U.S. patent number 6,280,604 [Application Number 09/523,645] was granted by the patent office on 2001-08-28 for electrode materials, systems and methods for the electrochemical detection of nitric oxide.
This patent grant is currently assigned to Duke University. Invention is credited to Barry W. Allen, Louis A. Coury, Claude A. Piantadosi.
United States Patent |
6,280,604 |
Allen , et al. |
August 28, 2001 |
Electrode materials, systems and methods for the electrochemical
detection of nitric oxide
Abstract
Nitric oxide-specific electrodes are useful for in situ
detection of nitric oxide in biomedical applications and have at
least a surface region capable of forming complexes with nitric
oxide. The nitric oxide complexes formed at the surface of the
electrodes apparently increase the concentration of nitric oxide
available for detection, leading to significantly improved relative
responses as compared to other known nitric oxide electrode
materials. The electrode has at least an exterior surface region
which contains ruthenium and/or at least one oxide of ruthenium.
The electrodes are pre-conditioned at a potential, or potentials,
different than the working potential of the electrode, followed by
further conditioning at the working potential. Direct response to
nitric oxide has been observed for ruthenium electrodes at or below
potentials about +675 mV vs. Ag/AgCl, while ruthenium electrodes
paradoxical response to nitric oxide has been observed at
potentials above +675 mV vs. Ag/Cl.
Inventors: |
Allen; Barry W. (Durham,
NC), Coury; Louis A. (Lafayette, IN), Piantadosi; Claude
A. (Durham, NC) |
Assignee: |
Duke University (Durham,
NC)
|
Family
ID: |
24085831 |
Appl.
No.: |
09/523,645 |
Filed: |
March 10, 2000 |
Current U.S.
Class: |
205/777.5;
204/291; 204/403.01; 204/403.06; 204/415; 205/778; 205/781;
205/792; 205/793; 600/309; 600/345; 600/353 |
Current CPC
Class: |
G01N
27/4045 (20130101) |
Current International
Class: |
G01N
27/30 (20060101); G01N 027/26 () |
Field of
Search: |
;205/777.5,778,781,792,793 ;204/403,415,291 ;600/309,345,353 |
References Cited
[Referenced By]
U.S. Patent Documents
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4158044 |
June 1979 |
Takabatake et al. |
4913780 |
April 1990 |
Habermann et al. |
5358889 |
October 1994 |
Emesh et al. |
5409591 |
April 1995 |
Baker et al. |
5466350 |
November 1995 |
Baker et al. |
5565075 |
October 1996 |
Davis et al. |
5603820 |
February 1997 |
Malinski et al. |
5806517 |
September 1998 |
Gerhardt et al. |
5980705 |
November 1999 |
Allen et al. |
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Foreign Patent Documents
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WO 93/215/18 |
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Oct 1993 |
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WO |
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WO 94/02845 |
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Feb 1994 |
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WO |
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WO 96/16593 |
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Jun 1996 |
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WO |
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|
Primary Examiner: Bell; Bruce F.
Attorney, Agent or Firm: Nixon & Vanderhye P.C.
Government Interests
GOVERNMENT GRANT STATEMENT
This invention was made with Government support under Grant No.
2PO1 HL42444-06 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is related to copending application Ser. No.
09/399,371, filed Sep. 20, 1999, still pending which in turn is a
continuation of application Ser. No. 08/942,354 filed on Oct. 1,
1997 (now U.S. Pat. No. 5,980,705), which in turn is based on, and
claims domestic priority benefits under 35 USC .sctn. 119(e) from,
U.S. Provisional Application Serial No. 60/027,355 filed on Oct. 2,
1996, the entire content of each application and patent being
expressly incorporated hereinto by reference.
Claims
What is claimed is:
1. An electrode for detecting nitric-oxide having a surface region
formed of a material which, at an electrochemical working potential
thereof, complexes with nitric oxide when exposed to a nitric
oxide-containing fluid, and which exhibits maximal nitric oxide
response after being held, prior to nitric oxide exposure, at a
pre-conditioning potential different from the working potential
thereof, followed by subsequent conditioning at the working
potential thereof.
2. The electrode of claim 1, wherein the material is at least one
selected from the group consisting of metals, metaloids,
electrically-conducting or electrically semiconducting oxides, and
mixtures thereof.
3. The electrode of claim 1, wherein the electrode does not exhibit
a maximal response to nitric oxide after being conditioned at its
working potential, but does exhibit a maximal response to nitric
oxide after being conditioned at both said pre-conditioning
potential and said working potential.
4. The electrode of claim 1, wherein the pre-conditioning potential
is greater than the working potential thereof.
5. The electrode of claim 1, wherein the pre-conditioning potential
is less than the working potential thereof.
6. The electrode of claim 1, wherein the pre-conditioning potential
is alternately changed between a first potential which is less than
the working potential thereof, and a second potential which is
greater than the working potential thereof.
7. The electrode subjected to a preconditioning potential according
to one or more preconditioning profiles as in any one of claims
4-6.
8. A method for the in vivo detection of nitric oxide comprising
placing an electrode according to any one of claims 1-6 at a site
within a patient or other living organism, and then determining the
nitric oxide present at said site by the electrochemical response
of said electrode.
9. The method of claims 8, wherein said step of determining the
nitric oxide present at said site includes subjecting the electrode
to a predetermined fixed potential which causes said electrode to
electrochemically respond directly to the presence of nitric oxide
at said site.
10. The method of claim 9, wherein said predetermined fixed
potential is less than about +675 mV vs. Ag/AgCl.
11. The method of claim 8, wherein said step of determining the
nitric oxide present in said sample includes subjecting the
electrode to a predetermined fixed potential which causes said
electrode to electrochemically respond indirectly to the presence
of nitric oxide at said site.
12. The method of claim 11, wherein said predetermined fixed
potential is greater than about +675 mV vs. Ag/AgCl.
13. The method of claim 8, wherein said step of determining the
presence of nitric oxide at said site includes subjecting the
electrode to a fixed potential sufficient to suppress oxidative
background current.
14. The method of claim 13, wherein said background current is
suppressed by allowing a film consisting essentially of nitrosyl
complexes or any structural isomers thereof to form on said
electrode surface.
15. A method of detecting nitric oxide in a biological sample
comprising bringing an electrode according to any one of claims 1-6
into contact with the biological sample, and then determining the
nitric oxide present in said sample by the electrochemical response
of said electrode.
16. The method of claim 15, wherein said step of determining the
nitric oxide present in said sample includes subjecting the
electrode to a predetermined fixed potential which causes said
electrode to electrochemically respond directly to the presence of
nitric oxide in said sample.
17. The method of claim 16, wherein said predetermined fixed
potential is less than about +675 mV vs. Ag/AgCl.
18. The method of claim 15, wherein said step of determining the
nitric oxide present in said sample includes subjecting the
electrode to a predetermined fixed potential which causes said
electrode to electrochemically respond indirectly to the presence
of nitric oxide in said sample.
19. The method of claim 18, wherein said predetermined fixed
potential is greater than about +675 mV vs. Ag/AgCl.
20. The method of claim 15, wherein said step of determining the
presence of nitric oxide at said site includes subjecting the
electrode to a fixed potential sufficient to suppress oxidative
background current.
21. The method of claim 20, wherein said background current is
suppressed by allowing a film consisting essentially of nitrosyl
complexes (or any structural isomers thereof) to form on said
electrode surface.
22. The electrode of claim 1, wherein said complex is a nitrosyl
complex or any structural isomer thereof.
23. The electrode of claim 1, wherein at least the surface region
consists essentially of ruthenium and/or at least one oxide of
ruthenium.
24. The electrode of claim 1, wherein at least the surface region
includes at least one oxide of ruthenium having the formula
RuO.sub.x, where x is a number greater than 0.
25. The electrode of claim 24, wherein x is a number greater than 0
up to and including about 2.
26. The electrode of claim 1, wherein the electrode response to
nitric oxide is enhanced by the presence of a chloride-containing
species on said electrode surface region thereof.
27. The electrode of claim 1, wherein the electrode response to
nitric oxide is enhanced by the formation on the electrode surface
region of an electrically-conductive oxide.
28. The electrode of claim 1, wherein the elctrode is comprised of
ruthenium and/or at least one oxide of ruthenium.
29. An electrode for the detection of nitric oxide in a biological
sample having a surface region formed of a material which, at an
electrochemical working potential thereof, complexes with nitric
oxide when exposed to a nitric oxide-containing fluid, and which
exhibits maximal nitric oxide response after being held, prior to
nitric oxide exposure, at a pre-conditioning potential different
from the working potential thereof, followed by subsequent
conditioning at the working potential thereof.
30. The electrode of claim 29, wherein the material is at least one
selected from the group consisting of metals, metaloids,
electrically-conducting or electrically semiconducting oxides, and
mixtures thereof.
31. The electrode of claim 29, wherein the electrode does not
exhibit a maximal response to nitric oxide after being conditioned
at its working potential, but does exhibit a maximal response to
nitric oxide after being held at both said pre-conditioning
potential and said working potential.
32. The electrode of claim 29, wherein the pre-conditioning
potential is greater than the working potential thereof.
33. The electrode of claim 29, wherein the pre-conditioning
potential is less than the working potential thereof.
34. The electrode of claim 29, wherein the pre-conditioning
potential is alternately changed between a first potential which is
less than the working potential thereof, and a second potential
which is greater than the working potential thereof.
35. The electrode subjected to a preconditioning potential
according to one or more preconditioning profiles as in any one of
claims 32-34.
36. The electrode of claim 29, wherein said complex is a nitrosyl
complex or any structural isomer thereof.
37. The electrode of claim 29, wherein at least the surface region
consists essentially of ruthenium and/or at least one oxide of
ruthenium.
38. The electrode of claim 37, wherein at least the surface region
includes at least one oxide of ruthenium having the formula
RuO.sub.x, where x is a number greater than 0.
39. The electrode of claim 38, wherein x is a number greater than 0
up to and including about 2.
40. The electrode of claim 29, wherein the electrode is comprised
of ruthenium and/or at least one oxide of ruthenium.
41. A method of making an electrode for the detection of nitric
oxide having a surface region formed of a material which, at an
electrochemical working potential thereof, complexes with nitric
oxide when exposed to a nitric oxide-containing fluid, which method
comprises conditioning at least the surface region of the electrode
by (i) subjecting the surface region to one or more
pre-conditioning potential(s) different from the working potential,
and then subsequently (ii) subjecting the surface region to
conditioning at the working potential.
42. The method of claim 41, wherein the material is at least one
selected from the group consisting of metals, metaloids,
electrically-conducting or electrically semiconducting oxides, and
mixtures thereof.
43. The method of claim 41, wherein the pre-conditioning potential
is greater than the working potential thereof.
44. The method of claim 41, wherein the pre-conditioning potential
is less than the working potential thereof.
45. The method of claim 41, wherein the pre-conditioning potential
is alternately changed between a first potential which is less than
the working potential thereof, and a second potential which is
greater than the working potential thereof.
46. A method of making an electrode which comprises subjecting the
electrode to a preconditioning potential according to one or more
preconditioning profiles as in any one of claims 43-45.
47. An electrode for the detection of nitric oxide made according
to any one of claims 41-45.
48. A nitric oxide detector system comprising an electrode having a
surface region formed of a material which complexes with nitric
oxide when exposed to a nitric oxide-containing fluid, a mechanical
support for the electrode, and electrical conductors to
electrically connect the electrode to an electrochemical
instrument.
49. The system of claim 48, wherein the material is at least one
selected from the group consisting of metals, metaloids,
electrically-conducting or electrically semiconducting oxides, and
mixtures thereof.
50. The system of claim 48 or 49, comprising plural electrodes, at
least one of which has a surface region formed of said material
which complexes with nitric oxide when exposed to a nitric
oxide-containing fluid.
51. The system of claim 50, wherein said mechanical support
includes a support tube sleeved over said plural electrodes.
52. The system of claim 51, further comprising a temperature probe
adjacent to said plural electrodes.
53. The system of claim 50, further comprising a catheter adjacent
to said plural electrodes.
54. The system of claim 50 wherein said support tube is sleeved
over said plural electrodes, said temperature probe and said
catheter.
55. A nitric oxide detector system comprising a probe assembly
which includes a plurality of individual electrodes, at least one
of which is formed of a material which complexes with nitric oxide
in response to contact with a nitric oxide-containing fluid and
generates a signal in response thereto, an electrochemical
instrument which receives said signal and outputs a signal
indicative of nitric oxide presence in said fluid, and instrument
cabling which operatively connects the individual electrodes to
said electrochemical instrument.
56. The system of claim 55, wherein the material is at least one
selected from the group consisting of metals, metaloids,
electrically-conducting or electrically semiconducting oxides, and
mixtures thereof.
57. The system of claim 48 or 55, wherein said at least one
electrode has a surface region formed of said material which, at an
electrochemical working potential thereof, complexes with nitric
oxide when exposed to a nitric oxide-containing fluid, and which
exhibits maximal nitric oxide response after being held, prior to
nitric oxide exposure, at a pre-conditioning potential different
from the working potential thereof, followed by subsequent
conditioning at the working potential thereof.
58. The system of claim 57, wherein the electrode does not exhibit
a maximal response to nitric oxide after being conditioned at its
working potential, but does exhibit a maximal response to nitric
oxide after being conditioned at both said pre-conditioning
potential and said working potential.
59. The system of claim 57, wherein the pre-conditioning potential
is greater than the working potential thereof.
60. The system of claim 57, wherein the pre-conditioning potential
is less than the working potential thereof.
61. The system of claim 57, wherein the pre-conditioning potential
is alternately changed between a first potential which is less than
the working potential thereof, and a second potential which is
greater than the working potential thereof.
62. A method of detecting a pathological condition which produces
or suppresses nitric oxide comprising bringing an electrode which
complexes with nitric oxide into nitric oxide detecting proximity
of a biological sample, measuring nitric oxide present in the
sample, whereby a pathological condition which produces or
suppresses nitric oxide can be determined.
63. The method of claim 62, wherein said electrode has a surface
region formed of a material which, at an electrochemical working
potential thereof, complexes with nitric oxide when exposed to a
nitric oxide-containing fluid, and which exhibits maximal nitric
oxide response after being held, prior to nitric oxide exposure, at
a pre-conditioning potential different from the working potential
thereof, followed by subsequent conditioning at the working
potential thereof.
64. The method of claim 62 or 63, wherein the material is at least
one selected from the group consisting of metals, metaloids,
electrically-conducting or electrically semiconducting oxides, and
mixtures thereof.
65. The method of claim 64, wherein the electrode does not exhibit
a maximal response to nitric oxide after being conditioned at its
working potential, but does exhibit a maximal response to nitric
oxide after being conditioned at both said pre-conditioning
potential and said working potential.
66. The method of claim 62 or 63, wherein the pre-conditioning
potential is greater than the working potential thereof.
67. The method of claim 62 or 63, wherein the pre-conditioning
potential is less than the working potential thereof.
68. The method of claim 62 or 63, wherein the pre-conditioning
potential is alternately changed between a first potential which is
less than the working potential thereof, and a second potential
which is greater than the working potential thereof.
Description
FIELD OF THE INVENTION
The present invention generally relates to the field of
electrochemical detection of nitric oxide and, in preferred
embodiments, includes materials for constructing electrodes,
microelectrodes, and ultramicroelectrodes (all hereinafter referred
to as "electrodes") that exhibit an increased response to nitric
oxide after a period of electrochemical conditioning and that
chemically increase the concentration of nitric oxide available for
detection at the electrode surface and/or catalytically increase
the rate constant for the electrolysis of nitric oxide.
In addition, the present invention relates to nitric oxide detector
systems that incorporate said nitric oxide electrodes, as well as
to the methods herein described for using such detector systems.
These systems and methods may be employed usefully to detect nitric
oxide in any biological or non-biological liquid sample and/or in
any biological system (e.g., near or within a whole organism,
tissue, group of cells, individual cell, sub-cellular component, or
in any fluid within, bathing or withdrawn from an organism).
BACKGROUND OF THE INVENTION
Nitric oxide (NO.) is a short-lived, free radical gas that has
recently been shown to play a fundamental role in biological
processes, resulting in a demand for accurate and precise
techniques for its detection.
Methods for detecting nitric oxide in biology and medicine include
spectrophotometry, chemiluminescence, and paramagnetic resonance.
But these techniques generally do not permit rapid measurement at
the site of NO. production and therefore are not ideal for
following the brief course of production and decay that
characterizes the metabolism of nitric oxide in biological samples
or systems. Electrochemical detection, however, can follow such
transient processes, and several electrodes have been developed for
the direct electrochemical detection of nitric oxide.
Recently issued U.S. Pat. No. 5,980,705 (incorporated by reference
hereinto) discloses certain novel electrodes and methods for
detecting nitric oxide. In general, such electrodes have a surface
region formed of a metal which complexes with nitric oxide when
exposed to a nitric oxide-containing fluid and exhibit maximal
nitric oxide response after being electrochemically
conditioned.
SUMMARY OF THE INVENTION
Broadly, the present invention relates to electrodes constructed
from materials that exhibit an enhanced response to nitric oxide
(after conditioning according to the above-cited U.S. Pat. No.
5,980,705 and/or conditioning and pre-conditioning as described
herein) and that may also meet one or more of the following four
criteria: 1. form electrically-conducting or
electrically-semiconducting oxides (see Example I); 2. exhibit an
enhanced response to nitric oxide in the presence of chloride (see
Example II); 3. exhibit an enhanced response to nitric oxide after
a period of electrochemical pre-conditioning followed by
conditioning (see Example III); 4. permit indirect detection of
nitric oxide (see Example IV).
The present invention also relates to detector systems that employ,
as one or more of their components, the novel nitric oxide
electrodes of this invention (see Example V). The detector systems
of this invention may usefully detect in vivo, in humans or
animals, normal processes or pathological conditions that result in
an increase or decrease in nitric oxide production, such as, for
example, bacteremia, septicemia, autoimmune diseases, stroke, heart
attack, tissue injury, surgical trauma, and vascular penetration
(see Example VI).
Most preferably, the electrodes of this invention may be prepared
from ruthenium, or have a coating prepared from ruthenium on a core
of supporting material. The ruthenium may be combined with one or
more other materials as may be desired. Alternatively, electrodes
of this invention may be prepared from materials or mixtures of
materials other than ruthenium, such that the resulting electrodes
exhibit an enhanced response to nitric oxide and also meet one or
more of the four criteria listed above. Such materials, in
principle, include all metals, metaloids, and non-metals, but most
particularly the six metals of the so-called platinum group
(elements having atomic numbers 44-46 and 76-78) and the transition
group metals, as well as metals, metaloids, and non-metals capable
of forming electrically-conducting or electrically-semiconducting
oxides, including oxides discussed in Tsuda, N. et al., (1990)
Electronic Conduction in Oxides. Berlin: Springer-Verlag, the
entire content of which is expressly incorporated hereinto by
reference.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Reference will hereinafter be made to the accompanying drawing
FIGURES, wherein:
FIG. 1A is a schematic view of an exemplary nitric oxide detector
system in accordance with the present invention;
FIG. 1B is an enlarged view of the distal end of the nitric oxide
electrode assembly depicted in FIG. 1A;
FIG. 2A is an amperogram showing the indirect response of a
ruthenium electrode observed according to Example IV;
FIG. 2B is a plot of the computed difference current between two
linear voltametric scans performed, respectively, in the presence
and absence of nitric oxide according to Example IV; and
FIG. 3 is a graphical plot of relative nitric oxide activity versus
time for the in vivo experiment conducted according to Example VI
below.
DETAILED DESCRIPTION OF THE INVENTION
The electrodes of this invention can be incorporated into a broad
variety of biomedical and/or clinical medical devices to facilitate
biomedical research and to enable the accurate and/or early
diagnosis and monitoring of various normal processes, medical
conditions and/or disease states in which nitric oxide plays a
role. For example, the electrodes of this invention may be
incorporated into catheters, needles, cannulas and the like for
insertion into a patient or experimental subject (e.g.,
intravenously, into a synovial capsule (joint capsule), into the
cerebrospinal fluid, or through a natural body orifice, such as the
urethra). The electrode may thus be brought into proximity to, or
contact with, a particular biological fluid, such as blood, urine,
synovial fluid and the like to allow for the real time detection in
vivo of nitric oxide therein. The electrodes of this invention may
therefore be employed usefully for the diagnosis and/or monitoring
of bacterial infections (e.g., sepsis), vascular competency,
autoimmune diseases (e.g., rheumatoid arthritis) and any other
disease or condition in which nitric oxide plays a role. Suffice it
to say here that the electrodes of this invention may also be
incorporated into patient monitoring equipment (e.g., blood drawing
systems) or laboratory bench devices (e.g. blood chemistry
analyzers).
The electrode for detecting nitric oxide described herein may thus
be fashioned or incorporated into a variety of devices for research
in the physical, chemical, biological and medical sciences, as well
as for veterinary and medical diagnosis or monitoring. The
electrode of this invention is not limited to the size or shape
described herein and therefore its configuration may be adapted to
virtually any specific end use application. For example, the
electrode may be made very small, with a maximum dimension of a few
micrometers (10.sup.-6 meters) or less, for measuring nitric oxide
activity in very small fluid samples (e.g. 10.sup.-6 liters or
less) in vitro; for insertion, in vivo, between individual living
cells in a tissue; or for insertion directly into living cells or
subcellular components.
This small size may, for example, be achieved by incorporating the
electrode onto a microelectronic device (e.g. on a microchip) or by
using abrasion, machining, heat and/or mechanical force to work the
electrode material into an appropriate configuration. The electrode
material may be electroplated, vapor-deposited, sputtered, printed,
silk screened, painted, or otherwise superimposed onto, or
incorporated in, an appropriate substrate. The electrode may be
encapsulated in glass, an organic polymer, or other material, or it
may be unencapsulated.
The electrode may be used invasively in biological research, in
veterinary research or diagnosis, and in medical research or
diagnosis, by being configured in such a way as to be able to be
placed within a living organism, or within living or non-living
components thereof, in a location in which nitric oxide activity is
to be determined. This may be accomplished by fashioning the
electrode into, or incorporating it into, catheters, needles,
syringes, surgical or medical instruments, micropipettes, drainage
tubes, prostheses, other monitoring devices (such as blood pressure
monitors), implants (such as cardiac pacemakers), dialysis
apparatus, microdialysis probes, plasmapheresis apparatus, to give
but a few examples.
The electrode may be used non-invasively by being fashioned into,
or incorporated into, devices for determining nitric oxide activity
in samples removed from living organisms. Such sample materials
include, but are not limited to, blood, urine, synovial fluid,
cerebrospinal fluid, ophthalmic fluids, lymph, extracellular fluid,
intracellular fluid, the fluid resulting from organ lavage,
surgical drainage fluid, dialysate, cell or tissue cultures or
culture media. Such non-invasive devices may be bench-top
instruments for use in research or diagnostic laboratories or in
bedside instruments for use in clinical or surgical medicine or in
field instruments for medical or veterinary practice.
The electrode of this invention may be used to make discrete
determinations of nitric acid activity, or may be used for
continuous, real-time monitoring or recording of nitric oxide
activity.
Electrochemical detector systems for nitric oxide in accordance
with the present invention may be embodied in a complete electrode
assembly connected to, or incorporated in, an electrochemical
instrument (e.g. a potentiostat) in order to permit measurements of
nitric oxide in any biological or non-biological liquid sample as
well as in a biological system (e.g. in or near a whole organism, a
tissue, a group of cells, an individual cell, sub-cellular
components, or in any fluid within an organism, bathing organisms
or withdrawn from organisms). Preferably, detector systems of this
invention include one or more electrodes, a mechanical support for
the electrodes, an electrochemical instrument, and electrical
conductors to connect the electrode(s) to the electrochemical
instrument. At least one of the constituent electrodes is an
electrode for detecting nitric oxide of the present invention.
One exemplary embodiment of a nitric oxide detector system 10 is
depicted schematically in accompanying FIG. 1A. As shown therein,
the detector system 10 generally includes a distal electrode
assembly 12 which is capable of being coupled operatively to a
shielded potentiostat 14 via shielded instrument cable 16 and
shielded plug 16a. A common connection to ground potential is
provided as depicted schematically in FIG. 1A. An optional
pre-amplifier (or current multiplier) 16b may be connected in
series between the plug 16a and the electrode assembly 12, if
needed or desired.
As is perhaps more clearly shown in FIG. 1B, the electrode assembly
12 includes-a nitric oxide electrode 20 (the working electrode), a
reference electrode 22, an auxiliary electrode 24, a temperature
sensor 26 and an open catheter 28 which provides a pathway for
delivery of medication, site irrigation, medical instrument
positioning or the like as may be needed and/or desired. The nitric
oxide electrode 20 is most preferably conditioned and/or
preconditioned and conditioned against the reference electrode and
by means of electrical current passed through the auxiliary
electrode, if present, in accordance with the techniques described
in the above-cited U.S. Pat. No. 5,980,705 or described herein. The
individual metal slugs comprising each electrode, which may be
formed of the same or different metals, are connected to electrical
leads 20-1, 22-1 and 24-1 which extend proximally through the
instrument cable 16. Furthermore, an exterior tube 20-2, 22-2 and
24-2 is sleeved over each of the electrodes 22, 24 and 26 and their
respective electrical leads 20-1, 22-1 and 24-1 so as to
structurally and electrically isolate one from another. An external
tube 30 is heat-shrunk around all of the electrodes 20, 22 and 24
as well as the temperature sensor 26 and catheter 28, if present.
In this regard, it will be understood that FIGS. 1A and 1B depict
such components as being loosely positioned within the external
housing tube 30 for ease of reference. In reality, the various
components of the probe assembly 12 are tightly retained within the
housing tube 30 so as to establish an integral, water-impermeable
probe unit.
The detector system 10 and electrode assembly 12 shown in FIGS. 1A
and 1B respectively may optionally be provided with additional
components as desired to further improve and/or enhance their
ability to detect nitric oxide or to adapt them to specific
applications. Such additional components may include, for example,
multiple working electrodes and/or reference and auxiliary
electrodes. A variety of mechanical supports can also be employed,
comprising industrial, technical, scientific, or medical tools,
instruments or devices (for example a medical cannula, catheter or
needle). Further, other devices or techniques, a representative few
of which are described below, may be incorporated into the complete
sensor to stabilize or enhance the nitric oxide response in the
presence of interfering chemical species, in conditions of changing
temperature or fluid flow, or in environments in which electrical
or electromagnetic noise or mechanical vibration are important
factors.
Semi-permeable membranes or coatings may be used to exclude from
the sensor chemical species that could be oxidized at the same
potential as nitric oxide or otherwise interfere with or block the
operation of the nitric oxide sensor.
Methods for compensating for changes in temperature of the fluid to
be analyzed for nitric oxide will be employed where such
temperature changes would cause a significant change in the nitric
oxide oxidation signal.
The rate of any electrochemical reaction generally changes with the
rate at which reactants and products are transported to and from
the electrode system. Thus, changes in fluid flow could cause a
change in the nitric oxide oxidation signal of the present
invention that could be misconstrued as a change in nitric oxide
activity. This can be minimized or eliminated by reducing the
dimensions of the nitric oxide-sensitive electrode (the working
electrode) since very small electrodes are less sensitive to
changes in fluid flow. The smaller nitric oxide signal resulting
from a smaller electrode can be overcome by using a number of very
small electrodes in an electrically-coupled array, such that the
combined surface areas of all the electrodes approximate the
surface area of a single, larger electrode.
Smaller electrodes generally exhibit a higher current densities
(current per unit area) than do larger electrodes and therefore can
enhance the ratio of signal (electrolysis current in the present
invention) to electrical or electromagnetic noise. Therefore for
several reasons, instead of a single large electrode, an array of
very small electrodes of equivalent total surface area may be used
in the complete nitric oxide sensor. The electrodes in such an
array must be spaced far enough from each other so that each
electrode does not perturb the NO concentration in the vicinity of
any of the others.
Ambient electrical or electromagnetic noise permeate electrical
conductors and cause extraneous current flow. In cases in which the
signals intentionally passed through the conductors are small, such
extraneous currents constitute serious interference. Enhancement of
the signal-to-noise ratio may be accomplished in the present
invention by incorporating an amplifier (or pre-amplifier) at or
near the electrodes, to strengthen the signal before it is
conducted through the cables that connect to the electrochemical
instrument.
Mechanical movement or vibration of electrical conductors can also
produce or alter electron flow and is therefore is another source
of extraneous current (triboelectric current). Triboelectric
currents can be minimized by use of special cables in which the
internal conductors are mechanically isolated from the outer sheath
of the cable. In addition, pre-amplifiers as well as opto-electric
components (in which electrical signals are converted to optical
signals and back again to electrical signals) can prevent
electrical currents induced in a cable (either electrically or
mechanically) from reaching the electrodes of the present
invention.
Passive electrical or magnetic shielding can also be used to
isolate extraneous electrical currents induced in a cable (either
electrically or mechanically) from the complete detector system of
the present invention.
A greater understanding of this invention will be gained by the
following non-limiting Examples.
EXAMPLE I
Unlike some other metals, ruthenium forms certain
electrically-conducting oxides (for example, ruthenium-dioxide,
RuO.sub.2). For this reason, ruthenium has been used to form
contacts in electrical switches in the field of electrical
equipment engineering [Vadimski, R. G. and Frankenthal, R. P.,
(1979) Ru and RuO.sub.2 as Electrical Contact Materials:
Preparation and Environmental Interactions. J. Electrochem Soc.:
Solid State Science and Technology, 126(11): p. 2017-2023]. The
present inventors found this behavior of certain ruthenium oxides
also facilitates the conduction of electrons in a ruthenium
electrode, in the field of electrochemical detection of nitric
oxide, and may therefore contribute to the enhanced response of the
ruthenium electrode to nitric oxide. Other metals, metaloids or
non-metals that form electrically conducting or semi-conducting
oxides, or mixtures of conductive, semi-conductive and
non-conductive oxides, may also prove useful for constructing
electrodes for the enhanced detection of nitric oxide.
EXAMPLE II
The chloride ion (Cl.sup.-) may be involved in the enhanced
response of some nitric oxide electrodes. The role of chloride
(Cl.sup.-) in the detection of nitric oxide by the electrode of the
present invention was investigated as follows.
A ruthenium working electrode was conditioned by being held at a
potential of +675 mV vs. Ag/AgCl. for two hours in 7.0 pH phosphate
buffer containing 150 mM sodium perchlorate (NaClO.sub.4), which
does not release chloride, Cl.sup.-, in solution. (This was instead
of phosphate buffered saline, PBS, containing 150 mM sodium
chloride, which does release Cl.sup.- in solution.) However, in
order to maintain the same reference potential as in PBS, the
Ag/AgCl reference electrode was immersed in PBS behind a
water-permeable ceramic frit.
The response of the ruthenium electrode to nitric oxide was greatly
diminished in phosphate buffered perchlorate solution (i.e., in the
absence of chloride), as compared to the response of the same kind
of electrode when exposed to nitric oxide in phosphate buffered
saline (i.e., in the presence of chloride). This diminished
response of ruthenium was comparable to that of platinum in
phosphate buffered saline (i.e., in the presence of chloride,
Cl.sup.-), and platinum does not show an enhanced response to
nitric oxide. These results demonstrate that Cl.sup.- is a factor
in the enhanced response of the ruthenium electrode to nitric
oxide.
EXAMPLE III
Electrode conditioning in the present invention consists in holding
the nitric oxide electrode at a fixed conditioning potential for a
period of time sufficient to enhance electrode performance and
then, without interruption, continuing to hold the electrode at the
same potential while the electrode is exposed to a fluid in which
nitric oxide is to be detected. To facilitate an understanding of
what follows, it is useful to distinguish the conditioning
potential from the working potential, the potential used for
detection of nitric oxide, even though they have the same value in
the case just described.
A useful variation of the conditioning procedure is accomplished by
applying to the electrode a preconditioning potential (or an
uninterrupted sequence of such potentials) that may be different
from the conditioning potential and/or the working potential.
As noted below, several pre-conditioning profiles were tried, at
potentials that were either higher or lower than the working
potential, or a combination of both. All of these experiments were
performed in chloride-containing buffer, PBS.
Profile A. A ruthenium electrode was pre-conditioned at +200 mV vs.
Ag/AgCl for 20 min. after which the potential was immediately
stepped up to +675 mV vs. Ag/AgCl (the conditioning potential) and
held at that potential for 2 hrs. before the electrode was used for
electrochemical measurement at +675 mV (the working potential). The
total of pre-conditioning time and conditioning time was,
therefore, 2 hours and 20 min.
Profile B. A ruthenium electrode was pre-conditioned at +800 mV vs.
Ag/AgCl for 20 min. after which the potential was immediately
stepped down to +675 mV vs. Ag/AgCl and held at that potential for
2 hrs. before the electrode was used for electrochemical
measurement at +675 mV. The total of pre-conditioning time and
conditioning time was again 2 hours and 20 min.
Profile C. A ruthenium electrode was pre-conditioned by
continuously alternating +200 mV vs. Ag/AgCl with +800 mV vs.
Ag/AgCl at 2 Hz. for 40 min. prior to conditioning at +675 mV vs.
Ag/AgCl for 2 hrs. The total of pre-conditioning time and
conditioning time was 2 hours and 40 min.
After profile A, the electrochemical response of ruthenium to
nitric oxide was consistently increased to the highest levels that
had previously been attained by conditioning the electrode solely
at the working potential.
After profile B, the electrochemical response of ruthenium to
nitric oxide was increased to higher levels than seen in profile A,
but electrode performance was not as consistent as with Profile A,
and the oxidation signal exhibited greater electrical noise.
After profile C, the electrochemical response of ruthenium to
nitric was consistently increased to higher levels than with
pre-conditioning Profile A and the signal was electrically
quiet.
For ruthenium, at least, pre-conditioning profiles A and C appear
to be superior to pre-conditioning profile B. Pre-conditioning
Profile A has the further advantage that it can be implemented with
simpler electrochemical instruments (potentiostats) that allow only
manual adjustment of the potential applied to the electrode. Other
pre-conditioning profiles may be used and are not limited to the
three profiles described above. Also three or more pre-conditioning
potentials may prove useful. These or other pre-conditioning
profiles may enhance the ability of materials other than Ru to
serve as electrodes for the detection of nitric oxide, including
other metals, metaloids and non-metals.
EXAMPLE IV
At fixed potentials above +675 mV vs. Ag/AgCl, the behavior of the
ruthenium electrode changed dramatically: the current response to
nitric oxide exhibited two peaks instead of one, and the second
peak was very large and, paradoxically, in the unexpected
direction. Specifically, at a fixed potential of +800 mV vs.
Ag/AgCl, an injection of 1.5 .mu.M NO. into PBS produces the
expected downward current peak, consistent with a flow of electrons
towards the working electrode, an oxidation. This initial peak was
immediately followed by a large upward-going current peak that was
not expected. Both peaks are shown in the amperogram of FIG. 2A.
The present inventors term the expected downward response the
direct response to nitric oxide, since it could arise from the
oxidation of nitric oxide without the intervention of any other
chemical processes; and they term the upward peak the indirect
response, since it could not arise directly from an oxidation or a
reduction of nitric oxide.
This upward-going response could also be seen in a plot of the
computed difference current between linear voltametric scans
performed, respectively, in the presence and absence of NO. as
evidenced in FIG. 2B. More specifically, the plot of FIG. 2B
represents the difference between two linear scan voltagrams, one
performed in PBS saturated with 1.5 .mu.M NO. and one performed in
PBS saturated with N.sub.2. As is evident therein, a large upward
peak is present between about +700 and +800 mV, vs. Ag/AgCl.
Although the indirect response appeared consistent with an
electrochemical reduction, there was no known component of the
analyte buffer employed in this example that would be reduced at
the high positive potentials used here. (Nitric oxide is reduced at
negative potentials.) While not wishing to be bound to any
particular theory at this time, the present inventors believe that
the indirect response is likely due to the suppression of the
oxidative background current (presumably chloride oxidation or the
electrolysis of water) after a film forms on the electrode surface
consisting essentially of nitrosyl-ruthenium complexes or
nitrosyl-chloro-ruthenium complexes (or structural isomers
thereof). Such a film could decrease the rate of oxidation of
Cl.sup.- or OH.sup.-. The resulting indirect signal is many times
larger than the oxidation signal, and could be used to detect NO.,
but has a slow time course.
The strong response of the ruthenium electrode to the direct
oxidation of NO. may be useful in sensors for biomedical
applications. The even stronger indirect (paradoxical) response may
be useful in applications in which a rapid response is not as
important.
EXAMPLE V
A complete electrode assembly, according to the present invention,
was constructed similarly to that shown in FIGS. 1A and 1B as
follows. A few turns of a forty-centimeter length of 30 gauge
copper wire were wrapped tightly around one end of a solid
cylindrical slug of ruthenium metal that was 1 cm. long and 0.5 mm
in diameter; the remaining length of the wire extended from the
slug to form an electrical lead. This process was repeated for
similar slugs of silver and platinum. Each slug was then inserted,
along with its electrical lead wire, into a separate, expanded tube
of the fluoropolymer FEP such that the slugs were completely
enclosed in the tubes, but 10 centimeters of the copper wire
protruded from one end of each tube. The triad of ruthenium,
silver, and platinum slugs, lead wires and FEP tubes was then
inserted into a single, larger expanded FEP tube so that the three
slugs were completely enclosed in the larger tube, aligned with
each other, and the three electrical leads protruded from one end
of the large FEP tube. This assembly was heated at a moderate
temperature to shrink the FEP around the slugs and leads. (See the
schematic representation thereof depicted in accompanying FIG.
1B.)
The electrode assembly was electrically shielded and then enclosed
in another expanded FEP tube, with lead wires and shielding
protruding at one end. Moderate heat was again applied to shrink
the FEP. The conductors of a shielded, three-conductor instrument
cable were then electrically connected to the protruding wires and
electrode shielding, and the other end of the cable was terminated
with a shielded plug. The electrode assembly and its junction with
the cable were inserted into an expanded tube of a different
fluoropolymer, PTFE. Intense heat was then applied to shrink the
outer layer of expanded PTFE and to fuse all inner layers of FEP,
resulting in a tightly encapsulated sensor and cable assembly. The
encapsulant was trimmed to expose the tips of the slugs, and the
sensor tip was then polished abrasively, cleaned, and dried.
The sensor tip was then exposed to hexamethyl-di-silazane and dried
in an oven. Reaction with silazane provides a hydrophobic surface
that excludes water from the microscopic spaces between the metal
slugs and the encapsulant. The sensor tip was again repolished,
cleaned and dried to remove the hydrophobic coating only from the
exposed surfaces of the metal disk electrodes. The sensor tip was
immersed in Nafion.TM. (Dupont), an anion-exclusion coating, then
dried again. The tip of the finished electrode was hydrated in
phosphate buffered saline prior to being used for detecting nitric
oxide. Therefore, while the invention has been described in
connection with what is presently considered to be the most
practical and preferred embodiment, it is to be understood that the
invention is not to be limited to the disclosed embodiment, but on
the contrary, is intended to cover various modifications and
equivalent arrangements associated with the spirit and scope of the
appended claims. For example, one or more catheter lumens may be
incorporated into the sensor assembly.
EXAMPLE VI
A complete detector system as described in Example V, above, was
used to detect nitric oxide in vivo, according to an experimental
animal protocol approved by the National Institutes of Health.
Specifically, an adult male baboon (Papio cynocephius) was
anesthetized and ventilated mechanically. An intravascular nitric
oxide sensor was inserted in a femoral vein. The working electrode
was pre-conditioned at +200 mV (vs. Ag/AgCl) for 20 minutes and
then conditioned at +675 mV (vs. Ag/AgCl) for two hours. Later,
heat killed bacteria (E. coli) were infused into a brachial vein.
Approximately twenty minutes later, the output of the nitric oxide
detector system, as shown in accompanying FIG. 3, demonstrated a
strong oxidation signal (shown in the upward direction here for
graphical purposes to signify an increase in nitric oxide),
consistent with the production of nitric oxide by the animal in
response to the experimental bacteremia. Other in vivo experiments,
using laboratory rats, with an approved protocol, showed similar
responses by the nitric oxide detector system after infusion of
lipopolysacharide (LPS), a component of bacteria. This response was
confirmed as a true nitric oxide response, since it was abolished
by injection of an inhibitor of biological nitric oxide production
(L-nitroarginine methyl ester) thirty minutes prior to infusion of
LPS.
These experiments demonstrate use of the nitric oxide detector
system of the present invention to give early warning of
bacteremia. This sensor could be incorporated on the tip of a
standard medical catheter (or other invasive device) to diagnose,
or monitor therapy for, bacteremia or other pathological conditions
that result in nitric oxide production. Such conditions include,
for example, bacteremia, septicemia, auto-immune diseases, stroke,
heart attack, tissue injury, surgical trauma, and vascular
penetration.
The nitric oxide electrode assembly can take any form needed for a
specific application, since metal can be deposited on supporting
substrates using a variety of methods, including
micro-photolithography, sputtering, chemical vapor deposition,
electroplating, screen printing, etc. Thus, the nitric oxide sensor
could be deployed in a linear fashion, as a stripe, band or helix
on a medical catheter. Such a linear or curvilinear nitric oxide
sensor could detect nitric oxide production in the tissues along
the route of catheter penetration, providing early warning of
infection reaching the patient from the catheter itself. A
contaminated catheter, or other medical device, thus detected could
be removed before systemic infection became established.
Alternatively, a non-invasive nitric oxide detector system
constructed according to the present invention includes an
electrode assembly and sample chamber enclosed within, or in
proximity to, the housing of the electrochemical instrument, with
provision for the introduction into the sample chamber of discrete
fluid samples in which nitric oxide activity is to be determined.
Such samples could, for example, be blood, or any other body fluid,
withdrawn from an experimental subject or a clinical or veterinary
patient. Such a detector system, would also be useful for
determining nitric oxide activity in non-biological samples.
While the invention has been described in connection with what is
presently considered to be the most practical and preferred
embodiment, it is to be understood that the invention is not to be
limited to the disclosed embodiment, but on the contrary, is
intended to cover various modifications and equivalent arrangements
included within the spirit and scope of the appended claims.
* * * * *