U.S. patent application number 14/348426 was filed with the patent office on 2017-09-21 for oxygenase-based biosensing systems for measurement of halogenated alkene concentrations.
This patent application is currently assigned to Colorado State University Research Foundation. The applicant listed for this patent is Colorado State University Research Foundation. Invention is credited to Brian C. Heinze, Kenneth F. Reardon.
Application Number | 20170269001 14/348426 |
Document ID | / |
Family ID | 51351470 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170269001 |
Kind Code |
A9 |
Reardon; Kenneth F. ; et
al. |
September 21, 2017 |
Oxygenase-Based Biosensing Systems For Measurement Of Halogenated
Alkene Concentrations
Abstract
A biosensing system that measures the concentration of
halogenated alkenes is disclosed.
Inventors: |
Reardon; Kenneth F.; (Fort
Collins, CO) ; Heinze; Brian C.; (Fort Collins,
CO) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Colorado State University Research Foundation |
Fort Collins |
CO |
US |
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Assignee: |
Colorado State University Research
Foundation
Fort Collins
CO
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Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20140234882 A1 |
August 21, 2014 |
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Family ID: |
51351470 |
Appl. No.: |
14/348426 |
Filed: |
October 1, 2012 |
PCT Filed: |
October 1, 2012 |
PCT NO: |
PCT/US12/58331 PCKC 00 |
371 Date: |
March 28, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13562592 |
Jul 31, 2012 |
9493806 |
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14348426 |
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12100308 |
Apr 9, 2008 |
9493805 |
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13562592 |
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61541421 |
Sep 30, 2011 |
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60922496 |
Apr 9, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 21/6428 20130101;
G01N 2021/7786 20130101; C12Y 114/00 20130101; G01N 21/7703
20130101; C12Q 1/26 20130101; C12Q 1/48 20130101; C12N 9/1088
20130101; C12N 9/0069 20130101; C12Y 113/00 20130101; C12N 9/0071
20130101; G01N 2021/7753 20130101; C12Q 1/34 20130101 |
International
Class: |
G01N 21/77 20060101
G01N021/77; C12Q 1/34 20060101 C12Q001/34; C12Q 1/48 20060101
C12Q001/48; C12Q 1/26 20060101 C12Q001/26 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with Government support under
contract number BES-0529048 awarded by the National Science
Foundation. The U.S. Government has certain rights in this
invention.
Claims
1. A biosensing system that measures the concentration of a
halogenated alkene in a solution, said biosensing system comprising
a first biocomponent that catalyzes the reaction of said
halogenated alkene, and a second biocomponent that catalyzes the
reaction of a halogenated alkene epoxide, and a transducer layer
that luminesces, and wherein said transducer layer is part of an
optode.
2. A method for measuring the concentration of a halogenated alkene
in a solution whereby a first biocomponent catalyzes the reaction
of said halogenated alkene and oxygen, and whereby a second
biocomponent catalyzes the reaction of a halogenated alkene epoxide
produced by said first biocomponent, and whereby a transducer layer
luminesces, and whereby said transducer layer luminescence is
altered by oxygen and/or hydrogen ions in said solution, and
whereby photons from the luminescence of said transducer layer
enter into a fiber optic cable and are transmitted to a
photomultiplier, and whereby said photomultiplier produces an
output signal that is coupled to an algorithm that transforms the
signal generated by said photomultiplier into an output correlated
to the concentration of said halogenated alkene in said
solution.
3. The method of claim 2 wherein: said first biocomponent is
selected from the group consisting of toluene ortho-monooxygenase,
toluene ortho-monooxygenase-Green, toluene ortho-monooxygenase
variant, and toluene dioxygenase; said second biocomponent is
selected from the group consisting of epoxide hydrolase,
glutathione synthetase, glutathione S-transferase and
gamma-glutamylcysteine synthetase; and said transducer layer
comprises a luminescent reagent selected from the group consisting
of RuDPP and fluorescein.
4.-5. (canceled)
6. The biosensing system of claim 1 wherein said first biocomponent
catalyzes the reaction of said halogenated alkene and oxygen and
said halogenated alkene epoxide is created by the reaction of said
first biocomponent with said halogenated alkene, wherein said
transducer layer luminescence is altered by oxygen and/or hydrogen
ions in said solution, and wherein photons from the luminescence of
said transducer layer enter into a fiber optic cable and are
transmitted to a photomultiplier, and wherein said photomultiplier
produces an output signal that is coupled to an algorithm that
transforms the signal generated by said photomultiplier into an
output correlated to the concentration of said halogenated alkene
in the solution.
7. The bio sensing system of claim 6 wherein said halogenated
alkene is selected from the group consisting of tetrachloroethene,
trichloroethene, dichloroethene, and monochloroethene.
8. The bio sensing system of claim 6 wherein said first
biocomponent is selected from the group consisting of toluene
ortho-monooxygenase, toluene ortho-monooxygenase-Green, and toluene
ortho-monooxygenase variant; said second biocomponent is selected
from the group consisting of epoxide hydrolase, glutathione
synthetase, glutathione S-transferase and gamma-glutamylcysteine
synthetase; and said transducer layer comprises a luminescent
reagent selected from the group consisting of RuDPP and
fluorescein.
9.-10. (canceled)
11. The biosensing system of claim 1 wherein said first
biocomponent and said second biocomponent comprise cells, and
wherein said cells contain enzymes selected from the group
consisting of oxygenases, monooxygenases, dioxygenases, toluene
dioxygenase, toluene ortho-monooxygenase, toluene
ortho-monooxygenase-Green, epoxide hydrolase, glutathione
synthetase, glutathione S-transferase and gamma-glutamylcysteine
synthetase and wherein said cells are immobilized within a matrix,
and wherein said matrix is in contact with said transducer
layer.
12. The bio sensing system of claim 11 wherein said cells are
alive.
13. The bio sensing system of claim 11 wherein said cells are
dead.
14. The bio sensing element of claim 11 wherein the nucleotide
coding sequence of said biocomponent enzymes are on a plasmid or
plasmids within a whole cell biocomponent or on a chromosome of a
whole cell biocomponent.
15. (canceled)
16. The bio sensing element of claim 11 wherein said optode is
selected from the group consisting of an oxygen optode and a pH
optode.
17. (canceled)
18. The biosensing system of claim 1, wherein said halogenated
alkene is trichloroethene, wherein said first biocomponent
catalyzes the reaction of trichloroethene and oxygen and said
second biocomponent catalyzes the reaction of trichloroethene
epoxide, and wherein said transducer layer luminescence is altered
by oxygen in said solution, and wherein photons from the
luminescence of said transducer layer enter into a fiber optic
cable and are transmitted to a photomultiplier, and wherein said
photomultiplier produces an output signal that is coupled to an
algorithm that transforms the signal generated by said
photomultiplier into an output correlated to the concentration of
trichloroethene in the solution.
19.-21. (canceled)
22. The method of claim 2, wherein said halogenated alkene is
trichloroethene wherein said first biocomponent selected from the
group consisting of toluene ortho-monooxygenase, toluene
ortho-monooxygenase-Green and, toluene ortho-monooxygenase variant,
and catalyzes the reaction of trichloroethene and produces
trichloroethene epoxide, and wherein said trichloroethene epoxide
is a substrate for a reaction catalyzed by said second biocomponent
selected from the group consisting of epoxide hydrolase,
glutathione synthetase, glutathione S-transferase and
gamma-glutamylcysteine synthetase, and wherein said transducer
layer luminescence is altered by oxygen in said solution, and
wherein said photomultiplier produces an output signal that is
coupled to an algorithm that transforms the signal generated by
said photomultiplier into an output correlated to the concentration
of said trichloroethene in said solution.
23.-43. (canceled)
44. The biosensing system of claim 1, wherein said first
biocomponent catalyzes the reaction of a halogenated alkene and
oxygen and said second biocomponent catalyzes the reaction of a
halogenated alkene epoxide created by the reaction of said first
biocomponent with said halogenated alkene, and wherein said
transducer layer comprises compounds and chemical complexes
containing ruthenium, and wherein said transducer layer
luminescence is altered by oxygen in said solution, and wherein
photons from the luminescence of said transducer layer enter into a
fiber optic cable and are transmitted to a photomultiplier, and
wherein said photomultiplier produces an output signal that is
coupled to an algorithm that transforms the signal generated by
said photomultiplier into an output correlated to the concentration
of said halogenated alkene in the solution.
45. (canceled)
46. A biosensing system that measures the concentration of a
halogenated alkene in a solution, said biosensing system comprising
a biocomponent that catalyzes the reaction of said halogenated
alkene, and a transducer layer that luminesces, and wherein said
transducer layer is part of an optode.
47.-53. (canceled)
54. The biosensing system of claim 46, wherein said biocomponent is
toluene ortho-monooxygenase, a toluene ortho-monooxygenase variant,
or toluene dioxygenase.
55. The biosensing system of claim 46, wherein said biocomponent
comprises a purified cell-free enzyme.
56. The biosensing system of claim 46, wherein said halogenated
alkene is trichloroethene.
57. The biosensing system of claim 46, wherein said transducer
layer comprises a luminescent reagent that is RuDPP or
fluorescein.
58. The biosensing system of claim 1, wherein said first and second
biocomponents comprise a purified cell-free enzyme.
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application No. 61/541,421
filed Sep. 30, 2011, which application is incorporated herein by
reference. This application is a continuation-in-part of U.S.
patent application Ser. No. 13/562,592 filed on Jul. 31, 2012 which
is a continuation-in-part of U.S. patent application Ser. No.
12/100,308, filed Apr. 9, 2008, which claimed the benefit of
priority to U.S. Provisional Patent Application Ser. Nos.
60/922,496, filed Apr. 9, 2007, and 61/024,453, filed Jan. 29,
2008, and which was a continuation-in-part of U.S. patent
application Ser. No. 10/478,822, now U.S. Pat. No. 7,381,538, which
was a national phase entry under 35 U.S.C. .sctn.371 of
PCT/US02/17407, filed Jun. 1, 2002, which claimed the benefit of
priority to U.S. Provisional Patent Application Ser. No.
60/295,211, filed Jun. 1, 2001.
BACKGROUND
[0003] Trichloroethene (TCE) and tetrachloroethene
(perchloroethene, PCE) are the most commonly used industrial
solvents and degreasers in the world. The annual U.S. consumption
of TCE was 245 million pounds in 2005, with a 4.5% per year
increase since then. As a consequence of its extensive use,
spillage and improper disposal have resulted, and thus TCE is one
of the most commonly found chemicals in contaminated sites. About
34% of the drinking water sources and most groundwater
contamination sites are estimated to contain TCE, and 75% of EPA
National Priority List hazardous waste sites and Superfund sites
have TCE pollution. TCE is a suspected carcinogen, as well as a
known kidney and liver toxin. In addition, TCE can be transformed
to vinyl chloride via microbial anaerobic dehalogenation in
groundwater, increasing the concerns regarding TCE contamination in
groundwater.
[0004] TCE concentration measurement using gas chromatography (GC)
is the most popular TCE detection method with good selectivity and
low limits of detection (LOD), as low as 0.02 .mu.g/L using EPA
method 8260b for volatile organic compounds, while absorption
spectroscopy based techniques (e.g., Fourier transform infrared
spectroscopy) can also detect trace amounts of TCE with short
acquisition times and high signal-to-noise ratios. However, these
methods are time-consuming and expensive, and additional
pretreatment steps are often required prior to sample analysis.
[0005] Biosensors have the potential to be excellent alternatives
or complements to traditional analytical chemical methods for
environmental monitoring. By integrating a biological process and
transduction, a biosensor is capable of real-time analysis with
simplicity of operation. In a biosensor system, enzymes have
benefits as the biocomponents due to their high sensitivity and
good specificity, while optical transduction has potential
advantages over electrical transduction in environmental monitoring
because of low signal losses over long distance as well as not
requiring a reference signal. Biosensors are often reagentless, and
can thus provide continuous, in-situ measurements as a
cost-effective alternative compared with traditional analytical
methods.
SUMMARY
[0006] In one aspect, a biosensing system is disclosed that
measures the concentration of a halogenated alkene in a solution
and comprises a first biocomponent that catalyzes the reaction of a
halogenated alkene, and a second biocomponent that catalyzes the
reaction of a halogenated alkene epoxide. The biosensing system
also includes a transducer layer that luminesces and produces
photons and is part of an optode.
[0007] In another aspect, a method for measuring the concentration
of a halogenated alkene in a solution is disclosed whereby a first
biocomponent catalyzes the reaction of a halogenated alkene and
oxygen, and a second biocomponent catalyzes the reaction of a
halogenated alkene epoxide produced by the first biocomponent; and
whereby a transducer layer luminesces and the luminescence of the
transducer layer is altered by oxygen in the solution; and whereby
the photons produced by the luminescence of the transducer layer
enter into a fiber optic cable and are transmitted to a
photomultiplier, whereby the photomultiplier produces an output
signal that is coupled to an algorithm that transforms the signal
generated by the photomultiplier into an output correlated to the
concentration of the halogenated alkene in the solution. In one
embodiment, the first biocomponent is selected from the group
consisting of toluene ortho-monoxygenase (EC 1.13.12), a toluene
ortho-monoxygenase variant, a toluene dioxygenase (EC 1.14.12.11),
and toluene ortho-monoxygenase-Green. In one embodiment, the second
biocomponent is selected from the group consisting of epoxide
hydrolase (EC 3.3.2.10), glutathione synthetase (EC 6.3.2.3),
glutathione S-transferase (EC 2.5.1.18) and gamma-glutamylcysteine
synthetase (EC 6.3.2.2). In one embodiment, the transducer layer is
RuDPP and/or fluorescein.
[0008] In an aspect of the disclosure, a biosensing system that
measures the concentration of halogenated alkenes in a solution
comprises a first biocomponent that catalyzes the reaction of a
halogenated alkene and oxygen and a second biocomponent that
catalyzes the reaction of a halogenated alkene epoxide created by
the reaction of the first biocomponent with the halogenated alkene.
The biosensing system also comprises a transducer layer that
luminesces and the luminescence of the transducer layer is altered
by oxygen in the solution; and the photons produced by the
luminescence of the transducer layer enter into a fiber optic cable
and are transmitted to a photomultiplier where the photomultiplier
produces an output signal that is coupled to an algorithm that
transforms the signal generated by the photomultiplier into an
output correlated to the concentration of the halogenated alkene in
the solution. In an embodiment, the halogenated alkene is selected
from the group consisting of tetrachloroethene, trichloroethene,
dichloroethene isomers, and monochloroethene. In one embodiment,
the first biocomponent is selected from the group consisting of
toluene ortho-monoxygenase, and toluene ortho-monoxygenase-Green,
and toluene dioxygenase. In one embodiment, the second biocomponent
is selected from the group consisting of epoxide hydrolase,
glutathione synthetase, glutathione S-transferase and
gamma-glutamylcysteine synthetase. In an embodiment, the transducer
layer is RuDPP and/or fluorescein.
[0009] In an aspect of the present disclosure, a biosensing element
is disclosed that measures the concentration of a halogenated
alkene in a solution. The tip comprises a first biocomponent that
catalyzes the reaction of the halogenated alkene and a second
biocomponent that catalyzes the reaction of a halogenated alkene
epoxide. The first biocomponent and said second biocomponent
comprise cells that contain enzymes selected from the group
consisting of oxygenases, monooxygenases, dioxygenases, toluene
ortho-monoxygenase-Green, toluene dioxygenase, epoxide hydrolase,
glutathione synthetase, glutathione S-transferase and
gamma-glutamylcysteine synthetase. The cells are immobilized within
a matrix that is in contact with a transducer layer. The transducer
layer is part of an optode. In one embodiment, cells are alive. In
an embodiment, cells are dead. In an embodiment, the transducer
layer is an optical transducer that interacts with oxygen. In
another embodiment, the transducer layer comprises RuDPP and/or
fluorescein.
[0010] In one aspect, a biosensing element that measures the
concentration of trichloroethene in a solution is disclosed. The
biosensing element comprises a first biocomponent that catalyzes
the reaction of trichloroethene and oxygen and a second
biocomponent that catalyzes the reaction of trichloroethene
epoxide. The biosensing element also comprises a transducer layer
that luminesces and the luminescence of the transducer layer is
altered by oxygen in the solution; and the photons produced by the
luminescence of the transducer layer enter into a fiber optic cable
and are transmitted to a photomultiplier wherein the
photomultiplier produces an output signal that is coupled to an
algorithm that transforms the signal generated by the
photomultiplier into an output correlated to the concentration of
trichloroethene in the solution. In one embodiment, the first
biocomponent is selected from the group consisting of toluene
ortho-monoxygenase, toluene ortho-monoxygenase-Green and toluene
dioxygenase. In another embodiment, the second biocomponent is
selected from the group consisting of epoxide hydrolase,
glutathione synthetase, glutathione S-transferase and
gamma-glutamylcysteine synthetase. In one embodiment, the
transducer layer is RuDPP.
[0011] In one aspect, a method is disclosed for measuring the
concentration of trichloroethene in a solution wherein a first
biocomponent selected from the group consisting of toluene
ortho-monoxygenase and toluene ortho-monoxygenase-Green catalyzes
the reaction of trichloroethene and produces trichloroethene
epoxide; and wherein the trichloroethene epoxide catalyzes the
reaction of a second biocomponent selected from the group
consisting of epoxide hydrolase, glutathione synthetase,
glutathione S-transferase and gamma-glutamylcysteine synthetase;
and wherein a transducer layer luminesces and the luminescence of
the transducer layer is altered by oxygen in the solution; and the
photons produced by the luminescence of the transducer layer enter
into a fiber optic cable and are transmitted to a photomultiplier
that produces an output signal that is coupled to an algorithm that
transforms the signal generated by the photomultiplier into an
output correlated to the concentration of trichloroethene or other
halogenated hydrocarbons in the solution.
[0012] In one aspect, a biosensing element is disclosed that
measures the concentration of trichloroethene in a solution. The
biosensing element comprises a first biocomponent that catalyzes
the reaction of trichloroethene and a second biocomponent that
catalyzes the reaction of trichloroethene epoxide. The first
biocomponent and the second biocomponent comprise cells that
contain enzymes from the group consisting of toluene
ortho-monoxygenase, toluene ortho-monoxygenase-Green, epoxide
hydrolase, glutathione synthetase, glutathione S-transferase and
gamma-glutamylcysteine synthetase. The cells are immobilized within
a matrix that is in contact with a transducer layer. The transducer
layer is part of an optode. In an embodiment, the cells are alive.
In another embodiment, the cells are dead. In an embodiment, the
transducer layer is a chemical transducer that interacts with
oxygen. In an embodiment, the transducer layer is an optical
transducer that interacts with oxygen.
[0013] In one aspect, a method for constructing biosensing systems
having a linear response to the concentration of an analyte in a
solution is disclosed wherein the biosensing system has an optode,
and the optode has a fiber optical cable having a first tip and a
second tip, and the first tip is covered by a transducer layer, and
the transducer layer is covered by a biocomponent layer, and the
biocomponent layer is covered by a porous layer, and the second tip
is coupled to a photon-detection device, and the photon-detection
device is coupled to a signal processing system, and the analyte
concentration in the solution, the depth of the biocomponent layer,
the depth of the porous layer, the diffusion coefficient of the
porous layer, the K.sub.m and V.sub.max of the reaction of the
analyte that is catalyzed by the biocomponent and the analyte are
selected such that the quotient between Da.sup.2 and 4.beta. is
from about 10 to about 1000. In one embodiment, the biocomponent is
toluene ortho-monooxygenase. In one embodiment, the biocomponent is
a toluene ortho-monooxygenase variant. In one embodiment, the
analyte is trichloroethene. In another embodiment, the biocomponent
has both a toluene ortho-monooxygenase variant and formate
dehydrogenase, and also has at least one enzyme selected from an
epoxide hydrolase, a glutathione synthetase, a glutathione
S-transferase and a gamma-glutamylcysteine synthetase. In one
embodiment, the transducer layer is RuDPP. In one embodiment, the
porous layer is track-etched polycarbonate.
[0014] In one aspect, a biosensing system for measuring the
concentration of an analyte in a solution is disclosed wherein the
biosensing system has an optode, and the optode has a fiber optical
cable having a first tip and a second tip, and the first tip is
covered by a transducer layer, and the transducer layer is covered
by a biocomponent layer, and the biocomponent layer is covered by a
porous layer, and the second tip is coupled to a photon-detection
device, and the photon-detection device is coupled to a signal
processing system, and the analyte concentration in the solution,
the depth of the biocomponent layer, the depth of the porous layer,
the diffusion coefficient of the porous layer, the K.sub.m and
V.sub.max of the reaction between the biocomponent and the analyte
are selected such that the quotient between Da.sup.2 and 4.beta. is
from about 10 to about 1000. In one embodiment, the biocomponent is
toluene ortho-monooxygenase. In one embodiment, the biocomponent is
a toluene ortho-monooxygenase variant. In one embodiment, the
analyte is trichloroethene. In another embodiment, the biocomponent
has both a toluene ortho-monooxygenase variant and formate
dehydrogenase, and also has at least one enzyme selected from an
epoxide hydrolase, a glutathione synthetase, a glutathione
S-transferase and a gamma-glutamylcysteine synthetase. In one
embodiment, the transducer layer is RuDPP. In one embodiment, the
porous layer is track-etched polycarbonate.
[0015] In an aspect, a biosensing system is disclosed that measures
the concentration of a halogenated alkene in a solution and
contains a biocomponent that catalyzes the reaction of the
halogenated alkene, and a transducer layer that luminesces and is
part of an optode.
[0016] In another aspect, a method for measuring the concentration
of a halogenated alkene in a solution is disclosed wherein a
biocomponent catalyzes the reaction of the halogenated alkene and
oxygen, and where a transducer layer luminesces, and the transducer
layer luminescence is altered by oxygen and/or hydrogen ions in the
solution, and the photons from the luminescence of the transducer
layer enter into a fiber optic cable and are transmitted to a
photomultiplier, and the photomultiplier produces an output signal
that is coupled to an algorithm that transforms the signal
generated by the photomultiplier into an output correlated to the
concentration of the halogenated alkene in the solution. In an
embodiment, the biocomponent is selected from the group consisting
of toluene ortho-monoxygenase, toluene ortho-monoxygenase-Green,
toluene ortho-monoxygenase variant, and toluene dioxygenase. In
another embodiment, the transducer layer is selected from the group
consisting of RuDPP and fluorescein.
[0017] In an aspect, a biosensing system is disclosed that measures
the concentration of halogenated alkenes in a solution and has a
biocomponent that catalyzes the reaction of a halogenated alkene
and oxygen, and a transducer layer that luminesces, and the
transducer layer luminescence is altered by oxygen and/or hydrogen
ions in said solution, and the photons from the luminescence of
said transducer layer enter into a fiber optic cable and are
transmitted to a photomultiplier, and the photomultiplier produces
an output signal that is coupled to an algorithm that transforms
the signal generated by said photomultiplier into an output
correlated to the concentration of said halogenated alkene in the
solution. In one embodiment, the biosensing system for halogenated
alkenes is selected from the group consisting of tetrachloroethene,
trichloroethene, dichloroethene, and monochloroethene. In another
embodiment, the biosensing system has a biocomponent that is
selected from the group consisting of toluene ortho-monoxygenase,
toluene ortho-monoxygenase-Green, toluene ortho-monoxygenase
variant, and toluene dioxygenase. In yet another embodiment, the
biosensing system has a transducer layer that is selected from the
group consisting of RuDPP and fluorescein.
BRIEF DESCRIPTION OF FIGURES
[0018] FIG. 1. Time course of a TOM-Green biosensing system
response to the addition of 0.61 mg/L TCE.
[0019] FIG. 2. TOM biosensing system signal as a function of
toluene concentration. Inset: biosensing system signals in the low
range of toluene concentrations (0-12 .mu.g/L).
[0020] FIG. 3. Activity retention of TOM-Green biosensing elements
stored at two temperatures in measurement solution (without
formate); each point represents the reading for a 92 .mu.M toluene
solution.
[0021] FIG. 4. Second signals as a percent of initial signals at
different TCE concentrations for all three types of TOM-Green
biosensing systems.
[0022] FIG. 5. Signal comparison with all three types of TOM-Green
biosensing systems at 2 .mu.g/L TCE.
[0023] FIG. 6. Signal comparison with all three types of TOM-Green
biosensing systems at 10 .mu.g/L TCE.
[0024] FIG. 7. Signal comparison with all three types of TOM-Green
biosensing systems at 50 .mu.g/L TCE.
[0025] FIG. 8. Graphical representation of Michaelis-Menten
equation relationships between enzyme reaction rate and substrate
concentration.
[0026] FIG. 9. Representation of optical enzymatic biosensing
element portion of a biosensing system for measuring analytes in
high concentrations.
[0027] FIG. 10. Response curve for biosensing system A. Biosensing
system A is a lactose biosensing system with a thin film of enzyme
immobilized on the surface.
[0028] FIG. 11. Response curve for biosensing system B. Biosensor
system B is a lactose biosensing system with a porous diffusive
barrier.
[0029] FIG. 12. Response curve for biosensing system C. Biosensing
system C is a lactose biosensing system having a less porous
diffusive barrier compared to the porous diffusive barrier used in
biosensing system B.
[0030] FIG. 13. System for providing design parameters used for
constructing biosensing elements.
[0031] FIG. 14. Schematic representation of a biosensing
system.
[0032] FIG. 15. Schematic representation of exemplary method for
using a biosensing system to measure the concentration of an
analyte in a solution.
[0033] FIG. 16. Response to trichloroethene of a biosensing system
with TOM Green enzyme expressed in E. coli TG-1 cells immobilized
on a pH optode using calcium alginate.
[0034] FIG. 17. Response to trichloroethene of a biosensing system
with toluene dioxygenase in Pseudomonas putida F1 with an oxygen
optode transducer.
DETAILED DESCRIPTION
[0035] Biosensing systems offer the potential of measurements that
are specific, continuous, rapid, and reagentless. Biosensing
elements of biosensing systems combine a biocomponent which is
coupled to a transducer to yield a device capable of measuring
chemical concentrations. A biocomponent may be any biological
detection agent. Examples of biocomponents include enzymes, whole
cells, microorganisms, RNA, DNA, aptamers and antibodies. The
biocomponent interacts with an analyte via a binding event and/or
reaction. The role of the transducer is to convert the biocomponent
detection event into a signal, usually optical or electrical. A
transducer is typically a physical sensor such as an electrode, or
a chemical sensor. The analyte normally interacts with the
biocomponent through a chemical reaction or physical binding. For
example, in the case of a biosensing system that uses an enzyme
biocomponent, the enzyme biocomponent would react with the analyte
of interest and a product or reactant of the enzyme catalyzed
reaction such as oxygen, ammonia, hydrochloric acid or carbon
dioxide, may be detected by an optical, electrochemical or other
type of transducer.
[0036] In one embodiment of the present disclosure, biosensing
systems contain a second biocomponent enzyme that catalyzes the
reaction of reactive products created by the reaction of a first
biocomponent enzyme with an analyte of interest. The second
biocomponent enzyme catalyzes the reaction of the reactive product
and prevents a decrease in activity of the first biocomponent
caused by the reactive product reacting with active site residues
or other residues that render the first biocomponent less active or
inactive.
[0037] In one embodiment, biocomponents of the biosensing system
are monooxygenases Enzyme Commission number (EC) 1.13 and/or
dioxygenases EC 1.14. In one embodiment, toluene
ortho-monooxygenase (TOM) and/or toluene ortho-monooxygenase-Green
(TOM-Green, a toluene ortho-monooxygenase variant) are used as a
biocomponent. In one embodiment, toluene diooxygenase (TDO) is used
as a biocomponent. Genes for the enzymes TOM and/or TOM-Green
and/or TDO may be cloned into plasmids and then introduced into
Escherichia coli (E. coli) or may also be cloned directly into the
chromosomal DNA of E. coli. The E. coli containing plasmids with
genes encoding TOM and/or TOM-Green and/or TDO may be used as
biocomponents. These genes may also be encoded naturally on plasmid
or chromosomal DNA in certain microorganisms that are useful as
biocomponents. In one embodiment, these genes may be introduced to
other suitable organisms such as other bacteria, archaea or
eukaryotes.
[0038] In one embodiment, biocomponents of the biosensing system
are monooxygenases Enzyme Commission number (EC) 1.13 and/or
dioxygenases EC 1.14. In one embodiment, toluene
ortho-monooxygenase (TOM) and/or toluene ortho-monooxygenase-Green
(TOM-Green, a toluene ortho-monooxygenase variant) are used as a
biocomponent. Genes for the enzymes TOM and/or TOM-Green may be
cloned into plasmids and then introduced into their native host,
such as Burkholderia cepacia G4, for example, or may also be cloned
directly into the chromosomal DNA of their native host. The native
hosts containing these plasmids with genes encoding TOM and/or
TOM-Green may be used as biocomponents. These genes may also be
encoded naturally on plasmid or chromosomal DNA in the native host
microorganisms that are useful as biocomponents.
[0039] Advantages in using biosensing systems for measuring
analytes include fast measurement, generally on the order of
minutes. This is a big advantage over traditional methods like GC
or HPLC in which a lot of time is spent in collection of the sample
and extraction of analytes from the sample.
[0040] Small size is another advantage of using biosensing systems.
Biosensing systems of the present disclosure have a compact design
and are therefore capable of measurements in confined places such
as needles and catheters in vivo and in conditions where weight is
critical like spacecraft or airplanes.
[0041] An advantage of using biosensing systems is that they can be
used to measure multiple analytes. Yet another advantage of using
biosensing systems is that they can be used in a continuous
real-time measurement. Biosensing systems disclosed herein may be
used in a reversible manner with extremely low signal loss.
Furthermore, biosensing systems are capable of measuring at depths
for applications such as groundwater monitoring. Biosensing systems
disclosed herein can make measurements in situ.
[0042] An important advantage is the ability of biosensing systems
to measure complex samples with no prior preparation of samples.
Biosensing systems can provide direct measurements in blood, food,
and waste water, for example. This is important as removal of the
sample from its environment (as in case of analyses by GC or HPLC)
can change its chemistry and can thereby lead to inaccurate
results. Also, this eliminates and simplifies sample separation
steps and reduces the cost of the process. Measurements using
biosensing systems can be made with minimum perturbations of the
sample.
[0043] Biosensing systems have high specificity and sensitivity for
measuring analytes of interest. Although most of the traditional
methods (GC or HPLC) are very sensitive, they require expensive,
laboratory-based hardware and trained operators. Other methods such
as solid-phase enzyme-linked immunoassay (ELISA) may have good
sensitivity but are generally not highly specific.
[0044] Another advantage for using biosensing systems of the
present disclosure is the low cost of mass production compared to
most of the traditional methods like GC or HPLC. Biosensing systems
of the present disclosure are easy to use compared to traditional
monitoring techniques such as gas chromatography, ion
chromatography and high-pressure liquid chromatography. Biosensing
systems using the proper biocomponents can also measure the
toxicity of chemicals whereas analytical methods such as GC and
HPLC can only measure concentration.
DEFINITIONS
[0045] Amperometric: Amperometric pertains to measurement of an
electrical current.
[0046] Halogenated alkene: A halogenated alkene is a hydrocarbon
chemical with at least one double bond and in which one or more
halogen atoms are substituted for hydrogen atoms. The halogen atoms
may be fluorine, chlorine, bromine, and/or iodine. Non-limiting
examples of halogenated alkenes include tetrachloroethene,
trichloroethene, dichloroethene and monochloroethene and isomers
thereof. Trichloroethene may also be referred to as
trichloroethylene. In general, a halogenated ethene compound may
also be referred to as a halogenated ethylene compound.
[0047] Dichloroethene: As used herein, "dichloroethene" includes
the isomers 1,1-dichloroethene, cis-1,2-dichloroethene, and
trans-1,2-dichloroethene. As used herein, the term "dichloroethene"
is synonymous with dichloroethenes. The term "dichloroethenes"
includes 1,1-dichloroethene, cis-1,2-dichloroethene,
trans-1,2-dichloroethene, and dichloroethene.
[0048] Halogenated hydrocarbon: A halogenated hydrocarbon is a
hydrocarbon chemical in which one or more halogen atoms are
substituted for hydrogen atoms. The halogen atoms may be fluorine,
chlorine, bromine, and/or iodine.
[0049] Oxygenases: An oxygenase is any enzyme that oxidizes a
substrate by transferring the oxygen from molecular oxygen
(O.sub.2) to it. The oxygenases form a class of oxidoreductases (EC
1); their EC number is EC 1.13 or EC 1.14. There are two types of
oxygenases, monooxygenases and dioxygenases.
[0050] Monooxygenase: Monooxygenases are enzymes that incorporate
one hydroxyl group into substrates in many metabolic pathways. The
oxygen atom in the hydroxyl originates from molecular oxygen
(O.sub.2). Generally, in the reaction catalyzed by monooxygenases,
two atoms of dioxygen are reduced to one hydroxyl group and one
H.sub.2O molecule by the concomitant oxidation of NAD(P)H.
Monooxygenases are a type of oxygenases.
[0051] Dioxygenase: Dioxygenases, or oxygen transferases, are
enzymes that incorporate both oxygen atoms from molecular oxygen
(O.sub.2) into the substrate of the reaction. Dioxygenases are a
type of oxygenases.
[0052] Toluene dioxygenase: Toluene dioxygenase is a class of
enzymes that belong to the family of oxidoreductases EC 1,
specifically to EC 1.14 and more specifically to EC 1.14.12.11.
Toluene dioxygenases, for example, catalyze the chemical reaction
of substrates toluene and NADH and H.sup.+ and O.sub.2 to the
products (1S,2R)-3-methylcyclohexa-3,5-diene-1,2-diol and
NAD.sup.+. Toluene dioxygenase is an oxidoreductase that acts on
paired electron donors with O.sub.2 as an oxidant and the
incorporation or reduction of oxygen. Toluene dioxygenase is
synonymous with toluene 2,3-dioxygenase.
[0053] Toluene ortho-monooxygenase: Toluene ortho-monooxygenase
(TOM) is an enzyme that belongs to the family of oxidoreductases EC
1, specifically to EC 1.13 and more specifically to EC 1.13.12. TOM
oxidizes many substrates, including o-xylene, m-xylene, p-xylene,
toluene, benzene, ethyl benzene, styrene, naphthalene,
trichloroethene as well as tetrachloroethene. TOM uses oxygen and
NADH as a cofactor to oxidize its substrate.
[0054] Toluene ortho-monooxygenase variant: Toluene
ortho-monooxygenase (TOM) variants refer generally to any variant
of TOM that has altered substrate binding kinetics, a faster
turnover rate or other improved enzymological parameters over
native TOM. One example of a TOM variant is TOM-Green, which has a
valine to alanine substitution (V106A) in the hydroxylase
alpha-subunit of TOM from Burkholderia cepacia G4.
[0055] NAD: NAD (nicotinamide adenine dinucleotide) used herein
includes the oxidized form NAD.sup.+ and the reduced form NADH. NAD
is a cofactor.
[0056] NADP: NADP (nicotinamide adenine dinucleotide phosphate)
used herein includes the oxidized form NADP.sup.+ and the reduced
form NADPH. NADP is a cofactor.
[0057] NAD(P)H: NAD(P)H is an inclusive term that embodies both the
reduced form of nicotine adenine dinucleotide, NADH, and the
reduced form of phosphorylated NADH, NADPH. NAD(P)H is a
cofactor.
[0058] FAD: FAD (Flavin Adenine Dinucleotide) used herein includes
FAD (fully oxidized form, or quinone form) that accepts two
electrons and two protons to become FADH.sub.2 (hydroquinone form).
FADH.sub.2 can then be oxidized to the semireduced form
(semiquinone) FADH by donating one electron and one proton. The
semiquinone is then oxidized once more by losing an electron and a
proton and is returned to the initial quinone form, FAD. FAD is a
cofactor.
[0059] FMN: FMN (Flavin Mononucleotide) used herein includes FMN
(fully oxidized form), or FMNH (semiquinone form), and FMNH.sub.2
(fully reduced form). FMN is a cofactor. In one embodiment, FMN is
a prosthetic group for oxidoreductases.
[0060] Cofactor: A cofactor used herein is a non-protein chemical
compound that is bound to a protein and is required for the
protein's biological activity. Non-limiting examples of cofactors
include: thiamine pyrophosphate, reduced and oxidized forms of
flavin adenine mononucleotide (FAD), reduced and oxidized forms of
flavin adenine mononucleotide (FMN), reduced and oxidized forms of
nicotinamide adenine dinucleotide (NAD), reduced and oxidized forms
of nicotinamide adenine dinucleotide phosphate (NADP), pyridoxal
phosphate, lipoamide, methylcobalamin, cobalamine, biotin, coenzyme
A, tetrahydrofolic acid, menaquinone, ascorbic acid, flavin adenine
dinucleotide, coenzyme F420, adenosine triphosphate, S-adenosyl
methionine, coenzyme B, coenzyme M, coenzyme Q, cytidine
triphosphate, glutathione, heme, methanofuran, molybdopterin,
nucleotide sugars, 3'-phosphoadenosine-5'-phosphosulfate,
pyrroloquinoline, quinine, tetrahydrobiopterin, and
tetrahydromethanopterin. Cofactors may also include metal ions such
as Ca.sup.2+, Zn.sup.2+, Fe.sup.2+, Fe.sup.3+, Mg.sup.2+,
Ni.sup.2+, Cu.sup.+, Cu.sup.2+, Mn.sup.2+, and iron-sulfur
clusters, for example.
[0061] Dehydrogenase: A dehydrogenase is an enzyme that oxidizes a
substrate by transferring one or more hydrides (H) to an acceptor,
usually NAD.sup.+/NADP.sup.+ or a flavin coenzyme such as FAD or
FMN.
[0062] Measurement solution: A measurement solution is a solution
in which an analyte may be dissolved to make a biosensor
measurement. A non-limiting example of a measurement solution is
0.15 M NaCl and 0.025 M CaCl.sub.2 at pH 7.0.
[0063] Biocomponent: A biocomponent binds, catalyzes the reaction
of or otherwise interacts with analytes, compounds, atoms or
molecules thereby generating an atom, molecule or compound.
Non-limiting examples of biocomponents include aptamers, DNA, RNA,
proteins, enzymes, antibodies, cells, whole cells, tissues,
single-celled microorganisms, and multicellular microorganisms. A
biocomponent may be a cell, microorganism, cell organelle or any
other membrane bound container that contains biocomponent enzymes
within. A biocomponent may be purified or otherwise substantially
isolated biocomponent enzymes. A biocomponent may be an unpurified
extract of cells containing biocomponent enzymes.
[0064] Analyte: An analyte is the substance or chemical constituent
that is desired to be detected or measured, such as the analyte
concentration. With enzymatic biosensors, the analyte itself is not
measured. Rather, a reaction of the analyte that is catalyzed by an
enzymatic biocomponent causes a change in the concentration of a
reactant or product that is measureable by the biosensing system.
An analyte may also be a substrate of an enzyme.
[0065] Transducer: A transducer is a substance that interacts with
the atoms, compounds, or molecules produced or used by the
biocomponent. The interaction of the transducer with the atoms,
compounds, or molecules produced or used by the biocomponent causes
a signal to be generated by the transducer layer. The transducer
layer may also generate a signal as an inherent property of the
transducer. The signal may be an electrical current, a photon, a
luminescence, or a switch in a physical configuration. In one
embodiment, the signal produced by the transducer is altered by a
reactant or product of the biocomponent or may also be altered by a
molecule such as oxygen.
[0066] Chemical transducer: A chemical transducer is a chemical
that interacts with an atom, molecule or compound and that
interaction causes the production of a proton, oxygen molecule,
luminescent event, photon or other atoms and molecules.
[0067] Optical transducer: An optical transducer is a material that
luminesces. An optical transducer interacts with an atom, molecule,
photon or compound and that interaction causes a change in the
intensity and/or lifetime of the fluorescence of the optical
transducer.
[0068] Physical transducer: A physical transducer is a material
that interacts with an atom, molecule, photon or compound and that
interaction causes a shift in its physical properties.
[0069] Biosensor: A biosensor measures the concentration of
compounds, atoms or molecules using a biocomponent. A biosensor may
also detect compounds, atoms or molecules using a biocomponent. A
biosensor may also measure the toxicity of compounds, atoms or
molecules using a biocomponent. A biosensor may alternatively be
referred to as a biosensing system and/or a biosensing element.
[0070] Biosensing system: A biosensing system contains a biosensing
element, a transducer, and a signal processing system. A biosensing
system may alternatively be referred to as a biosensor system.
Biosensing system may alternatively refer to various parts of the
biosensing system such as the biosensing element, for example. A
biosensing system may also contain a biosensing element, an optode,
and a signal processing system.
[0071] Biosensing element: A biosensing element detects analytes. A
biosensing element comprises one or more biocomponents and a
transducer. In certain embodiments, a biosensing element comprises
one or more biocomponents, a transducer and/or an optode.
[0072] Crosslinking: Crosslinking is the process of linking a
biocomponent to a matrix. Crosslinking may be through chemical
bonds, ionic interactions, physical entrapment or other modes and
methods of linking a biocomponent to a matrix.
[0073] Matrix: A matrix is an interlacing, repeating cell, net-like
or other structure that embodies the biocomponents. The
immobilization material is an example of a matrix. A matrix may be
a polymer.
[0074] Immobilization material: Immobilization material is the
substance, compound or other material used to immobilize the
biocomponent onto the biosensing element transducer layer. The
immobilization material may be a matrix or may be less ordered than
a matrix. The immobilization material may be a polymer such as
cellulose acetate, polycarbonate, collage, acrylate copolymers,
poly(ethylene glycol), polytetrafluoroethylene (PTFE), agarose,
alginate, polylysine, alginate-polylysine-alginate microcapsule,
algal polysaccharides, agar, agarose, alginate, and carrageenan,
polyacrylamide, polystyrene, polyurethane and other naturally
occurring and synthetic polymers.
[0075] Polymer: Polymers as used herein include any natural or
synthetic polymer including cellulose acetate, polycarbonate,
collage, acrylate copolymers, poly(ethylene glycol),
polytetrafluoroethylene (PTFE), agarose, alginate, polylysine,
alginate-polylysine-alginate microcapsule, algal polysaccharides,
agar, agarose, alginate, and carrageenan, polyacrylamide,
polystyrene, polyurethane and other naturally occurring and
synthetic polymers. Polymers may be used to create a diffusivity
barrier between the bulk solution and a biocomponent of a
biosensing system. A polymer may be a porous layer.
[0076] Optode: An optode is a sensor device that measures the
concentration of a specific substance usually with the aid of a
transducer. An optode can be an optical sensor device that
optically measures the concentration of a specific substance
usually with the aid of a transducer. In one embodiment, for
example, an optode requires a transducer, a polymer to immobilize
the transducer and instrumentation such as optical fiber, a light
source, detectors and other electronics. Optodes can apply various
optical measurement schemes such as reflection, absorption, an
evanescent wave, luminescence (for example fluorescence and
phosphorescence), chemiluminescence, and surface plasmon resonance.
Optodes may be fiber optical cable, planar wave guides or other
surfaces conducive to the propagation of total internally
reflecting light waves. An optode may be an optical transducer such
as a photon detector.
[0077] pH sensor: A pH sensor measures the concentration of
hydrogen ions in a solution.
[0078] pH optode: A pH optode is an optode that has a detection
element that interacts with hydrogen ions. Examples of detection
elements that interact with hydrogen ions are fluorescein,
fluoresceinamine and other fluorescein-containing compounds. In an
embodiment, for example, a pH optode based on luminescence has a
luminescent reagent that is pH responsive.
[0079] Luminescence: Luminescence is a general term which describes
any process in which energy is emitted from a material at a
different wavelength from that at which it is absorbed.
Luminescence may be measured by intensity and/or by lifetime decay.
Luminescence is an umbrella term covering fluorescence,
phosphorescence, bioluminescence, chemoluminescence,
electrochemiluminescence, crystalloluminescence,
electroluminescence, cathodoluminescence, mechanoluminescence,
triboluminescence, fractoluminescence, piezoluminescence,
photoluminescence, radioluminescence, sonoluminescence, and
thermoluminescence.
[0080] Fluorescence: Fluorescence is a luminescence phenomenon in
which electron de-excitation occurs almost spontaneously, and in
which emission from a luminescent substance ceases when the
exciting source is removed. Fluorescence may be measured by
intensity and/or by lifetime of the decay.
[0081] Fluorescein: Fluorescein is a fluorophore. In water,
fluorescein has an absorption maximum at 494 nm and emission
maximum of 521 nm. As used herein, the term "fluorescein" includes
isomers, analogs and salts of fluorescein including, but not
limited to, fluoresceinamine, resorcinolphthalein, C.I. 45350,
solvent yellow 94, D & C yellow no. 7, angiofluor, Japan yellow
201, soap yellow, uranine, D&C Yellow no. 8 and fluorescein
isothiocyanate.
[0082] Phosphorescence: Phosphorescence is a luminescence
phenomenon in which light is emitted by an atom or molecule that
persists after the exciting source is removed. It is similar to
fluorescence, but the species is excited to a metastable from which
a transition to the initial is forbidden. Emission occurs when
thermal energy raises the electron to a from which it can
de-excite. Phosphorescence may be measured by intensity and/or by
lifetime of the decay.
[0083] Oxygen sensor: An oxygen sensor measures, or is responsive
to, the concentration of oxygen in a solution.
[0084] Oxygen optode: An oxygen optode is an optode that has a
transducer layer that interacts with oxygen. An example of a
transducer layer that interacts with oxygen is
tris(4,7-diphenyl-1,10-phenanthroline)Ru(II) chloride, also known
as RuDPP.
[0085] Photon-detection device: A photon-detection device is a
class of detectors that multiply the current produced by incident
light by as much as 100 million times in multiple dynode stages,
enabling, for example, individual photons to be detected when the
incident flux of light is very low. Photon-detection devices may be
vacuum tubes, solid photomultipliers or other devices that interact
with incident light, and amplify or otherwise process the signal
and/or photons produced by that interaction. Alternative
embodiments of a photon-detection device include an image sensor,
CCD sensors, CMOS sensors, photomultiplier tubes, charge coupled
devices, photodiodes and avalanche photodiodes.
[0086] Signal processing system: A signal processing system
processes the signal from a biosensing system into information that
can be displayed to an end user. An example of a signal processing
system is a photon-detection device that detects the photons from
the output of a photo optical cable of the optode of the biosensing
system. The output of the photon-detection device is coupled to the
input of a converter or sampler device such as a signal processor
or a transimpedance amplifier. The output of the converter or
sampler device is coupled to the input of a microprocessor that
processes the output of the converter or sampler device into an
output corresponding to the concentration of an analyte within the
solution that was measured by the biosensing system. The output of
the microprocessor is then communicated to an end user, for example
by displaying the concentration on a screen.
[0087] Image sensor: An image sensor is a device that converts an
optical image to an electric signal. Examples of image sensors
include charge-coupled devices (CCD) or complementary
metal-oxide-semiconductor (CMOS) active pixel sensors.
[0088] Sampler device: A sampler device reduces a continuous signal
to a discrete signal. A common example is the conversion of a sound
wave or light wave (a continuous signal) to a sequence of samples
(a discrete-time signal).
[0089] Avalanche photodiode: An avalanche photodiode (APD) is a
highly sensitive semiconductor electronic device that exploits the
photoelectric effect to convert light to electricity. APDs can be
thought of as photodetectors that provide a built-in first stage of
gain through avalanche multiplication.
[0090] Converter: A converter is a current-to-voltage converter,
and is alternatively referred to as a transimpedance amplifier. A
converter is an electrical device that takes an electric current as
an input signal and produces a corresponding voltage as an output
signal. In another embodiment a converter may be a
voltage-to-current converter.
Biocomponents
[0091] Biocomponents react with, bind to or otherwise interact with
an analyte. Reactive biocomponents produce or react with atoms,
molecules or compounds that interact with the transducer.
[0092] Enzymes are proteins that can serve as biocomponents that
catalyze reactions of their substrates. Substrates may be analytes.
The products or reactants of the enzymatic reactions are usually
measured by the biosensing system. In one embodiment, the products
of the substrates that react with the analyte may themselves be
acted upon and thereby produce additional products which may be
measured by the biosensing system. Therefore, a biosensing system
may measure primary, secondary or even higher orders of products
caused by an initial reaction or binding of the analyte with the
biocomponent.
[0093] Generally, enzymes for use in biosensing systems may be
disposed within whole cells or extracted from cells and purified.
Whole cells and microorganisms are also biocomponents and are
generally less expensive than purified enzymes and may provide an
environment for longer enzyme stability. The cells and organisms
used as biocomponents may or may not be living (able to replicate).
Whether or not the cells are living, diffusion mechanisms and
membrane-bound pumps may still be active that allow for the
exchange of analytes and other compounds with the environment of
the cell. It is often advantageous to use dead cell or dead
microorganisms or substantially purified enzymes as a biocomponent
at least because the proteolytic enzymes and pathways operating in
a living cell generally cease to function and the enzymes, for
example, that are responsible for binding or reacting with the
analytes therefore last longer than they would in a living cell.
Another advantage of using dead cells or microorganisms or
substantially purified enzymes is that if the biosensing system is
used in-situ, such as in-line testing of milk being produced at a
factory, there can be no contamination of the sample with cells or
microorganisms that may infect or adulterate the sample.
[0094] Purified enzymes may be used as a biocomponent in biosensing
systems. The use of cell-free enzyme preparations may reduce the
impact of unwanted side reactions. Often, the extraction, isolation
and purification of a particular enzyme can be expensive.
Additionally, enzymes may lose their activity when separated from
their intracellular environment that provides structural proteins,
co-factors, consistent pH levels, buffers and other factors that
contribute to the molecular integrity of the enzyme. However, some
enzymes are more robust than others. For example, enzymes isolated
from extremophilic organisms such as hyperthermophiles, halophiles,
and acidophiles often are more resistant to being exposed to
environments substantially different from those found inside of a
cell or microorganism. Extracellular enzymes are also usually more
robust than enzymes that are membrane bound or solely exist within
the cytosol.
[0095] An enzyme's resistance to becoming inactivated due to
environmental factors, or even by the nature of the reaction that
they catalyze, may be increased through mutagenic techniques. Such
techniques are well known in the art and include various
incarnations of changing the coding nucleotide sequence for the
protein through various techniques. The proteins produced by
expressing the mutagenic nucleotide sequences may then be tested
for resistance to environmental factors and/or increased reactivity
with substrates. Such an increase in reactivity may be due to
advantageous binding specificity and/or increased kinetics of the
binding and/or reaction catalyzed by the enzyme.
[0096] Methods of choosing cells and microorganisms that increase
the response of the biosensing system may also be used to create
biosensing systems that possess increased sensitivity, have quicker
response times and last longer. Such techniques include directed
evolution and using micro-assays to determine an increase in the
production amount and/or rate of production of the molecules and/or
atoms that react with the transducer layer.
Transducers
[0097] A transducer is a device that produces a measurable signal,
or change in signal, upon a change in its chemical or physical
environment. Transducers suited for biosensing systems that use
enzymes as the biocomponent are those that interact with the
reactants and/or products of the biocomponent and send a signal
that is processed into a measurement reading. The nature of the
interaction of the biological element with the analyte has a major
impact on the choice of transduction technology. The intended use
of the biosensing system imposes constraints on the choice of
suitable transduction technique.
[0098] Amperometric transducers work by maintaining a constant
potential on the working electrode with respect to a reference
electrode, and the current generated by the oxidation or reduction
of an electroactive species at the surface of the working electrode
is measured. This transduction method has the advantage of having a
linear response with a relatively simple and flexible design. Also,
the reference electrode need not be drift-free to have a stable
response. Since the signal generated is highly dependent on the
mass transfer of the electroactive species to the electrode surface
there can be a loss in sensitivity due to fouling by species that
adsorb to the electrode surface. As a result of fouling, use of
amperometric transducers is restricted where continuous monitoring
is required. Enzymes, particularly oxidoreductases, are well suited
to amperometric transduction as their catalytic activity is
concerned with electron transfer.
[0099] Electroactive species that can be monitored at the electrode
surface include substrates of a biological reaction (e.g., O.sub.2,
NADH), final products (e.g., hydrogen peroxide for oxidase
reactions, benzoquinone for phenol oxidation) and also
electrochemical mediators that can directly transfer electrons from
the enzyme to the working electrode surface (e.g. hexacyanoferrate,
ferrocene, methylene blue).
[0100] Potentiometric transducers work by having a potential
difference between an active and a reference electrode that is
measured under the zero current flow condition. The three most
commonly used potentiometric devices are ion-selective electrodes
(ISEs), gas-sensing electrodes and field-effect transistors (FETs).
All these devices obey a logarithmic relationship between the
potential difference and the activity of the ion of interest. This
makes the sensors have a wide dynamic range. One disadvantage of
this transducer is the requirement of an extremely stable reference
electrode. Ion selective electrodes are commonly used in areas such
as water monitoring. FETs are commercially attractive as they can
be used to make miniaturized sensors, but manufacturing cost of
FETs are high. Examples of potentiometric sensors are for
acetaldehyde and cephalosporins, where the sensing electrode
measures pH. Other examples are sensors used to measure creatinine,
glutamine and nitrate with the sensing electrode detecting NH.sub.3
gas.
[0101] Conductimetric transducers are often used to measure the
salinity of marine environments. Conductance is measured by the
application of an alternating current between two noble metal
electrodes immersed in the solution. Due to specific enzyme
reactions, they convert neutral substrates into charged products,
causing a change in the conductance of the medium. This method can
be used to make more selective and informative sensors by using
multi-frequency techniques.
[0102] Optical transducers use optical phenomena to report the
interaction of the biocomponent and the analyte. The main types of
photometric behavior which have been exploited are ultraviolet and
visible absorption, luminescence such as fluorescence and
phosphorescence emission, bioluminescence, chemiluminescence,
internal reflection spectroscopy using evanescent wave technology
and laser light scattering methods.
[0103] One embodiment of an optical transducer uses luminescent
reagents. In optical transducers that use luminescent reagents, a
luminescent substance is excited by incident light, and as a result
it emits light of longer wavelength. The intensity and/or lifetime
decay of emitted light changes when an atom, molecule or compound
binds or otherwise interacts with the luminescent substance. The
atom, molecule or compound may be a reactant or product of the
biocomponent. Thus, if a reactant or product of the biocomponent
catalyzes the reaction of the luminescent transducer and affects
the intensity and/or lifetime decay of the light emitted by the
transducer layer, the change in the measurement of the intensity
and/or lifetime decay can be measured as a response to a particular
analyte. There are several luminescent reagents that may be useful
as optical transducers. Examples include
Tris(4,7-diphenyl-1,10-phenanthroline)Ru(II) chloride, also known
as RuDPP, for oxygen sensors, trisodium
8-hydroxy-1,3,6-trisulphonate fluorescein, fluoresceinamine and
other compounds containing fluorescein for pH sensors, fluoro
(8-anilino-1-naphthalene sulphonate) for Na.sup.+ ion sensor and
acridinium- and quinidinium-based reagents for halides.
[0104] Chemiluminescent and bioluminescent sensors work on
principles similar to fluorescent sensors. Chemiluminescence occurs
by the oxidation of certain substances, usually with oxygen or
hydrogen peroxide, to produce visible light. Bioluminescence is,
for example, the mechanism by which light is produced by certain
enzymes, such as luciferase.
[0105] Calorimetric transducers use the heat generated from
biological reactions and correlate it with the reaction conditions.
In order to measure such small amounts of heat liberated during the
reaction, a very sensitive device is required. In the calorimetric
technique a very sensitive, electrical resistance thermometer is
used to detect temperature changes down to 0.001.degree. C. This
method is advantageous, as it is independent of the chemical
properties of the sample. calorimetric transduction has been used
in a wide range of areas, including clinical chemistry,
determination of enzyme activity, monitoring gel filtration,
chromatography, process control and fermentation.
[0106] An acoustic transducer uses materials such as piezoelectrics
as a sensor transducer due to their ability to generate and
transmit acoustic waves in a frequency-dependent manner. The
optimal resonant frequency for acoustic-wave transmission is highly
dependent on the physical dimensions and properties of the
piezoelectric crystal. Any change in the mass of the material at
the surface of the crystal will cause quantifiable changes in the
resonant frequency of the crystal. There are two types of
mass-balance acoustic transducers: bulk wave and surface acoustic
wave. Acoustic transduction is a relatively cheap technique but it
has the disadvantage of having low sensitivity with non-specific
binding. This technique is commonly used to measure the
concentration of volatile gases and vapors. A piezoelectric
immunobiosensor for measuring an analyte of interest in drinking
water may use a piezoelectric crystal coated with polyclonal
antibodies that bind to that analyte. When the analyte molecules
come into contact with the antibodies, they bond with the
antibodies causing a change in the crystal mass, which in turn
leads to a shift in the oscillation frequency and produces a
measurable signal that can be measured and correlated to the
concentration of the analyte of interest within the sample.
Optical and Signal Processing Systems
[0107] In an embodiment, biosensing systems of the present
disclosure have a biocomponent, a transducer, a photon-detection
device, and a signal-processing system. A signal processing system
processes the signal from a photon-detection device into
information that can be displayed to an end user. An example of a
signal processing system is a microprocessor that accepts an input
signal from a photon-detection device that is coupled to a
biosensing element. The signal processing system then uses a
software program that encodes an algorithm. The algorithm used by
the software transforms the data provided by the input signal and
provides an output signal that correlates to a numerical display of
the concentration of an analyte that the biosensing system
detected.
[0108] In an embodiment of the present disclosure, a biosensing
system comprises biocomponent attached to a fiber optic pH optode,
lens focusing system, photomultiplier (PMT), analog/digital (A/D)
converter and a microprocessor. The biosensing element may be
coupled to a polymethylmethacrylate (PMMA) optical fiber optic. The
length of this connecting optical fiber may vary from 1 mm to well
over 1 km. In an embodiment, the other end of this cable is
attached to a light emitting diode (LED). In another embodiment,
the other end of this cable is attached to a metal casing
containing a 5 W halogen lamp or other light source and a lens
focusing system. The light source should be able to operate at high
temperatures, having a very short warm-up time in order to reach a
constant power output. In one embodiment, light from the halogen
lamp is first passed through a bandpass filter such as a 480-nm
bandpass filter, for example. The light is then collected,
paralleled and focused to the tip of fiber optic cable using a lens
focusing system. An embodiment of the lens focusing system
comprises spheric, aspheric, and convex lenses, and a dichroic
mirror. Light from the lamp that radiates in opposite directions to
the lens system may be refocused by the spheric lens and paralleled
by the aspheric lens.
[0109] When light, for example light at 480 nm, is incident on a
sensing tip coated with PVA/fluoresceinamine dye, fluorescence
occurs. In an embodiment, this light is then passed back through a
520 nm bandpass filter or other bandpass filter having a frequency
of light that is either blue or red shifted in comparison to the
incident light wavelength, paralleled by focusing lens and then
directed by the dichroic mirror onto the window of a single channel
photo-detection device. The change in intensity and/or lifetime
decay properties of the light can be measured. The photon detection
device processes this light and the output potentiometric signal is
sent to a computer interface using a connector block where it was
converted into a digital signal by a data acquisition card. The
final output is observed on a computer using software such as
LabView software or other algorithmic software that interprets the
signals from the sensing tip and processes them into correlating
concentration measurements of the atom, compound, molecule or
analyte of interest.
Immobilization of the Biocomponent
[0110] In order to construct a biosensing system, the biocomponent
of the biosensing element of the biosensing system needs to be
bound to or otherwise in contact with the transducer. This can be
achieved by immobilizing the biocomponent on to the transducer. The
viability of a biosensing system depends on the processing and type
of material used for immobilizing the biocomponent. The material
used for immobilizing the biocomponent may be referred to as a
matrix, matrix material or as an immobilizing material.
[0111] Biocomponents may be very sensitive to the immobilizing
process as well as the material that is used for immobilization.
The pH, ionic strength, and any other latent chemistries of the gel
matrix should be compatible with the biocomponent. The reactants
and products of the reaction carried by the biocomponent should not
affect the material used for immobilization. The biocomponent
should be effectively immobilized and there should not be any
leakage of the biocomponent from the matrix during the active
lifetime of the biosensing system. The immobilization material
should be non-toxic and non-polluting. The material should have
proper permeability to allow sufficient diffusion of substrates,
products and gases. The matrix material should allow for sufficient
cell activity and cell density. The immobilization material should
protect the biocomponent from biotic and abiotic environmental
stresses that would lower biocomponent activity or lifetime.
Techniques of Immobilization
[0112] In one embodiment, adsorption is used to immobilize the
biocomponent. Many substances adsorb enzymes, cells, microorganisms
and other biocomponents on their surfaces, e.g., alumina, charcoal,
clay, cellulose, kaolin, silica gel and collagen. Adsorption can be
classified as physical adsorption (physisorption) and chemical
adsorption (chemisorption). Physisorption is usually weak and
occurs via the formation of van der Waals bonds or hydrogen bonds
between the substrate and the enzyme molecules. Chemisorption is
much stronger and involves the formation of covalent bonds.
Adsorption of the biocomponent may be specific through the
interaction of some moiety, link or other reactive component of the
biocomponent or may be non-specific.
[0113] In another embodiment, microencapsulation is used to
immobilize the biocomponent. In this method, a thin microporous
semipermeable membrane is used to surround the biocomponent.
Because of the proximity between the biocomponent and the
transducer and the very small thickness of the membrane, the
biosensing element response is fast and accurate, and there is
always an option of bonding the biocomponent to the fiber optical
portion of the biosensing system via molecules that conduct
electrons, such as polypyrrole, for example. The membrane used for
microencapsulation may also serve additional functions such as
selective ion permeability, enhanced electrochemical conductivity,
mediation of electron transfer processes, or controlling the
sensitivity of the response of the biosensing system. Examples of
membranes that may be used for microencapsulation immobilization of
biocomponents are cellulose acetate, polycarbonate, collage,
acrylate copolymers, poly(ethylene glycol) and
polytetrafluoroethylene (PTFE). Additional materials that may be
used are agarose, and alginate and polylysine, which together form
an alginate-polylysine-alginate microcapsule.
[0114] In another embodiment, entrapment is used to immobilize the
biocomponent. In this method, cells are physically constrained
(entrapped) to stay inside a three-dimensional matrix. The
materials used for entrapment must allow uniform cell distribution,
biocompatibility and good transport of substrates, cofactors and
products. Both natural and synthetic materials (like alginate,
agarose and collagen) may be used for entrapment.
[0115] In another embodiment, hydrogels are used to immobilize the
biocomponent. Hydrogels provide a hydrophilic environment for the
biocomponent and they require only mild conditions to polymerize.
Hydrogels are capable of absorbing large quantities of water which
can facilitate enzymatic biocomponent reactions such as hydrolysis.
Both natural and synthetic hydrogels may be used such as algal
polysaccharides, agar, agarose, alginate, and carrageenan,
polyacrylamide, polystyrene and polyurethane.
[0116] Alginate, a hydrogel, provides a good, biocompatible
microenvironment for the biocomponent and has a gentle
encapsulation process. It is a naturally occurring linear polymer
composed of .beta.-(1,4) linked D-mannuronic acid and
a-(1,4)-L-guluronic acid monomers. Commercially, alginate is
obtained from kelp, but bacteria such as Azotobacter vinelandii,
several Pseudomonas species and various algae also produce it. When
alginate is exposed to Ca.sup.2+ ions, a cross-linking network is
formed by the bonding of Ca.sup.2+ ions and polyguluronic portions
of the polymer strand by a process known as ionic gelation. The
gelation process is temperature-independent. Complete gelling time
without biocomponents may be from about 1 minute to greater than
about 30 minutes. Gelling time usually increases with an increase
in biocomponent density and decreases with an increase in
CaCl.sub.2 concentration.
[0117] In another embodiment, sol-gels may be used to entrap
biocomponents into silicate networks. Sol-gels may require milder
polymerization processes and create matrices that exhibit good mass
transport and molecular access properties particularly for
electrochemical and optical transduction modes.
[0118] In another embodiment, cross-linking is used to immobilize
the biocomponent. Cross-linking chemically bonds the biocomponent
to solid supports or to other supporting materials such as a gel.
Bifunctional agents such as glutaraldehyde, hexamethylene
diisocyanate and 1,5-dinitro-2,4-difluorobenzene may be used to
bind the biocomponent to the solid support such as a matrix, for
example. Cross-linking produces long-term stability under more
strenuous experimental conditions, such as exposure to flowing
samples, stirring, washing, etc.
[0119] In another embodiment, covalent bonding is used to
immobilize the biocomponent. Covalent bonding uses a particular
group present in the biocomponent, which is not involved in
catalytic action, and attaches it to the matrix, transducer layer,
membrane or fiber optical surface through a covalent bond. The
radicals that take part in this reaction are generally nucleophilic
in nature (e.g., --NH.sub.2, --COOH, --OH, --SH and imidazole
groups).
Stabilization
[0120] Biosensing systems of the present disclosure are stable and
long-lived, can stand prolonged storage and can also perform
consistently when used for extended periods. Biocomponents may be
stabilized through various means, depending upon the type of
biocomponent and transducer used.
[0121] In one embodiment, the biocomponent may be stabilized
through molecular modification. Molecular modification improves the
stability of enzymes, and other biocomponents, through changing
certain amino acids or nucleotides in the peptide or nucleic acid
sequence, respectively. Molecular modifications may increase the
temperature stability of various enzymes by modifying the amino
acids at the catalytically active enzyme reaction site or at
structurally sensitive amino acid sequences, through site-directed
mutagenesis.
[0122] Another method for improving the stability of biocomponents,
such as enzymes, is through glycosylation. Since glycosylated
proteins are very stable, grafting or otherwise bonding
polysaccharides or short chains of sugar molecules onto protein
molecules usually improves the stability of the biocomponent.
[0123] In one embodiment, the biocomponent may be stabilized
through cross-linking. Cross-linking of the biocomponent may occur
through covalent bonding, entrapment, encapsulation and other
immobilization techniques or processes. These immobilization
processes can improve enzyme stability by reducing the
biocomponent's mobility and thereby reducing degradation of its
three-dimensional structure. In addition, cross-linking prevents
the loss of biocomponents from the matrix in which they are
immobilized. Using the entrapment method discussed above, the loss
of biocomponents may further be reduced by the addition of certain
gel-hardening agents such as glutaraldehyde, polyethyleneimine,
hexamethylenediamine and formaldehyde.
[0124] In another embodiment for stabilizing the biocomponent,
freeze drying, also known as lyophilization, may be used. Freeze
drying is a method for long-term preservation of microorganisms and
enzymes. It involves removal of water from frozen bacterial
suspensions by sublimation under reduced pressure. The
lyophillization is performed in the presence of cryoprotective
agents such as glycerol and DMSO, which reduce the damage caused
during freezing and during thawing. Lyophillized biocomponents, for
example dried cells, are stable to degradation by keeping the
lyophilized biocomponents below 4.degree. C., and away from oxygen,
moisture and light. Even after prolonged periods of storage, such
as about 10 years, lyophillized biocomponents may then be
rehydrated and restored to an active. Two examples of lyophilizing
techniques used on biocomponents include centrifugal freeze-drying
and prefreezing.
[0125] In another embodiment, the biocomponents by be stabilized
through heat shocking. Heat shocking involves heating vacuum-dried
cells at a high temperature (about 300.degree. C., for example) for
a very short time (about 2-3 minutes, for example). With the proper
combination of temperature and heating time, biocomponents such as
whole cells and microorganisms can be killed but still retain an
active enzyme system that may be used to detect a compound of
interest. These dead cells and microorganisms can be kept for a
long time away from moisture without any requirement of
nutrients.
[0126] In another embodiment, the addition of carbohydrates and
other polymers will stabilize the biocomponents. Carbohydrates used
to stabilize biocomponents include polyalcohols and various sugars
such as trehalose, maltose, lactose, sucrose, glucose and
galactose, for example. This stabilization may occur due to the
interaction of polyhydroxyl moieties from the polyalcohols and/or
sugars with water with the biocomponents, thus increasing
hydrophobic interactions and keeping the biocomponents in a stable
conformation.
[0127] In an additional embodiment, stabilization of the
biocomponents may occur through freezing the biocomponents. When a
biocomponent is frozen, the metabolic activities may be reduced
considerably. Storage of the biosensing system, and/or biosensing
element at temperatures at which the biocomponents remain frozen
may increase the stability and life-time of the biosensing
system.
Biosensing Elements
[0128] Several biosensing system designs are disclosed herein
including biosensing elements on the tip of a fiber optical cable,
and biosensing elements displaced upon a surface, for example. The
biosensing system may be based on an optical pH or optical oxygen
sensor. Oxygenases may be used alone as the biocomponent or in
conjunction with other biocomponents. The biosensing elements may
be separate from one another or combined into the same tip or
biosensing element.
[0129] Some biosensing systems are made using food-grade enzymes
and materials. These biosensing systems are advantageously used for
measuring analytes in food products.
[0130] In an embodiment, the disclosures presented herein are a set
of biosensing system designs based on optical transduction. Optical
enzymatic biosensing system designs using an optical signal
transaction are more robust and less susceptible to chemical
interference than electrochemical (e.g., amperometric) methods. In
one embodiment, optical pH and optical oxygen sensors (optodes)
employ fluorophores that are sensitive to either protons (H.sup.+
ions) or molecular oxygen. Optical enzymatic biosensing elements
are formed by combining a transducer and/or optode with a
biocomponent that catalyzes a reaction with the analyte and results
in altered pH or oxygen.
Biosensing System Measurement at High Analyte Concentrations
[0131] Some biosensing system applications may require the
measurement of relatively high analyte concentrations. Without
certain modifications, these concentrations may be high enough to
saturate the response of the biocomponent, meaning that all of the
binding sites of an antibody or all of the enzymatic reaction sites
are occupied. Under these saturating conditions, the biosensing
system response is no longer dependent upon the analyte
concentration and no measurement can be made.
[0132] One embodiment of the present disclosure is for optical
enzymatic biosensing systems for the measurement of analytes at
high concentrations. Optical enzymatic biosensing systems for the
measurement of analytes at high concentrations and the concepts
disclosed herein are broadly applicable for the measurement of many
different kinds of analytes in solutions such as the measurement of
halogenated alkenes, for example.
[0133] Optical enzymatic biosensing systems may use biosensing
elements that may be constructed as thin enzyme-containing films
deposited or placed over an optical transducer layer. The response
of these biosensing systems (signal as a function of analyte
concentration) is governed by the rate of the enzymatic reaction
and the manner in which that rate depends on the analyte
concentration. For most enzymes, this relationship is the
saturation type shown in FIG. 8 in which the rate depends nearly
linearly on analyte concentration at low concentrations but becomes
independent of concentration at high concentrations. For a
biosensing system that has a biosensing element with a thin-layer
biocomponent, this means that the biosensing system response
becomes saturated and consequently it is not possible to
distinguish one high concentration value from another.
[0134] To describe this high concentration range more accurately,
it is convenient to use the Michaelis-Menten equation, which
relates the enzymatic reaction rate R.sub.enz to the concentration
of the analyte (C.sub.A) as
R.sub.enz=kC.sub.EC.sub.A/K.sub.M+C.sub.A in which k and K.sub.M
are parameters of the enzymatic reaction rate (depending on the
enzyme and the analyte) and C.sub.E is the concentration of enzyme.
The combined term kC.sub.E is frequently presented as V.sub.max,
the maximum reaction rate ("velocity"). The Michaelis-Menten
equation has been found to accurately describe many different
enzyme-catalyzed reactions.
[0135] When analyte concentrations are low enough that C.sub.A is
much less than K.sub.M, the Michaelis-Menten equation approximately
reduces to a first-order (linear) dependence of the reaction rate
on the analyte concentration, R.sub.enz=(V.sub.max/K.sub.M)C.sub.A
This linear response is the desired operating condition for a
biosensing system. However, for thin-film enzymatic biocomponent
biosensing systems, this range extends only to values of C.sub.A
that are small relative to K.sub.M; "small" can be interpreted as
10% or less. At higher analyte concentrations, the relationship of
the enzymatic reaction rate to the analyte concentration, and thus
the relationship of the biosensing system response to the analyte
concentration, becomes increasingly nonlinear. Once the analyte
concentration becomes much larger than K.sub.M such that
C.sub.A+K.sub.M=C.sub.A, the enzymatic reaction rate and the
biosensing system response become essentially independent of
C.sub.A. Modifying the Michaelis-Menten equation for this case of
C.sub.A>>K.sub.M yields R.sub.enz=V.sub.max.
[0136] The analysis above is based on the assumption that the
analyte concentration in the vicinity of the biocomponent enzyme
molecules ("local" concentration) is the same as in the solution in
which the biosensing element is placed ("bulk solution"
concentration). However, this situation can be manipulated such
that the local concentration is lowered such that it falls within
the linear measurement range. The local concentration can be
related to the bulk solution concentration by either calculating
the reaction-diffusion behavior of the system or through
experimental calibration procedures.
[0137] A solution to extend the linear (useful) measurement range
of optical enzymatic biosensing systems beyond that available with
thin-film designs is to add a mass transfer (diffusion) barrier.
This diffusion barrier may take the form of a polymer coating, a
membrane, or any other material through which the analyte passes
more slowly than through the measurement medium. An effective
diffusion barrier could also be created by increasing the thickness
of the enzyme layer. Biosensing systems that have an increased
thickness of the enzyme layer are generally referred to as
thick-film biosensing systems. Linear measurement ranges can be
extended through the use of thick-film biosensing system designs.
The rates of analyte mass transfer and reaction remain coupled in
thick-film biosensing system designs. Thus, at some analyte
concentration, the rate of mass transfer is high enough that the
analyte concentration near the enzymes exceeds the linear reaction
rate range and the biosensing system no longer has a direct, linear
response to the analyte concentration.
[0138] In one embodiment, biosensing systems of the present
disclosure use a design scheme for the construction of optical
enzymatic biosensing systems capable of measurements at high
analyte concentrations. This is based on the combination of a high
mass transfer resistance and a high enzyme concentration, so that
the analyte concentration near the transducer/fluorophore layer
always remains in the linear reaction rate (and biosensing system
response) range.
[0139] For any given concentration of any particular analyte, the
appropriate ranges of the mass transfer coefficient of the analyte
or substrate from the bulk solution to the enzyme layer, and the
reaction rate parameters of the enzyme layer can be determined
according to Equation 1: ((((Da+1-.beta.).sup.2)/4.beta.)>>1.
Where .beta.=the substrate concentration in the bulk solution
divided by the K.sub.M of the enzyme for the substrate; and where
Da is (h.sub.eV.sub.maxh.sub.p)/(D.sub.pK.sub.M) where h.sub.e is
the thickness of the enzyme layer which is embedded within a
matrix; h.sub.p is the thickness of a porous polymeric or ceramic
material which sits atop the enzyme layer; where D.sub.p is the
diffusion coefficient of the polymer coating, see FIG. 9.
[0140] Therefore, by using Equation 1, the calculations provide
specific design parameters such as the thickness of the enzymatic
(detection) and mass transfer resistance layers such that a linear
biosensing system response is obtained for a given concentration,
see FIG. 4.
[0141] As an example of different embodiments of biosensing systems
of the present disclosure, a series of biosensing systems were
constructed with different membranes or no membrane covering the
enzyme layer. The analyte concentration that was measured was
lactose, but this series of biosensing systems is representative
for any analyte or substrate, such as halogenated alkenes, for
example. In one embodiment, biosensing system A, the biosensing
system has only a thin film of enzyme that is immobilized on the
surface of the biosensing system that is exposed to the solution.
In another embodiment, biosensing system B, the biosensing system
has a porous layer placed over the same thickness of enzyme layer
as was used in biosensing system A. In another embodiment,
biosensing system C, the same thickness of enzyme layer as
biosensing systems A and B has a membrane layer placed over it that
is less porous than the porous layer of biosensing system B.
[0142] Biosensing systems B and C have a membrane material
consisting of track-etched polycarbonate with a pore size of 0.015
.mu.m. Additional mass transfer resistance was provided for
biosensing system C by casting a polyurethane coating on top of the
porous layer material.
[0143] The response of biosensing system A to a series of lactose
standards is show in FIG. 5. From FIG. 10 it is seen that the
biosensing system response begins to saturate at concentrations
above 1.01 mM lactose. Signal saturation is due to the presence of
analyte at concentrations that exceed the K.sub.M of the
enzyme.
[0144] Biosensing system B has the addition of a diffusive barrier
on top of the enzyme layer. This diffusive barrier extended the
linear range of biosensing system B into higher concentration
ranges, see FIG. 11. For biosensing system B, a porous
polycarbonate membrane was immobilized on top of the enzyme layer
to act as barrier to analyte mass transfer, which resulted in a
lower analyte concentration in the enzyme layer compared to that in
bulk solution.
[0145] Biosensing system C used a less porous polycarbonate
membrane relative to the membrane of biosensing system B. This
decrease in the porosity of the diffusive barrier resulted in the
ability to measure lactose at even higher concentrations relative
to biosensing system B, see FIG. 12. The linear range of biosensing
system C was extended into this higher concentration regime as a
direct result of the increased mass transfer resistance of the less
porous diffusive barrier.
[0146] FIG. 13 shows one exemplary embodiment of a system 100 that
is used to provide the appropriate design parameters for
constructing biosensing elements used in biosensing systems that
have a linear response in a given range of an analyte concentration
in a solution. System 100 uses a computer 110 that has a
microprocessor 120 that contains software 130 that processes input
data 140 to provide output data 150 that contains the appropriate
design parameters used for constructing biosensing elements used in
biosensing systems that have a linear response in a given range of
an analyte concentration in a solution. Output data 150 is
displayed upon a screen or saved in a memory storage device or may
be transmitted to another memory device or display device.
Constructing the Biosensing System and/or Biosensing Element
[0147] In an embodiment, the biosensing element is constructed by
putting an immobilized biocomponent within a matrix and coupling
that biocomponent-containing matrix onto a transducer. In another
embodiment, a biosensing system is created by bonding, affixing or
otherwise causing the biocomponent to be in contact with an
optode.
[0148] An embodiment of biosensing system of the present disclosure
is depicted in FIG. 14. FIG. 14 depicts a biosensing system 10.
Biosensing system 10 includes a biocomponent 20 that is displaced
within a matrix 22. Matrix 22 is in direct contact with a
transducer 30. Transducer 30 is in direct contact with an end of a
bifurcated optical cable 50. Biocomponent 20 and transducer 30
comprise a biosensing element 40. Bifurcated optical cable 50
transmits light from a light source 70 through a filter 80. The
light that is transmitted through filter 80 is transmitted through
bifurcated optical cable 50 at a first light wavelength 82.
Transducer 30 interacts with first light wavelength 82 and
luminesces at a second light wavelength 90. Second light wavelength
90 is transmitted through bifurcated optical cable 50 and is
detected by a photon-detection device 60 that produces a signal
that is sent to a signal processing system 62. Signal processing
system 62 contains software 64 that uses an algorithm for
determining the concentration of an analyte in a solution based on
the luminescence of transducer 30 at second wavelength 90.
Method of Using the Biosensing System and/or Biosensing Element
[0149] FIG. 15 shows one exemplary method 200 for using a
biosensing system to measure the concentration of an analyte in a
solution. In step 202, method 200 is implemented by generating
light of a first wavelength 82 by light source 70 as it passes
through filter 80 and travels down bifurcated optical cable 50 to
interact with transducer 30 of biosensing element 40. In step 204,
method 200 is further implemented by placing biosensing element 40
at the end of a bifurcated optical cable 50 into a solution. In
step 206, an analyte diffuses into matrix 22 and catalyzes the
reaction of biocomponent 20. In step 208, the product of the
reaction of the analyte with biocomponent 20 produces or uses
oxygen and/or hydrogen ions that interact with transducer 30 to
affect the amount of fluorescence at a second light wavelength 90
of transducer 30. In step 210, the second light wavelength 90 is
transmitted through bifurcated optical cable 50 and detected by
photon-detection device 60. In step 212, photon-detection device 60
detects and multiplies the signal of second light wavelength 90 and
sends a signal to signal processing system 62. In step 214, signal
processing system 62 has software 64 that uses an algorithm that
transforms the signal from photon-detection device 60 into an
output that can be read as a numerical representation of the
concentration of the analyte in the solution that biosensing
element 40 was placed into in step 204.
TOM-Green Biosensing Element
[0150] Presented herein is a fiber optic enzymatic biosensing
system for the fast and simple measurement of TCE concentration
and/or the concentration of other halogenated alkenes. In an
embodiment of the present disclosure, biosensing systems use TOM as
a biocomponent. In another embodiment, oxygenases that use other
halogenated alkenes as a substrate may be used as a
biocomponent.
[0151] TOM is an enzyme involved in the ortho-hydroxylation of
toluene and is a member of EC group 1.13. TOM can catalyze the
first steps in aerobic TCE dehalogenation with oxygen and reduced
nicotinamide adenine dinucloetide (NADH). In these first steps of
aerobic TCE dehalogenation, a very active TCE epoxide intermediate
is formed. The epoxide formed during the course of the reaction
often leads to the inactivation of TOM. In an embodiment of the
present disclosure, other enzymes are used to degrade the TCE
epoxide into less reactive species thereby preventing the
inactivation of TOM by the TCE epoxide and prolonging the useful
life of the biosensing elements of the biosensing systems.
[0152] In one embodiment, biosensing systems of the present
disclosure use a variant of TOM, TOM-Green, as the biocomponent.
TOM-Green was created through using a DNA shuffling technique. In
one embodiment, TOM-Green is created by making a V106A substitution
in the hydroxylase alpha-subunit of TOM from Burkholderia cepacia
G4. TOM-Green has an initial TCE degradation rate that is twice
that of native TOM. In an embodiment, TCE measurement with this
biosensing system is performed on the basis of the measurement of
oxygen consumption during the oxidation reaction.
[0153] In an embodiment of the present disclosure, calcium alginate
gel may be used to immobilize whole cells containing TOM-Green on a
fiber optic oxygen sensor such as an oxygen optode. The oxygen
optode is based on a phosphorescent indicator chemical that
exhibits reduced light emission intensity by molecular oxygen via
dynamic quenching. One example of an indicator chemical that
exhibits reduced light emission intensity by molecular oxygen via
dynamic quenching is RuDPP although other chemicals that fluoresce
or phosphoresce and whose fluorescence or phosphorescence are
quenched by molecular oxygen may be used as well. Other forms of
oxygen-interacting fluorophores and oxygen optode technologies may
also be used in the biosensing systems of the present disclosure,
for example, oxygen-interacting fluorophone containing systems that
measure changes in the fluorescence lifetime of a fluorophore. As a
result of the enzymatic reaction, the oxygen concentration within
the alginate layer decreases with the presence of TCE, which is
often apparent as an increase in phosphorescence detection. TCE
epoxide toxicity may be evaluated by using two types of Escherichia
coli cells with the same TOM-Green expressing plasmid but different
secondary plasmids, each with a unique epoxide toxicity mitigation
mechanism.
Demonstration of the TOM-Green Biosensing System for TCE
Measurement
[0154] A 0.1 mL aliquot of 25 mg/L aqueous TCE solution was
injected into 4.0 mL of measurement solution in which the
biosensing element of the biosensing system was immersed. The
proposed detection mechanism is that the reaction between TCE and
oxygen is catalyzed by the intracellular TOM-Green enzyme
immobilized on the biosensing element, and that this reaction
consumes oxygen in the solution (as well as NADH inside the cells).
As a result, the decrease of oxygen in the alginate layer then
causes an increase in the phosphorescence intensity of the
immobilized RuDPP because of reduced quenching by oxygen. The
biosensing system reading is defined as the measured
phosphorescence intensity at a single condition (e.g., measurement
solution without analyte at 1 mg/L dissolved oxygen), while the
difference between the readings before and after TCE added is
termed the biosensing element or biosensing system signal.
[0155] The signal of a biosensing system with whole cells of E.
coli TG1 pBS(Kan)TOM-Green was 2000 counts with a response time of
4 h (FIG. 1), as the result of TCE concentration increase from zero
to 0.61 mg/L. When the biosensing system reading reached a steady
value (variation less than or equal to the system noise), the
remaining TCE concentration in the vial was found to be
0.60.+-.0.03 mg/L by GC-MS. This indicates that TCE detection
inside the biosensing system is based on a steady-balance between
diffusion and reaction of TCE and oxygen in the biosensing element
region, rather than the depletion of TCE in the sample.
[0156] In one example, a TOM-Green biosensing system was
constructed using E. coli TG-1 cells engineered to express
TOM-Green enzyme that were immobilized on a pH optode using calcium
alginate, see FIG. 16.
Characterization of TOM-Green Biosensing System
Reproducibility
[0157] Biosensing systems were tested with 5 .mu.g/L TCE solutions
in order to evaluate reproducibility. The biosensing system signal
reproducibility had a relative standard deviation (RSD)=12.8% for
n=9, within a batch. In addition, biosensing elements made in
different batches were also tested under the same conditions to
evaluate batch-to-batch reproducibility. The results showed that
these biosensing systems were also consistent with a 11% RSD for
biosensing elements made from five different batches.
Effects of Cell Concentration
[0158] E. coli TG1 pBS(Kan) TOM-Green cells were immobilized at
different concentrations in calcium alginate to validate the effect
of enzyme concentration on biosensing system performance.
Triplicate measurements were made for each of three different
cell-to-alginate w/w ratios (3:1, 2:1, and 1:1). All of these
biosensing elements were tested with 5 to 20 .mu.g/L TCE and no
significant differences in the signal were observed (p<0.01).
This result indicates that the oxygen concentration gradient from
the alginate layer to the bulk solution is unaffected by cell
concentration in the range studied.
[0159] Similarly, the biosensing system response time was not
dependent upon the cell concentration on the biosensing element. In
an embodiment, the measurements with a TCE-based biosensing element
require about 2 h each.
Calibration Curve and Limit of Detection
[0160] A series of TCE solutions from 50 .mu.g/L to 4 mg/L were
measured with TOM-Green biosensing systems. Each biosensing element
was used only once, and each concentration point was measured in
triplicate. The biosensing system signal increased monotonically
with TCE concentration and the overall calibration curve was
nonlinear over this range. A linear region was observed from 1.2 to
9.8 .mu.g/L TCE with R.sup.2=0.962 (FIG. 2). The limit of detection
(LOD), calculated as three times the standard deviation of the
noise obtained from control experiments, was equal to 1.2 .mu.g/L,
less than the EPA Maximum Contaminant Level Goal for TCE (5
.mu.g/L) in National Primary Drinking Water Regulations.
[0161] The LOD of the TOM-Green biosensing system for TCE is low,
having a linear detection range at levels corresponding to
environmentally relevant values.
Accuracy
[0162] Water samples from two lakes (Horsetooth Reservoir and City
Park Lake, Fort Collins, Colo.) were spiked with TCE to quantify
the biosensing system performance in real environmental matrices.
In each case, three different TCE concentrations were used, chosen
to span the linear measurement range of the biosensing system of
this particular embodiment of the present disclosure. The
concentrations measured by the TOM-Green biosensing system and the
GC/MS method are compared in Table 1, shown below. The average
difference between the biosensing system and GC/MS measurements was
0.1.+-.0.2 .mu.g/L with a confidence interval (CI) of 95%, n=18,
indicating that the TOM-Green biosensing systems provide accurate
and reliable measurement for TCE in these aqueous matrices.
TABLE-US-00001 TABLE 1 Comparison of TCE measurements in spiked
water samples. TCE concentration (.mu.g/L) TOM-Green Sample
Biosensing System GC-MS Spiked in Horsetooth Reservoir water High
9.8 .+-. 0.2 9.8 .+-. 0.1 Medium 4.9 .+-. 0.1 4.8 .+-. 0.1 Low 1.1
.+-. 0.1 1.2 .+-. 0.1 Spiked in City Park Lake water High 9.8 .+-.
0.1 9.7 .+-. 0.1 Medium 4.8 .+-. 0.1 4.8 .+-. 0.1 Low 0.8 .+-. 0.2
1.2 .+-. 0.1
Selectivity
[0163] TOM-Green has been reported to catalyze the reaction of
several chlorinated and aromatic chemicals in addition to TCE via a
similar hydroxylation mechanism. Therefore, toluene, benzene, and
TCE were chosen to evaluate the selectivity of the TOM-Green
biosensing system. All of these analytes were measured at a
concentration of 1 mg/L. The biosensing system signal was largest
for TCE (2280.+-.80 counts), followed by toluene (570.+-.60
counts), and then benzene (40.+-.10 counts). This trend is
consistent with data from a previous study in which TOM-Green was
found to have a higher degradation rate for TCE than for other
analytes. The 1 mg/L TCE concentration registered the highest
biosensing system signal, suggesting that the signal increases
monotonically when TCE concentration increases.
Effects of Temperature and pH on Biosensing System Signal
[0164] Hydrogen ion concentration, as measured by pH, and
temperature are two crucial factors in environmental monitoring,
since both enzyme activity and mass transfer rates of TCE and
oxygen could be affected. In addition, the phosphorescence
properties of RuDPP are also temperature dependent. To quantify the
effect of pH on the TOM-based biosensing system signal, sets of
three biosensing systems were tested in measurement solutions
buffered at pH 5.0, 6.0, or 7.0, spanning a common pH range in
typical groundwater aquifer. The signals corresponding to 5 .mu.g/L
TCE at different pH values were 290.+-.20 counts (pH=5), 280.+-.30
counts (pH=6), and 300.+-.40 (pH=7), indicating that the
measurements of the TOM-based biosensing system were independent of
pH in this range. Similarly, the signals of a set of three
biosensing systems at three temperatures were investigated. The
signals of these biosensing systems to 5 .mu.g/L TCE were 270.+-.50
counts at 15.degree. C., 290.+-.20 counts at 20.degree. C. and
430.+-.30 counts at 30.degree. C.
Activity Retention
[0165] Biosensing systems of the present disclosure retain activity
with use or storage or prolonged periods of time and through
multiple uses. To investigate the retention of activity among
biosensing systems, two groups of biosensing elements were stored
in a measurement solution without TCE at 4.degree. C. or 20.degree.
C. At various intervals, biosensing elements were transferred from
the storage solution and used to measure 10 .mu.g/L TCE. For both
storage temperatures, the biosensing system performance declined
over time, and eventually no detection of TCE was recorded.
Biosensing elements stored at 4.degree. C. retained activity over a
longer period than those stored at 20.degree. C. (FIG. 3). Thus,
NADH starvation or enzyme denaturation may be responsible for the
deteriorating biosensing system activity over time, especially at
higher temperature.
[0166] In one embodiment of the present disclosure, NADH may be
regenerated within a biocomponent cell through a co-enzyme system.
In another embodiment, NADH may be made available to the
biocomponent via capillary action or pumping of a delivery
tube.
[0167] In one embodiment, the retention of activity of TOM is
increased through the regeneration of NADH. NADH regeneration via
an external supply of formate can partially replenish biocomponent
TOM activity since intracellular formate dehydrogenase can reduce
the NAD+ to NADH by the oxidation of formate. NADH regeneration can
also be accomplished by providing formate dehydrogenase as an
additional biocomponent on the same biosensing element or on a
different biosensing element that is part of the same biosensing
system; and supplying formate to the formate dehydrogenase in order
to regenerate the supply of NADH and/or NADPH. Regeneration
experiments were conducted to test the extent of regeneration via
this formate scheme using TOM-Green biosensing systems.
[0168] In comparison with controls having no formate regeneration
between repeated measurements, the signal from regenerated
TOM-Green biosensing elements showed 2.+-.3% increase at a TCE
concentration of 50 .mu.g/L, 2.+-.4% increase at a TCE
concentration of 10 .mu.g/L and 5.+-.5% increase at a TCE
concentration of 2 .mu.g/L, indicating that NADH regeneration has a
smaller effect than TCE epoxide on the biocomponent cells, see
FIGS. 5-7.
Mitigation of TCE Epoxide Toxicity
[0169] In one embodiment, biosensing systems disclosed herein
contains at least two biocomponents, at least a first biocomponent
that react directly or indirectly with an analyte of interest and
at least a second biocomponent that mitigages the damage caused by
the product or by-product of the reaction catalyzed by the first
biocomponent. Damage caused by the product or by-product of the
reaction can be mitigated by enzymes including epoxide hydrolase,
glutathione synthetase, glutathione S-transferase and
gamma-glutamylcysteine synthetase or other enzymes that quench or
otherwise react with products or by-products of biocomponent
reactions.
[0170] In another embodiment, biosensing systems disclosed herein
contain at least a first biocomponent that reacts directly or
indirectly with an analyte of interest.
[0171] TCE epoxide is electrophilic and may directly or indirectly
react with various intracellular biological molecules such as DNA,
RNA, lipids, proteins, and other small molecules. The reactions
often result in the inactivation of enzymes, cells or other
biocomponents. E. coli cells with TOM-Green plasmid and
gamma-glutamylcysteine synthetase (GSHI), EC number 6.3.2.2, and/or
epoxide hydrolase (EchA), EC numbers 3.3.2, 3.3.2.3, 3.3.2.9,
3.3.2.10, plasmids were developed to mitigate the damage created by
TCE epoxide.
[0172] Biosensing systems were made with E. coli TOM-Green, E. coli
TOM-Green/GSHI, and E. coli TOM-Green/EchA. Biosensing systems in
each group were made in a single batch and tested with 50 .mu.g/L
TCE ("high" concentration), 10 .mu.g/L TCE ("medium"
concentration), and 2 .mu.g/L TCE ("low" concentration), in
triplicate, while each biosensing system was tested three times at
the same TCE concentration. At high TCE concentration, the E. coli
TOM-Green biosensing system was inactivated after the first use,
while the second measurement of E. coli TOM-Green/GSHI and E. coli
TOM-Green/EchA biosensing systems retained about 50% of their
initial signals, and the third measurement had about 10% of the
initial signals, see FIG. 4. At a medium TCE concentration range,
the second measurement signals were about 75-80% that of first
measurement in the case of the TOM-Green/GSHI and TOM-Green/EchA
biosensing systems, while the TOM-Green biosensing systems retained
about 30% activity after the first use. In the low concentration
range, the TCE toxicity effect was less obvious since all three
kinds of biosensing systems shared the same range of activity
retention after first time usages. The TOM-Green/GSHI had a higher
biosensing system signal than TOM-EchA in all conditions.
Demonstration of the Toluene Dioxygenase Biosensing System for TCE
Measurement
[0173] In one embodiment, a biosensing system having toluene
dioxygenase in Pseudomonas putida F1 as a biocomponent and an
oxygen optode transducer was used to measure the concentration of
TCE, see FIG. 17.
[0174] In another embodiment, a biosensing system may be
constructed using substantially purified toluene dioxygenase as a
biocomponent.
EXAMPLES
Bacterial Strains and Growth Conditions
[0175] The various biocomponent enzymes of the biosensing systems,
TOM-Green, TOM-Green/EchA, and TOM-Green/GSHI were expressed in E.
coli strain TG1. E. coli cultures were grown aerobically on agar
plates made from Luria-Bertani (LB) medium with 20 g/L Bacto-agar
(Difco) and 100 mg/L kanamycin (plus 50 mg/L chloramphenicol in the
case of TOM-Green/EchA and TOM-Green/GSHI) at 30.degree. C. for 24
h. A culture tube containing 2 mL LB medium supplemented with same
concentrations of antibiotics was inoculated from an individual
colony on an agar plate and shaken overnight at 30.degree. C. and
200 rpm, then transferred to a flask containing 200 mL of the same
LB-Kan medium and shaken at 30.degree. C. and 200 rpm. The cell
concentration was measured as culture absorbance at 600 nm (optical
density at 600 nm, OD.sub.600) with a spectrophotometer
Spectronic.RTM. 20 Genesys.TM., Thermo Electron Corporation. IPTG
solution was prepared with deionized water and added to the culture
with a final concentration of 1 mM in the early exponential growth
phase (OD600 of 0.6) to induce TOM-Green, TOM-Green/EchA and
TOM-Green/GSHI expression. The culture was harvested 4 h after IPTG
was added, centrifuged, and resuspended in 20 mL of a solution
containing 10 mM phosphate-buffered saline at pH 7.4 and stored at
4.degree. C. until further use.
Exemplary Biosensing Element
[0176] A biosensing element consisting of a layer of whole cells
immobilized over an oxygen optode was constructed from a 25-cm
section of polymethylmethacrylate (PMMA) optical fiber terminated
with a straight tip (ST) connector. The fiber jacket was detached
from 1 mm of the distal end (non-connector terminated) and then
polished with 2000-grit and 3 .mu.m polishing film (part of a fiber
optic tool kit, IF-TK4-RP2, Industrial Fiber Optics) to minimize
potential signal loss due to scattering. One mg of the
oxygen-sensitive RuDPP was dissolved into 1 mL chloroform and mixed
with 200 mg silicone gel (clear RTV silicone, Permatex, Inc.). A 1
.mu.L aliquot of this mixture was then added to the polished fiber
tip. The RuDPP gel layer was affixed to the optical fiber end as
soon as the chloroform evaporated. Previously stored E. coli whole
cells containing plasmids encoding enzymes such as TOM, TOM-Green,
epoxide hydrolase, glutathione synthetase, glutathione
S-transferase, and/or gamma-glutamylcysteine synthetase, were
centrifuged and mixed with sodium alginate solution (2.5% w/w) in a
cell-to-alginate ratio (wet cell mass: alginate solution) of 1:1
w/w. A 2-.mu.L aliquot of the cell-alginate mixture was placed on
the tip of each oxygen optode and immobilized after immersing the
optode in 0.47 M calcium chloride solution for 30 min at 0.degree.
C. All biosensing elements were stored at 0.degree. C. in a
solution of 0.15 M NaCl and 0.025 M CaCl.sub.2 at pH 7.0, the
"measurement solution".
Biosensing System Measurement Protocols
[0177] In one embodiment, biosensing system experiments were
performed in 5 mL glass vials containing 4 mL of measurement
solution saturated with air at room temperature with a small
magnetic stir bar for rapid mixing. The biosensing element was
immersed in this solution, sealed in the glass vial with a rubber
septum, and shielded from external light sources. Aliquots of 0.1
mL of a TCE solution of 0.1 to 4 mg/L were injected into the
measurement solution after the sensor had produced a steady output.
A steady output is defined as the time when the variation in the
output was no larger than the peak-to-peak noise for a period of at
least 5 min.
[0178] In another embodiment, biosensing systems may be used for
continuous measurements.
TCE Concentration Measurement by Gas Chromatography
[0179] To assess the accuracy of the TCE concentration data
obtained from the biosensing systems, GC analysis was performed via
a modification of EPA Method 8260b. After a biosensing system
measurement, 0.75 mL of aqueous solution was collected from the
measurement vial and transferred into a 2 mL glass screw-top GC
vial containing 0.75 mL of chloroform. The GC vial was then capped
with a Telfon-coated septum and mixed on a rotating wheel for 15
min. One .mu.L of the chloroform phase was injected into a Hewlett
Packard 5890 gas chromatograph equipped with a HP model 5971A mass
spectrometric (MS) detector. A calibration curve of the GC-MS total
ion count peak area vs. the TCE concentration in solution was
obtained using dilutions of the 200 mg/L TCE standard solution. The
GC calibration curve was linear over the range of TCE
concentrations from 1 to 1000 .mu.g/L (R.sup.2=0.973).
Preparation of Biosensing Element Using Dry-Heated Cells
[0180] In order to prepare dry heated cells, cells stored at
4.degree. C. in phosphate-buffered saline solution were centrifuged
at 15,000.times.g for 3 minutes and were washed twice with
distilled water. These cells were suspended in a small quantity of
water (3 mL of stored cell suspension were washed and then
suspended in 0.5 mL of water). This suspension was put in a 10-mL
beaker and water was completely removed by vacuum drying at
35.degree. C. It took about an hour to dry these cells. The dried
cells were then scratched off from the surface of beaker using a
spatula. The beaker was then covered with aluminum foil and left in
the oven at a constant temperature of 270.degree. C. and for a
given period of time (30 sec, 60 sec, etc.). These dry heated cells
looked like a highly porous solid and had a light orange color.
These dry-heated cells (.about.0.003-0.004 g) were also immobilized
using the same entrapment method. However it was found that when
these cells were directly mixed with 4% (w/v) of alginate, there
were a lot of small bubbles in the cell-alginate mixture. Since it
was important to eliminate these bubbles in order to obtain a
stable response, these cells were first suspended in 10 .mu.L of
NaOH (pH 7.0) in a 1.5 mL-vial and then 8% (w/v) of alginate was
added to it (from about 0.3 to about 0.5 g/g of dry wt. of cells to
wt. of alginate). This mixture was used to make the biosensing
element.
Preparation of Biosensing Element Using Chloramphenicol-Treated
Cells
[0181] Cells stored at 4.degree. C. in phosphate-buffered saline
were centrifuged at 15,000.times.g for 2 minutes and the pellet was
then washed twice with saline (9 g/L of NaCl [pH 7.1]) containing
50 .mu.g/mL of chloramphenicol. Next, sodium alginate (4% w/v in
water) containing either 50 or 200 .mu.g/mL of chloramphenicol was
added and mixed well with the cell pellet. This cell and alginate
mixture was kept for 5 minutes at room temperature before it was
used to make the biosensing element.
Preparation of Biosensing Element Using Protease Inhibitor Treated
Cells
[0182] Cells stored at 4.degree. C. in phosphate-buffered saline
were centrifuged at 15,000.times.g for 2 minutes and the pellet was
then washed twice with saline (9 g/L of NaCl [pH 7.1]) containing 5
.mu.L of protease inhibitor cocktail in 1 mL of saline solution.
This cocktail was prepared by adding 215 mg of lyophilized protease
inhibitor in a solution containing 1 mL of DMSO (Dimethyl
sulfoxide) and 4 mL of deionized water. The cocktail had a broad
specificity for the inhibition of serine, cysteine, aspartic and
metalloproteases, and aminopeptidases. It was stored at -20.degree.
C. in the freezer. These cells were then mixed with Na-alginate
solution (4% w/v) containing 200 .mu.L of cocktail per mL of
alginate solution. The cell-alginate mixture was left for about 5
minutes at room temperature before it was used for making the
biosensing element. The ratio of the weight of wet cells to the
weight of alginate used in the experiment was 0.72 g/g.
Preparation of Biosensing Element with a Poly-L-Lysine Coating
[0183] The alginate bead was coated with poly-L-lysine (PLL) by
preparing the biosensing element with a biocomponent as described
above. The Ca-alginate bead on the biosensing element was then
washed twice with saline solution (9 g/L of NaCl in water). Then
the biosensing element was immersed in 10 mL of 0.4% (w/v) of
poly-L-lysine.HCl solution, stored at 4.degree. C. inside the
refrigerator) in saline for 30 minutes at 30.degree. C.
Oxygen Sensor Biosensing Element Construction
[0184] In one embodiment, the optode used in the biosensing element
is an oxygen optode. An oxygen optode is a sensor based on optical
measurement of the oxygen concentration. In one embodiment, a
chemical film is glued to the tip of an optical cable and the
fluorescence properties of this film depend on the oxygen
concentration. Fluorescence is at a maximum when there is no oxygen
present. When an O.sub.2 molecule comes along it collides with the
film and this quenches the photoluminescence. In a given oxygen
concentration there will be a specific number of O.sub.2 molecules
colliding with the film at any given time, and the fluorescence
properties will be stable.
[0185] In one example, a biosensing element for measuring the
concentration of oxygen consisted of a layer of immobilized whole
cells over an oxygen optode, which was constructed from a 25-cm
section of PMMA optical fiber terminated with a straight tip (ST)
connector. The fiber jacket was detached from 1 mm of the distal
end (non-connector terminated) and then polished with 2000-grit and
3 .mu.m polishing film (part of a fiber optic tool kit, IF-TK4-RP2,
Industrial Fiber Optics) to minimize potential signal loss due to
scattering. One mg of the oxygen-sensitive phosphorophore RuDPP,
which is classified as phosphorophores since its longer decay
lifetime than typical fluorophores, was dissolved into 1 mL
chloroform and mixed with 200 mg silicone gel (clear RTV silicone,
Permatex, Inc.). A 1-.mu.L aliquot of this mixture was then added
to the polished fiber tip. The RuDPP gel layer was affixed to the
optical fiber end as soon as the chloroform evaporated. Previously
stored E. coli whole cells (with plasmids which may encode for TOM,
TOM-Green, epoxide hydrolase, glutathione synthetase, glutathione
S-transferase, and/or gamma-glutamylcysteine synthetase, for
example) were centrifuged and mixed with sodium alginate solution
(2.5%) in a cell-to-alginate ratio (wet cell mass: alginate
solution) of 1:1 w/w unless otherwise specified. 2 .mu.L of the
cell-alginate mixture was placed on the tip of each oxygen optode
and immobilized after immersing the optode in 0.47 M calcium
chloride solution for 30 min at 0.degree. C. All biosensing
elements were stored at 0.degree. C. in a measurement solution of
0.15 M NaCl and 0.025 M CaCl.sub.2 at pH 7.0.
Oxygen and pH Biosensing System Instrumentation
[0186] The oxygen biosensing system instrumentation consisted of
two separate optoelectronic modules: a 470-nm LED and a 450/60 nm
optical bandpass filter (Chroma Technologies) as the excitation
light source, and a computer-controlled Ocean Optics USB4000-FL
spectrometer with 10 nm resolution for detection. The 470-nm
excitation light was delivered through one leg of a bifurcated
optical fiber assembly that has two 1-mm fibers side-by-side in the
common end (Ocean Optics, Inc.), which was connected with the
biosensing system via an ST connector. The phosphorescent emission
light (peak at 620 nm) from the biosensing element was directed
back into the detector through the other leg of the bifurcated
optical fiber and measured by the spectrometer (sensitivity of
approximately 60 photons/count at 600 nm). The spectrometer output
from 615 nm to 625 nm was integrated over 200 ms and five such
values were averaged to yield one measurement value per second. The
change in the intensity of the emission light over time correlates
to the oxygen concentration change in the RuDPP layer of the
biosensing element. Alternatively, the fluorescence lifetime of a
fluorophore, such as RuDPP, may be measured and correlated to an
oxygen concentration.
[0187] In another embodiment, the fluorescence lifetime of a
fluorophore in a pH optode may be measured and correlated to a
change in hydrogen ion concentration. Alternatively, a pH optode
can measure the change in the intensity of the emission light of a
fluorophore, such as fluorescein, over time and correlate that
change in intensity to the hydrogen ion concentration and thus to
the concentration of an analyte of interest.
[0188] The above examples, embodiments, definitions and
explanations should not be taken as limiting the full metes and
bounds of the invention.
* * * * *