, that is they are capable of interacting with
different species of analytes to produce distinct agents and/or
changes. It will also be appreciated by one of ordinary skill in
the art that so-called artificial receptors can be utilized in the
present invention.
[0058] Thus, in certain embodiments, the present invention
contemplates the use of receptors as cross-reactive recognition
agents, including, but not limited to, chemosensors, phosphorescent
chemosensors, cryptands, carcerands, hemicarcerands,
hemicarceplexes, carceplexes, spherands, hemispherands,
cryptahemispherands, coraplexes, velcraplexes, cyclophanes, cyclic
oligonucleotides, cyclic ureas, cyclic peptides, nanotubes,
discrete aggregates, clefts and polyaza clefts, macrolactams,
macrobicyclics, macrocyclics, macrotricyclics, calix[n]arenes,
crown ethers, cyclodextrins, hemispherands, cages, chlorophyls,
cavitands, cavitand dimers, catenanes, grids, polymers, double and
triple helicates, porphryns, viruses, self-assembling enzymes, DNA,
RNA, peptides, proteins, micelles, fibers and discs. Each of these
agents, as described herein, and equivalents thereof, as utilized
in the inventive system, is capable of interacting with one or more
analytes of interest and producing a detectable agent and/or change
that uniquely identifies each of the one or more analytes. In but
one example, a broadly selective receptor (e.g., cross-reactive)
could be monitored for analyte binding by a change in fluorescence
(a fluorescent probe is utilized to monitor the binding of the
guest), FTIR (fourier transform infared spectroscopy), NMR (nuclear
magnetic resonance spectroscopy), vapor pressure osmometry, or any
other suitable method to monitor a change in binding.
[0059] It will be appreciated by one of ordinary skill in the art
that a specific cross-reactive recognition agent can selected to
tailor the inventive system to the specific analytes being
analyzed. Exemplary systems for use in the inventive cross-reactive
sensor are described in the following: "Handbook of Biosensors and
Electronic Noses: Medicine, Food, and Environment", Kress-Rogers,
Ed., CRC Press, New York, 1997; "Biosensors: Fundamentals and
Applications" Turner, A.; Karube, I.; Wilson, G., Eds., Oxford
University Press, Oxford, 1987; and "Introduction to Bioanalytical
Sensors", Cunningham, A., Ed., John Wiley & Sons, Inc., New
York, 1998, and the entire contents of each reference are hereby
incorporated by reference.
Detection of Analytes
[0060] After interaction with the cross-reactive recognition agent
to produce a distinct agent and/or change, this agent and/or change
is capable of either being monitored or analyzed directly, or a
transducer element agent may also be employed to facilitate
analysis. As used herein, the terms "transducer" or "energy
transducer" are meant to include agents that are capable of
relaying information about each detectable agent and/or change
generated by the recognition event for each of the species of
liquid analyte of interest. It will be appreciated by one of
ordinary skill in the art that a variety of transducer agents can
be utilized, and that transducer agent is selected for the ability
to relay information about the agent and/or change generated by the
cross-reactive recognition event.
[0061] For example, as discussed above, the hydrolysis of esters
can be detected by pH sensitive dyes (due to the production of
protons). Additionally, amino acids can be detected using L-amino
oxidase or D-amino acid oxidase using a pH indicator or O.sub.2
indicator; alcohols can be detected using alcohol dehydrogenase or
alcohol oxidase using a pH sensor, NADH-indicator, or O.sub.2
indicator; and proteins can be detected using proteases and a pH
sensor or competition assay with a protein that is reactive and
carries covalently attached dye molecules which increase in
fluorescence as the reaction progresses.
[0062] In general, suitable transducers are selected from the group
consisting of electrochemical transducer, optical transducer,
thermal transducer, and acoustic transducer, to name a few. In
certain preferred embodiments, electrochemical transducers are
utilized, preferably those involving an energy transduction mode
selected from the group consisting of amperometric, conductimetric,
impedimetric, potentiometric, and potentiometric stripping
analysis. In other preferred embodiments, the optical transducers
are utilized, preferably those involving an energy transduction
mode selected from the group consisting of absorbance,
chemiluminescence, electrogenerated chemiluminescence,
fluorescence, fluorescence lifetime, fiber optic waveguides,
near-field microscopy, near-field spectroscopy, near-infared,
planar waveguides, surface enhanced raman, and surface plasmon
resonance. In certain particularly preferred embodiments of the
present invention, the optical transducer comprises a pH sensitive
dye, most preferably pH sensitive dyes selected from the group
consisting of fluorescein, carboxyfluorescein, SNAFL, SNARF,
LysoSensor Green DND-189, Oregon Green, NERF, LysoSensor
Yellow/Blue DND-160, HPTS (pyranine), BCECF, BCPCF, and Bodipy, or
oxygen sensitive dyes comprises Ru(bipy).sub.3Cl.sub.2,
Ru(4,7-diphenyl-1,10-phen).sub.3(Cl).sub.2 and
trans-1-(2'-methoxyvinyl)pyrene. Other fluorescent probes can also
be utilized as transducer agents according to the present
invention, many of which are described in "Molecular Probes:
Handbook of Fluorescent Probes and Research Chemicals", Seventh
Edition, Richard P. Haughland, 1999, the entire contents of which
are hereby incorporated by reference. As depicted in the examples
described below, the use of pH sensitive fluorescent dyes enables
the detection of relative fluorescence over time and thus the
differences in reaction rates can be measured, thus uniquely
identifying desired analytes (for examples, esters). As depicted in
FIGS. 2 and 3, five substrates initially tested are distinguishable
by two esterases using principal component analysis. Additionally,
FIG. 3 depicts distinguishable regions corresponding to specific
analytes. In still other embodiments, acoustic transducers are
utilized preferably those involving an energy transduction mode
selected from the group consisting of acoustic plate mode, flexural
plate mode, surface acoustic wave, surface transverse wave, and
thickness shear mode. In yet other embodiments thermal transducers
are utilized, which preferably employ atic or heat
transduction.
[0063] These and other suitable transducers are more generally
described with respect to particular systems in "Introduction to
Bioanalytical Sensors", A. J. Cunningham, John Wiley & Sons,
New York: 1998, the entire contents of which are hereby
incorporated by reference. Additionally, this reference describes
the methods of analysis and data interpretation for specific
transducers for a variety of systems, each of which can be adapted
to the present invention. Thus, the present invention additionally
provides a system for analysis comprising 1) the sensor system
described in detail above, and 2) a processing unit for the
acquisition and analysis of data. In certain preferred embodiments,
this processing unit is capable of measuring measuring
fluorescence. It will be appreciated that any processing unit may
be utilized that is appropriate for the particular transducer
employed, or, for the case of direct analysis, a processing unit
that is capable of processing and interpreting data directly from
the agent and/or change produced upon interaction of the
cross-reactive recognition element and the analyte to provide a
familiar readout (e.g. ppm, pH, relative fluorescence, to name a
few).
[0064] Furthermore, the present invention also provides a method of
analysis comprising: 1) contacting one or more analytes of interest
with a cross-reactive sensor system as described above, and 2)
analyzing the agents and/or change associated with the interaction.
It will be appreciated that this agent and/or change is either
analyzed directly, or with the help of a transducer. In certain
embodiments, a processing unit (e.g., fluorescence detector can be
used to monitor relative fluorescence over time) is utilized for
the analysis of the agent and/or change associated with the
interaction of the cross-reactive recognition element and the
analytes of interest. One example of a technique for analysis that
can be utilized in the method of the present invention can be found
in "Identification of Multiple Analytes Using an Optical Sensor
Array and Pattern Recognition Neural Networks" Anal. Chem. 1997,
69, 4641-4648, the entire contents of which are hereby incorporated
by reference. One of ordinary skill in the art will also realize
that, in addition to a step of processing using a processing unit,
an optional step may include further data analysis, e.g., using a
chemoinformatic step (for example, via computational analysis) to
sort, process, or further analyze the data obtained.
Uses
[0065] As will be appreciated by one of ordinary skill in the art,
the ability to detect desired liquid analytes is very useful in a
range of disciplines. For example, the inventive system may be
utilized for medical/biochemical applications, specifically for the
analysis of such agents, including, but not limited to, drugs (for
example, cocaine), glucose, blood gas, neurotransmitters (for
example, acetylcholine), DNA sequence, pH and electrolytes. Other
uses include, but are not limited to, environmental analysis (for
the analysis of such harmful agents as PCBs, pesticides, heavy
metals, herbicides) and bioprocessing technology (for the analysis
of pH, sugars, Mab Production, dissolved gases, recombinant DNA
processes, alcohols). For example, industries involved in
pharmaceuticals, food processing and recombinant DNA technology
need effective sensors for monitoring various processes. One of
ordinary skill in the art will realize that the inventive system
may be utilized in a variety of disciplines requiring analysis of
liquid analytes, and are not limited to those applications
discussed above.
EQUIVALENTS
[0066] The representative examples which follow are intended to
help illustrate the invention, and are not intended to, nor should
they be construed to, limit the scope of the invention. Indeed,
various modifications of the invention and many further embodiments
thereof, in addition to those shown and described herein, will
become apparent to those skilled in the art from the full contents
of this document, including the examples which follow and the
references to the scientific and patent literature cited herein. It
should further be appreciated that the contents of those cited
references are incorporated herein by reference to help illustrate
the state of the art.
[0067] The following examples contain important additional
information, exemplification and guidance which can be adapted to
the practice of this invention in its various embodiments and the
equivalents thereof.
EXEMPLIFICATION
Example 1
Enzyme-Based Sensor Array
[0068] As described above, the present invention provides an
inventive sensor system that utilizes cross-reactive recognition
elements, wherein each of the cross-reactive recognition elements
is capable of interacting with more than one species of liquid
analyte of interest, whereby each of the one or more cross-reactive
recognition elements reacts in a different manner with each of the
one or more species of liquid analytes of interest to produce a
detectable agent for each analyte of interest. This detectable
agent can then be analyzed directly, or a transducer agent can also
be provided to relay the chemical information for analysis. In one
embodiment of the present invention, an enzyme-based sensor array
is utilized, as will be described in more detail below.
[0069] The present example utilizes a cross reactive sensor array
that is generated from a group of esterases that react to a broad
range of ester analytes, where each ester elicits a response from
multiple esterases. As described herein, one advantage to the
cross-reactive sensor array is that only a few sensors are needed
to distinguish a wide variety of analytes since a pattern
recognition program can differentiate the many combinations of
responses. The present example further illustrates the inventive
approach, which employs analyte-related enzymes that all catalyze
the same type of reaction, but have different and somewhat
overlapping specificities. In this way, the specificity of the
sensor array is restricted to a certain class of substrates. We
utilize the cross-reactivity of enzymes in combination with a
pattern recognition scheme to identify the specific molecule
present.
[0070] Enzymes catalyze reactions required for biological processes
and exhibit intrinsic specificity. In their recognition of
substrates, many enzymes are selective; for example, L-glutamate
oxidase oxidizes only L-glutamate. Other enzymes are
class-selective, such as L-amino acid oxidase, which catalyzes the
oxidation of a range of L-amino acids with varying kinetics. The
incorporation of class-selective enzymes into an enzymatic array
bioassay format exploits the enzyme's inherent cross-reactive
nature.
[0071] Esterases catalyze the hydrolysis of esters to carboxylic
acids (see equation below).
##STR00002##
A fluorescent pH indicator, fluorescein, was added to the reaction
mixture to measure the change in acidity resulting from the
hydrolysis reaction in a 96-well microtiter plate format. The
fluorescence response was monitored over time to give a temporal
pH-induced fluorescence pattern. Esterases were utilized for these
initial demonstrations of the enzymatic array assay because they
are commercially available, relatively stable, and react with a
wide range of esters. Esterases have been used to test ester
chirality (Janes et al., Chem. Eur. J., 1998, 4, 2324) and as
synthetic tools for efficiently hydrolyzing a variety of esters
(Ohno et al., Org. React, 1989, 37, 1).
General Procedure for Enzyme-Based Sensor Array:
[0072] In but one example of an inventive sensor array system, a
cross-reactive esterase is utilized as the recognition element.
[0073] First, solutions of desired substrates (certain exemplary
substrates are depicted in FIGS. 4 and 5) were made from 100 mM of
substrate in CH.sub.3CN (or buffer, depending on the solubility of
the ester). The analyte concentration was chosen to be at most
one-fifth of the Michaelis constant (kM) for the esterases. All
solvents and substrates were purchased from Aldrich, Sigma, and
Fluka Chemical Companies and used as received.
[0074] All substrates were purchased from Sigma or Fluka.
Subsequently, a solution having the following components is
generated:
[0075] 420 .mu.L of 100 mM substrate in CH.sub.3CN;
[0076] 470 .mu.L CH.sub.3CN;
[0077] 600 .mu.L of a fluorescein dye solution in buffer; and
[0078] 10510 .mu.L 0.01 mM Phosphorous Buffer Solution to
generate:
[0079] 12000 .mu.L total substrate solution
Second, enzyme solutions were assembled to the desired activity
levels as shown below and exemplary enzyme based biosensors are
listed below and were purchased from Sigma and Fluka:
[0080] Esterase from Rabbit liver
[0081] Esterase from Porcine liver
[0082] Acetylcholine Esterase from Electrophorus electricus
[0083] Cholesterol Esterase from Hog pancrease
[0084] Esterase from Hog liver
[0085] Esterase from Horse liver
[0086] Esterase from Mucor miehei (denoted fungi)
[0087] Esterase from Bacillus sp. (denoted bacteria-1)
[0088] Esterase from Bacillus thermoglucosidasius (denoted
bacteria-2)
Additionally, depending on the activity of the above esterases, the
solutions were assembled as such:
TABLE-US-00001 Cholesterol 35 U/mg 5.65 mg/mL Acetylcholine 850
U/mg 0.073 mg/mL Bacillus sp. 0.1 U/mg 6.0 mg/mL Bacillus th. 0.1
U/mg 6.0 mg/mL Hog liver 220 U/mg 1.3 mg/mL Horse liver 0.7 U/mg
2.5 mg/mL Mucor 1.0 U/mg 5.7 mg/mL Porcine 150 U/mg 1 .mu.L/mL
Rabbit 80-120 U/mg 2 .mu.L/mL
[0089] The microtiter plate assay provides a rapid and reproducible
system to measure the hydrolysis reactions. In the present example,
the microtiter plate assay contained the nine esterases in
different columns and the ester analytes in the different rows.
Each esterase catalyzed hydrolysis reaction was addressed
individually to monitor the kinetics by scanning each well
independently. After preparation of the desired solutions, to the
microtiter plate was first pipetted (using a pipetman) 100 .mu.L of
the substrate solution into each well. Then, a 5 .mu.L aliquot of
the enzyme solution was pipetted into the well, resulting in a
volume in the 96 well microtiter plate (per well) of 105 .mu.L,
which equals to 29 .mu.M substrate and 30-0.04 .mu.g of enzyme, or
at most a concentration of substrate of one-fifth the Km. The plate
was allowed to shake for 10 seconds before the first reading. The
reader reads 15 second intervals while shaking before each read.
The plate was monitored for 90 seconds by a Molecular Devices,
Spectra Max Gemini, fluorescence microtiter plate reader. The
reader scans each row and column of the microtiter plate reading
each well individually. Therefore, each enzyme was monitored
individually and the enzyme's kinetics were measured at the same
point in time. The reader displays the resulting changes in
fluorescence in graph form, the slopes of which are used for the
computational analysis program, MATLAB. The MATLAB program analyzes
the individual interactions and separates them into clusters, which
can indicate the ability of the sensor to distinguish the
analytes.
[0090] The nine lyophilized esterases (Sigma and Fluka) were used
as received and chosen based on their availability and wide range
of specific activities. Twenty-three esters, ranging from simple
aliphatic esters to multi-functional chiral esters, were chosen as
analytes. Exemplary analytes include
[0091] Ethyl propionate (EP)
[0092] Ethyl benzoate (EB)
[0093] Ethyl valerate (EV)
[0094] Ethyl acetate (EA)
[0095] Ethyl butyrate (BA)
[0096] Propyl butyrate (PB)
[0097] Isopropyl nicotinate (IN)
[0098] Isopropyl acetate (IA)
[0099] Methyl 2-methyl butyrate (MMBU)
[0100] methyl butyrate (MBU)
[0101] Methyl benzoate (MB)
[0102] Methyl 2-methyl glycidate (MMG)
[0103] Methyl nicotinate (MNI)
[0104] Methyl 6-methyl nicotinate (MMNI)
[0105] Methyl cyclohexane carboxylate (MC)
[0106] L-alanine methyl ester (LM)
[0107] D-alanine methyl ester (DM)
[0108] t-butyl acetate (TA)
[0109] Hexyl acetate (HA)
[0110] 2-naphthyl acetate (NA)
[0111] Acetylcholine chloride (AC)
[0112] Phenyl acetate (PA)
[0113] Propyl acetate (PRA)
The esters vary in the placement of functional groups close to the
reaction center and include representatives ranging from methyl,
ethyl and propyl esters, as well as acetates.
[0114] FIGS. 6-16 depict the relative fluorescence versus time for
exemplary substrates (FIGS. 4 and 5) upon interaction with
different types of cross-reactive esterases. Furthermore, FIGS.
17-19 depict the principal component analysis for different sets of
esterases and analytes of interest, establishing distinguishable
regions for specific analytes, allowing identification of the
specific analytes.
[0115] The differing hydrolytic susceptibility of the esters to the
esterases resulted in reactivity rate patterns, which were used to
distinguish the esters. Among all of the esters, phenyl acetate
(PA) is hydrolyzed the fastest. These patterns of reactivity
provide a means to distinguish PA from other substrates.
[0116] Of the nine esterases examined, acetylcholine esterase
hydrolyzes all twenty-three esters. Rabbit esterase reacts with all
of the esters except the simple aliphatic esters. In order to
exemplify the discriminating ability of the current system,
D-alanine (DM) and L-alanine (LM) methyl esters were included as
analytes. DM and LM are both hydrolyzed by acetylcholine esterase
and bacteria 1 esterase, while bacteria 2 esterase, hydrolyzes only
LM and not DM. These differences in reactivities provide a
"fingerprint" of each ester. (See FIG. 26).
[0117] The sensitivity of the assay is further illustrated in FIGS.
20 and 21. The reaction rates of bacteria esterase 1 with ethyl
valerate and methyl methyl nicotinate are quite distinguishable,
having Vmax and Km values of 2.times.10.sup.-7 Ms.sup.-1 and 1 mM
and 4.times.10.sup.-7 Ms.sup.-1 and 2.6 mM, respectively.
[0118] The esterase array was further tested for assay
reproducibility. The hydrolysis reaction slopes of three esters,
PA, methyl butyrate (MB), and ethyl butyrate (BA) were measured
initially and after three months. The initial slope range and
standard deviation for the three esters with acetylcholine esterase
were PA (-1.4.+-.0.2).times.10.sup.2, MB
(-1.3.+-.0.01).times.10.sup.2, and BA (-2.9.+-.1.6).times.10.sup.3
respectively; after three months the slopes were PA
-1.5.times.10.sup.3, MB -1.3.times.10.sup.2, and BA
-3.2.times.10.sup.2. The slopes, therefore, lie within the initial
ranges after three months. FIG. 22 further illustrates this point
by showing that the slope of the reaction in fluorescence
units/time does not vary from experiment to experiment over a
number of days.
[0119] The hydrolysis reaction initial slopes were used as input
for principal component analysis (PCA). The individual interactions
were analyzed and separated into clusters for which the tightness
of the clusters indicates the array's ability to distinguish the
analytes. By combing the response patterns of all nine esterases
for the twenty-three analytes from four independent assays, a
confusion matrix was compiled from the PCA data. The rate curves
for the esterase from fungi are shown in FIG. 23. The confusion
matrix compares the calculated versus actual ester identity and is
90% correct using 98% of the data's variance.
TABLE-US-00002 TABLE 1 The confusion matrix results: column number
indicates number of esters identified incorrectly. 0 1 2 3 4 EA;
PA; NA (IA) PRA BA (EA(2); MB; IN; (DM(2); TB MMBU; MNI; EV; (EV;
LM) MBU) MMG; MBU; AC; IA; PB; EB; MC; EP; DM; LM, MMNI; HA; MMBU
Abbreviation in parenthesis indicates the PCA identification of the
incorrect ester.
[0120] As seen in Table 1, four of the twenty-three esters were
misidentified. For three of the four misidentified esters, no clear
structural basis exists to cause the esters to be misclassified in
PCA, however, their hydrolysis reaction slopes are similar. The
fourth ester, BA was misidentified four times-twice as ethyl
acetate (EA) and once each as MB and methyl 2-methyl butyrate
(MMB). Without being limited to any particular theory, we propose
that the completely incorrect assignment of BA is based on
structural similarities, as twice it was misidentified as an ethyl
ester and twice as a methyl ester of butyrate. These results are
further illustrated in the PCA confusion matrix of FIG. 24, which
shows the data for twenty-three different esters; the PCA confusion
matrix of FIG. 25, which illustrates separately the confusion
matrix data for esters EA, BA, MB, and MMB; and the PCA confusion
matrix of FIG. 26, which illustrates separately the data for the
esters LM and DM. These results indicate that the identification of
the esters, LM, and DM (FIG. 26) is 100% accurate.
[0121] For mixture analysis, concentration runs of four esters, PA,
ethyl valerate (EV), methyl nicotinate (MNI) and methyl 6-methyl
nicotinate (MMNI) were performed with the nine esterases. The rates
of the esterase reaction were plotted on a Lineweaver-Burk plot to
determine the Km and Vmax and were used to identify subsequent
mixtures of the four esters. For example, an equal volume mixture
of PA/MMNI gave a reaction rate of 1.1.times.10 Ms.sup.-1 for
bacteria esterase 1, while the reaction rates of the individual
components added to 1.3.times.10 Ms.sup.-1. For all of the
esterases, the esterase reaction of the mixture is a linear
combination of the individual esters because the concentrations of
the esters are well below the Km for the esterase.
[0122] In conclusion, the esterases' inherent cross-reactivity was
incorporated into the array and the resulting nine-esterase array
was able to distinguish over twenty individual analytes. The
ability to distinguish such a diverse group of analytes using a
limited suite of sensing materials demonstrates the utility of the
approach. The microtiter plate format is an efficient and
reproducible method for performing such analysis.
Example 2
Enzyme-Based Sensor Array
[0123] The present enzyme based sensor array is amenable to many
different types of cross-reactive recognition elements, including
cross-reactive recognition elements that are enzymes. To illustrate
this point we used enzyme proteases to distinguish various protein
substrates. Specifically, the present example demonstrates that
different proteins can be distinguished by cross-reactive
degradation using non-specific proteases. The sensor array consists
of seven different proteases: proteinase K, chymotrypsin, papain,
carboxypepsidase A, substilisin, protease (staphylococcus), and
protease VII. Each protease has the capability of cleaving a
particular peptide analyte to yield predictable and identifiable
fragments, which are used to identify the peptide analyte. For
example, reaction of chymotrypsin with a particular nine amino acid
peptide analyte to yield three distinguishable primary amine
products is shown below.
##STR00003##
The resulting primary amines can be easily measured by any suitable
amine reactive dye.
[0124] A typical reaction mixture consisted of, for example, 1
.mu.g of protein analyte and 0.5 units of protease. The reaction
mixture was incubated with shaking at room temperature for one
hour. Once the digestion was complete, 5 .mu.l of
o-phthaldialdehyde (OPA) dye solution was added, which reacts with
all primary amines. The reaction of OPA with a primary amine is
illustrated below.
##STR00004##
Reaction with OPA is allowed to proceed for two minutes before
reading the plate. Those skilled in the art will readily appreciate
that this reaction can easily be modified to suit different
combinations of enzyme proteases and their substrates.
[0125] Those skilled in the art will further appreciate that any
protein analyte can be used in a protease reaction. Exemplary
protein analytes include catalase, fumarase, pepsin, glucose
oxidase, cocarboxylase, alcohol oxidase, hemocyanin,
.beta.-lactoglobulin, tyrosinase, yeast enzyme concentrate, bovine
albumin, galactose oxidase, lectin, peroxidase, lysozyme,
acetylcholine esterase, aldehyde dehydrogenase, glutaminase,
alcohol dehydrogenase, sheep albumin, human albumin, horse albumin,
goat albumin, casein, alkaline phosphitase, urease, aconitase,
hexokinase, avidin, cystatin, s-acetyl coenzyme A synthetase,
papain, and IgG.
[0126] FIG. 30 depicts a PCA confusion matrix using 99% of the
variance. Specifically, the calculated protein identity was
compared with the actual identity of the protein to assess the
accuracy of the assay. The results obtained were 86% correct for
the 29 proteins tested. FIG. 31 shows a PCA confusion matrix for
the various albumin protein analyte family members tested. The
results in FIG. 31 reveal that 70% of the incorrect assignments
were due to the albumins. This could be due to the similarity in
structure and sequence of the albumin proteins.
Example 3
Immobilization of Cross-Reactive Recognition Elements
[0127] The present invention further provides an inventive sensor
array system wherein the cross-reactive recognition element is
immobilized onto a solid support made of different materials, such
as polymer, silica, glass, metal and the like. For example, where
the cross-reactive recognition element is an enzyme, the enzyme can
be immobilized onto a solid support. In one embodiment, the
cross-reactive recognition element is immobilized onto a bead. In
general, one type of cross-reactive recognition element is
immobilized on one bead. Other solid supports include columns,
plates, vials, tubes, slides, pellets, disks, strips, wafers,
electrical leads, electrodes, wires, fibers, gels, or particles
such as cellulose beads, controlled pore-glass beads, silica gels,
polystyrene beads optionally cross-linked with divinylbenzene and
optionally grafted with polyethylene glycol and optionally
functionalized with amino, hydroxy, carboxy, or halo groups,
grafted co-poly beads, polyacrylamide beads, latex beads,
dimethylacrylamide beads optionally cross-linked with
N,N'-bis-acryloyl ethylene diamine, or glass particles coated with
hydrophobic polymer, to name a few. Those skilled in the art will
recognize that any of the cross-reactive recognition elements
described herein may be immobilized onto a solid support using
methods standard in the art.
[0128] According to the present invention, solid supports are
synthesized to which the cross-reactive recognition element may be
attached. In general, this may be accomplished by cross-linking the
solid support to the cross-reactive recognition element of
interest. For example, a bead may be attached to a cross-linker,
which may then be cross-linked to a cross-reactive recognition
element, e.g., an enzyme, for use in a sensor array. The beads in
the sensor array may then be contacted with the analyte(s), as
described herein above. Some examples of beads include microbeads
(e.g., 3 .mu.m, 5 .mu.m, and 10 .mu.m in diameter) and nanobeads
(e.g., 100 nm, 90, nm and 10 nm in diameter), which may be made of
silica, metal, or any standard polymer. The cross-linker may be
incorporated into the bead at the time of bead synthesis, or
alternatively, may be added to the bead after bead synthesis.
[0129] As but one example, amine reactive cross-linkers may be used
to generate amine funtionalized beads. A cross-reactive recognition
element, e.g., an enzyme, may be attached to the bead by
cross-linking the amine on the bead to the amine on the
cross-reactive recognition element. Some examples of amine reactive
cross-linkers include disuccinimidyl suberate (DSS), disuccinimidyl
tartrate (DST), dimethyl 3,3'-dithiobispropionate (DTBP), 3,3;
dithiobis[sulfosuccinimidyl-2HCl]-propionate, disuccinimidyl
glutarate (DSG), dithiobis[succinimidyl propionate] (DSP), dimethyl
saberimidate.2HCl (DMS), dimethylpimelimadate.2HCl (DMP), dimethyl
adipimidate.2HCl (DMA), 1,5 difluoro-2,4-dinitrobenzene (DFDNB),
bis [sulfosuccinimidyl] (Bs.sup.3),
bis[2-succinimidyloxycarbonyloxyethyl]sulfone (BSOCOES), and
bis-[.beta.-(4-azidosalicylamido) ethyl]disulfide (BASED), all of
which are available from Pierce Chemical Company, Rockford,
Ill.
[0130] As will be appreciated by those skilled in the art, the bead
and the cross-reactive recognition element may be attached by
cross-linkers other than amine reactive cross-linkers. For example,
the bead and the cross-reactive recognition element may be attached
by reaction of a carboxylic acid and an amine, a sulfhydryl and an
amine, or an arginine and an amine. Such cross-linkers are also
available from Pierce Chemical Company, Rockford, Ill.
[0131] The synthesis reaction involves contacting a cross-linker of
choice, for example, an amine reactive cross-linker, with the solid
support, e.g., beads, and an enzyme in a vial, which is shaken for
30 minutes. 1 .mu.M glycine solution is then added to the vial to
abate any reactive group and the beads are washed repeatedly. Those
skilled in the art will further appreciate that the cross-reactive
recognition element may be attached to the solid support
simultaneously with the synthesis of the solid support.
[0132] Once synthesized, the beads containing the cross-reactive
recognition elements are placed in complimentary wells of an etched
optical imaging fiber from Illumina (San Diego, Calif.) (1 mm
diameter with 3 or 5 .mu.m wells). The fiber tip is then coated
(e.g., spin coated) with a colorless polymer layer (e.g.,
acrylamide generated by the standard reaction of an amine reactive
cross linker (bis(acrylamine)) with acrylamide) to secure the beads
to their respective complimentary wells. The polymer layer further
slows down the dissociation of H.sup.+ into solution so that the
dye can be used to measure the reaction.
[0133] FIGS. 27-29 illustrate reaction rates with each of
twenty-three different esters obtained using beads having
immobilized thereon one of three different esterases; rabbit
esterase, bacteria esterase, and porcine esterase to demonstrate
the success of this approach.
* * * * *