U.S. patent application number 10/443269 was filed with the patent office on 2003-10-30 for hemoglobin sensor.
Invention is credited to Beck, Thomas, Chatelier, Ron, Hodges, Alastair.
Application Number | 20030201177 10/443269 |
Document ID | / |
Family ID | 28794859 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030201177 |
Kind Code |
A1 |
Hodges, Alastair ; et
al. |
October 30, 2003 |
Hemoglobin sensor
Abstract
The present invention relates to a device and method for
measuring hemoglobin in a fluid sample. The device comprises a
disposable electrochemical cell, such as a thin layer
electrochemical cell, containing a reagent capable of being reduced
by hemoglobin. A suitable fluid sample that may be analyzed
according the present invention is whole blood. If the hemoglobin
to be analyzed is present in red blood cells, a lysing agent may be
added to the sample to release the hemoglobin prior to
analysis.
Inventors: |
Hodges, Alastair; (Blackborn
South, AU) ; Chatelier, Ron; (Bayswater, AU) ;
Beck, Thomas; (North Richard, AU) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
28794859 |
Appl. No.: |
10/443269 |
Filed: |
May 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10443269 |
May 21, 2003 |
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09616512 |
Jul 14, 2000 |
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09616512 |
Jul 14, 2000 |
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09314251 |
May 18, 1999 |
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6174420 |
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09314251 |
May 18, 1999 |
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09068828 |
Mar 15, 1999 |
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6179979 |
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09314251 |
May 18, 1999 |
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08852804 |
May 7, 1997 |
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5942102 |
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Current U.S.
Class: |
204/403.01 ;
204/403.14 |
Current CPC
Class: |
C12Q 1/006 20130101;
G01N 33/84 20130101; C12Q 1/004 20130101 |
Class at
Publication: |
204/403.01 ;
204/403.14 |
International
Class: |
G01N 027/26 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 15, 1996 |
US |
PCTAU9600723 |
Nov 15, 1996 |
PCT/AU96/00724 |
Claims
What is claimed is:
1. A device for measuring an amount of hemoglobin in a sample, the
device comprising an electrochemical cell having a sensing chamber,
a first electrode, a second electrode wherein the second electrode
is a distance of less than about 500 microns from the first
electrode, an aperture for admitting the sample into the sensing
chamber, and a reagent contained within the sensing chamber wherein
the reagent is capable of being reduced by hemoglobin to generate
an electrical signal indicative of the amount of hemoglobin.
2. The device of claim 1, wherein the device may only be used for a
single test.
3. The device of claim 1, wherein the first electrode comprises a
sensing electrode.
4. The device of claim 1, wherein the first electrode comprises a
material selected from the group consisting of platinum, palladium,
carbon, indium oxide, tin oxide, gold, iridium, copper, steel,
silver, and mixtures thereof.
5. The device of claim 1, wherein the first electrode is formed by
a technique selected from the group consisting of sputtering, vapor
coating, screen printing, thermal evaporation, ink jet printing,
ultrasonic spraying, slot coating, gravure printing and
lithography.
6. The device of claim 1, wherein the second electrode comprises a
counter electrode.
7. The device of claim 1, wherein the second electrode comprises a
metal in contact with a metal salt.
8. The device of claim 7, wherein the metal in contact with a metal
salt is selected from the group consisting of silver in contact
with silver chloride, silver in contact with silver bromide, silver
in contact with silver iodide, mercury in contact with mercurous
chloride, and mercury in contact with mercurous sulfate.
9. The device of claim 7, the electrochemical cell further
comprising a third electrode.
10. The device of claim 9, wherein the third electrode comprises a
reference electrode.
11. The device of claim 10, wherein the third electrode comprises a
metal in contact with a metal salt.
12. The device of claim 11, wherein the metal in contact with the
metal salt is selected from the group consisting of silver in
contact with silver chloride, silver in contact with silver
bromide, silver in contact with silver iodide, mercury in contact
with mercurous chloride, and mercury in contact with mercurous
sulfate.
13. The device of claim 1, wherein the second electrode comprises a
reference electrode.
14. The device of claim 1, wherein the reagent is selected from the
group consisting of dichromate, vanadium oxides, permanganate,
electroactive organometallic complexes, quinones, and
dichlorophenolindophenol.
15. The device of claim 1, wherein the reagent comprises
ferricyanide.
16. The device of claim 1, the sensing chamber further comprising a
buffer, wherein the buffer is contained within the sensing
chamber.
17. The device of claim 16, wherein the buffer is selected from the
group consisting of phosphates, carbonates, alkali metal salts of
mellitic acid, and alkali metal salts of citric acid.
18. The device of claim 1, further comprising a red blood cell
lysing agent.
19. The device of claim 18, wherein the lysing agent is selected
from the group consisting of ionic detergents, nonionic detergents,
proteolytic enzymes, and lipases.
20. The device of claim 18, wherein the lysing agent comprises
saponin.
21. The device of claim 18, wherein the lysing agent is selected
from the group consisting of sodium dodecyl sulfate, cetyl
trimethylammonium bromide, and polyethoxylated octylphenol.
22. The device of claim 1, wherein the sample comprises fluid whole
blood.
23. The device of claim 1, the sensing chamber further comprising a
support, the support contained within the sensing chamber.
24. The device of claim 23, wherein the support is a material
selected from the group consisting of mesh, nonwoven sheet, fibrous
filler, macroporous membrane, sintered powder, and combinations
thereof.
25. The device of claim 23, wherein the reagent is contained within
or supported on the support.
26. The device of claim 23, further comprising a buffer, wherein
the buffer is contained within or supported on the support.
27. The device of claim 23, wherein the lysing agent is contained
within or supported on the support.
28. The device of claim 1, wherein the second electrode is mounted
in opposing relationship to the first electrode.
29. The device of claim 1, wherein the second electrode is at a
distance of less than about 150 microns from the first
electrode.
30. The device according to claim 1, wherein the second electrode
is at a distance of from about 50 microns to about 150 microns from
the first electrode.
31. The device of claim 1, further comprising an interface for
communication with a meter.
32. The device of claim 31, wherein the interface communicates a
voltage or a current.
33. The device of claim 1, wherein the electrochemical cell
comprises a thin layer electrochemical cell.
Description
RELATED APPLICATION
[0001] This application is a continuation of application Ser. No.
09/616,512, filed Jul. 14, 2000, which is a continuation-in-part of
application Ser. No. 09/314,251, filed May 18, 1999, now U.S. Pat.
No. 6,174,420. application Ser. No. 09/314,251 is a continuation of
application Ser. No. 08/852,804, filed May 7, 1997, now U.S. Pat.
No. 5,942,102, and a continuation of application Ser. No.
08/068,828, filed Mar. 15, 1999, now U.S. Pat. No. 6,179,979.
application Ser. No. 08/852,804 is the national phase under 35
U.S.C. .sctn.371 of prior PCT International application Ser. No.
PCT/AU96/00723 which has an International filing date of Nov. 15,
1996, which designated the United States of America, and which was
published by the International Bureau in English on May 22, 1997,
and claims the benefit of Australian Provisional Patent application
Ser. No. PN 6619, filed Nov. 16, 1995. application Ser. No.
08/068,828 is the national phase under 35 U.S.C. .sctn.371 of prior
PCT International Application No. PCT/AU96/00724 which has an
International filing date of Nov. 15, 1996, which designated the
United States of America, and which was published by the
International Bureau in English on May 22, 1997, and claims the
benefit of Australian Provisional Patent application Ser. No. PN
6619, filed Nov. 16, 1995. The contents of each patent and
application as recited above to which priority is claimed is hereby
incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a device and method for
measuring the level of hemoglobin in a blood sample. The device
comprises a disposable electrochemical cell containing an agent
which lyses red blood cells and a reagent capable of being reduced
by hemoglobin.
BACKGROUND OF THE INVENTION
[0003] Hemoglobin is a respiratory molecule found in red blood
cells. It is responsible for transporting oxygen from the lungs to
body cells and for transporting carbon dioxide from body cells to
the lungs. Hemoglobin has a molecular weight of 68,000 and contains
four polypeptide chains. Each chain binds to a heme group which
consists of a tetrapyrrole ring chelated to an Fe.sup.+2 ion. hi
the lungs, the iron atoms of the hemoglobin molecule reversibly
combine with an oxygen molecule, which is then transported to body
cells as blood circulates. The oxygen is released from the
hemoglobin molecule in the tissues, then the oxygen-free hemoglobin
molecule picks up carbon dioxide which is transported back to the
lungs, where it is released.
[0004] Hemoglobin is produced in cells in the bone marrow that
become red blood cells. Certain illnesses result in a deficiency of
hemoglobin, such as anemia and sickle cell disease. Still other
diseases, such as polycythemia or erythrocytosis, result in
excessive levels of hemoglobin. Therefore, as an aid in the
diagnosis or monitoring of such diseases, a method and device for
determining the concentration of hemoglobin in whole blood is
desirable.
[0005] Numerous methods and devices for the determination of
hemoglobin are known. These methods include both direct analysis,
i.e., analysis without prior modification of the hemoglobin, and
indirect analysis. An example of a direct analysis method is the
Tallquist Method, wherein a measurement of the transmission or
reflection optical density of the red color imparted by
oxyhemoglobin, one form of hemoglobin, is obtained. An example of
an indirect analysis method is Drabkin's Method. In this method,
the iron in hemoglobin is oxidized with a ferricyanide to form
methemoglobin, which is converted with a cyanide to
cyanmethemoglobin, which is then measured spectrometrically. Both
of these methods have the disadvantage of requiring expensive
analytical instrumentation and complicated sample preparation.
Therefore, a quick, simple, and inexpensive device and method for
measuring hemoglobin that overcomes the deficiencies of prior art
methods is desirable.
SUMMARY OF THE INVENTION
[0006] The present invention provides a device and method for
measuring hemoglobin with a disposable sensing element, suitable
for a single use, that can be combined with a meter to give a
robust, fast, and easy to use test that is amenable to field as
well as laboratory use. In particular, the invention relates to the
use of an electrochemical sensor that utilizes a redox agent that
reacts with hemoglobin to produce an electrochemically detectable
signal. The method of the present invention measures total
hemoglobin, oxygenated plus unoxygenated, in contrast to
spectrophotometric methods wherein the hemoglobin must be converted
to a single form in a separate chemical step, e.g., oxidation of
hemoglobin containing Fe.sup.+2 to methemoglobin containing
Fe.sup.+3. Measurement of hemoglobin by the method of the present
invention is not dependent upon the extent of glycosylation or
oxygenation of the hemoglobin present in the sample.
[0007] In a first aspect of the present invention, a device for
detecting a presence or an absence of hemoglobin in an aqueous
sample is provided, the device including an electrochemical cell
having a sensing chamber, a first electrode, a second electrode, an
aperture for admitting the sample into the sensing chamber, and a
reagent contained within the sensing chamber wherein the reagent is
capable of being reduced by hemoglobin to generate an electrical
signal indicative of the presence or absence of hemoglobin. The
electrochemical cell may be designed to be disposed of after use in
a single experiment.
[0008] In one aspect of this embodiment, first electrode is a
sensing electrode. The sensing electrode may be platinum,
palladium, carbon, indium oxide, tin oxide, gold, iridium, copper,
steel, silver, or mixtures thereof. The first electrode may be
formed by a technique including sputtering, vapor coating, screen
printing, thermal evaporation, ink jet printing, ultrasonic
spraying, slot coating, gravure printing or lithography.
[0009] In another aspect of this embodiment, the second electrode
is a counter electrode. The second electrode may be a metal in
contact with a metal salt, for example, silver in contact with
silver chloride, silver in contact with silver bromide, silver in
contact with silver iodide, mercury in contact with mercurous
chloride, and mercury in contact with mercurous sulfate. The second
electrode may also be a reference electrode.
[0010] In another aspect of this embodiment, the electrochemical
cell further includes a third electrode, which may be a reference
electrode. The third electrode may include a metal in contact with
a metal salt, for example, silver in contact with silver chloride,
silver in contact with silver bromide, silver in contact with
silver iodide, mercury in contact with mercurous chloride, and
mercury in contact with mercurous sulfate.
[0011] In another aspect of this embodiment, the reagent may
include dichromate, vanadium oxides, permanganate, electroactive
organometallic complexes, quinones, dichlorophenolindophenol, and
ferricyanide. A buffer, such as a phosphate, carbonate, alkali
metal salt of mellitic acid, or alkali metal salt of citric acid,
may be contained within the sensing chamber. The sensing chamber
further includes a red blood cell lysing agent, for example, one
selected from ionic detergents, nonionic detergents, proteolytic
enzymes, lipases, saponin, sodium dodecyl sulfate, cetyl
trimethylammonium bromide, or polyethoxylated octylphenol.
[0012] In another aspect of this embodiment, the sample includes
whole blood.
[0013] In another aspect of this embodiment, the sensing chamber
further includes a support contained within the sensing chamber,
for example, mesh, nonwoven sheet, fibrous filler, macroporous
membrane, sintered powder, or combinations thereof. The reagent,
red blood cell lysing agent, and/or buffer may be contained within
or supported on the support.
[0014] In another aspect of this embodiment, the second electrode
is mounted in opposing relationship a distance of less than about
500 microns from the first electrode; less than about 150 microns
from the first electrode; or less than about 150 microns and
greater than about 50 microns from the first electrode.
[0015] In another aspect of this embodiment, the device includes an
interface for communication with a meter. The interface may
communicate a voltage or a current. The electrochemical cell may be
a thin layer electrochemical cell.
[0016] In a second aspect of the present invention, a method for
detecting a presence or an absence of hemoglobin in an aqueous
sample is provided, the method including providing a device
including an electrochemical cell having a sensing chamber, a first
electrode, a second electrode, an aperture for admitting the sample
into the sensing chamber, and a reagent contained within the
sensing chamber, wherein the reagent is capable of being reduced by
hemoglobin to generate an electrical signal indicative of the
presence or absence of hemoglobin; providing an aqueous sample;
allowing the sample to flow through the aperture and into the
sensing chamber, such that the first and second electrodes are
substantially covered; and obtaining an electrochemical measurement
indicative of the presence or absence of hemoglobin present in the
sample.
[0017] In one aspect of this embodiment, the electrochemical cell
is designed to be disposed of after use in a single experiment, or
may be a thin layer electrochemical cell. The electrochemical
measurement may be an amperometric measurement, a potentiometric
measurement, a coulometric measurement, or a quantitative
measurement.
[0018] In a third aspect of the present invention, a method is
provided for measuring hemoglobin in a fluid whole blood sample,
the whole blood sample containing red blood cells, the red blood
cells containing hemoglobin, wherein the method includes providing
a device including an electrochemical cell having a sensing
chamber, a first electrode, a second electrode, an aperture for
admitting the sample into the sensing chamber; a reagent contained
within the sensing chamber and capable of being reduced by
hemoglobin; and a red blood cell lysing agent contained within the
sensing chamber; placing the whole blood sample in the sensing
chamber, whereby the lysing agent contained within the sensing
chamber releases hemoglobin from the red blood cells, whereby the
hemoglobin thus released reduces the reagent; and obtaining an
electrochemical measurement indicative of the level of hemoglobin
present in the whole blood sample.
[0019] In one aspect of this embodiment, the electrochemical cell
is designed to be disposed of after use in a single experiment, or
may be a thin layer electrochemical cell.
[0020] In one aspect of this embodiment, the method further
includes obtaining an electrochemical measurement indicative of the
presence or absence of hemoglobin in the sample by applying a
negative potential to the first electrode; measuring a current
generated by reaction of the reagent and hemoglobin; analyzing the
current to give a result, the result including a time required for
substantial lysis of the red blood cells or a derived final value
for the current; calculating a percentage of the reaction completed
as a function of time based on the result,of the analyzing step;
reversing the potential on the first electrode; measuring a
transient current; and determining a diffusion coefficient and a
concentration of a reduced form of the reagent based on the
transient current.
[0021] In a fourth aspect of the present invention, a method of
manufacture of a device for detecting the presence or absence of
hemoglobin in an aqueous sample is provided, the device including
an electrochemical cell having a sensing chamber, a first
electrode, a second electrode, an aperture for admitting the sample
into the sensing chamber, and a reagent contained within the
sensing chamber, and wherein the reagent is capable of being
reduced by hemoglobin to generate an electrical signal indicative
of the presence or absence of hemoglobin, the method including
forming an aperture extending through a sheet of electrically
resistive material, the aperture defining a side wall of the
sensing chamber; mounting a first layer having a first electrode to
a first side of the sheet and extending over the aperture whereby
to define a first sensing chamber end wall, the first electrode
facing the first side of the sheet; mounting a second layer having
a second electrode to a second side of the sheet and extending over
the aperture whereby to define a second sensing chamber end wall in
substantial overlying registration with the first layer, the second
electrode facing the second side of the sheet, whereby the sheet
and layers form a strip; providing an aperture in the strip to
permit entry of sample into the sensing chamber; and providing a
reagent capable of being reduced by hemoglobin, wherein the reagent
is contained within the sensing chamber.
[0022] In one aspect of this embodiment, the method further
includes the step of providing a vent in the strip to permit the
escape of air displaced from the sensing chamber as sample fills
the sensing chamber.
[0023] In another aspect of this embodiment, the aperture is of a
rectangular cross-section.
[0024] In another aspect of this embodiment, at least one of the
electrodes includes a noble metal, such as palladium, platinum, and
silver. At least one of the electrodes may be a sputter coated
metal deposit. The electrodes may be adhered to the sheet, for
example, by an adhesive such as a heat activated adhesive, pressure
sensitive adhesive, heat cured adhesive, chemically cured adhesive,
hot melt adhesive, and hot flow adhesive.
[0025] In another aspect of this embodiment, the method may include
the step of providing a buffer and/or a red blood cell lysing agent
contained within the sensing chamber. The reagent and/or buffer may
be printed onto at least one wall of the sensing chamber. A support
contained within the sensing chamber may also be provided, such as
mesh, fibrous filler, macroporous membrane, sintered powder, or
combinations thereof. The reagent may be supported on or contained
within the support.
[0026] In another aspect of this embodiment, at least the sheet or
one of the layers includes a polymeric material selected from
polyester, polystyrene, polycarbonate, polyolefin, and mixtures
thereof, or polyethylene terephthalate.
[0027] In another aspect of this embodiment, the second electrode
is mounted in opposing relationship a distance of less than about
500 microns from the first electrode; less than about 150 microns
from the first electrode; or less than about 150 microns and
greater than about 50 microns from the first electrode.
[0028] In another aspect of this embodiment, the electrochemical
cell is designed to be disposed of after use in a single
experiment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows a plan view of an electrochemical cell.
[0030] FIG. 2 shows a cross-section view on line 10-10 of FIG.
1.
[0031] FIG. 3 shows an end-section view on line 11-11 of FIG.
1.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0032] The following description and examples illustrate a
preferred embodiment of the present invention in detail. Those of
skill in the art will recognize that there are numerous variations
and modifications of this invention that are encompassed by its
scope. Accordingly, the description of a preferred embodiment
should not be deemed to limit the scope of the present
invention.
[0033] Other methods and devices for obtaining electrochemical
measurements of fluid samples are discussed further in copending
U.S. patent application Ser. No. 09/616,433, filed on Jul. 14,
2000, entitled "IMMUNOSENSOR," copending U.S. patent application
Ser. No. 09/615,691, filed on Jul. 14, 2000, entitled "ANTIOXIDANT
SENSOR," copending U.S. patent application Ser. No. 09/616,556,
filed on Jul. 14, 2000, entitled "ELECTROCHEMICAL METHOD FOR
MEASURING CHEMICAL REACTION RATES," each of which is incorporated
herein by reference in its entirety.
[0034] The Sample
[0035] In preferred embodiments, a method and device for measuring
hemoglobin levels in a fluid whole blood sample is provided. If the
whole blood sample is not in liquid form, i.e., dried blood, it can
be analyzed after solid sample is mixed into a suitable fluid,
e.g., water. Whole blood contained within a solid tissue sample may
be analyzed after extraction using techniques well-known in the
art.
[0036] Prior to its analysis, hemoglobin must be released from the
red blood cells in which it is contained. This may be accomplished
by pretreating the whole blood sample with a lysing agent prior to
its introduction into the electrochemical cell. Alternatively, a
lysing agent may be contained within the electrochemical cell
itself. Other agents may also be used to pretreat the sample. For
example, pH may be adjusted to a desired level by means of a buffer
or neutralizing agent, or a substance that renders interfering
species nonreactive may be added.
[0037] The Electrochemical Cell
[0038] The electrochemical cell of the present invention is
disposable and designed for use in a single experiment. In a
preferred embodiment, the electrochemical cell is a thin layer
sensor such as that disclosed in U.S. Pat. No. 5,942,102
(incorporated herein by reference in its entirety). As herein used,
the term "thin layer electrochemical cell" refers to a cell having
closely spaced electrodes such that reaction products from the
counter electrode arrive at the working electrode. In practice, the
separation of electrodes in such a cell for measuring glucose in
blood will be less than 500 microns, and preferably less than 200
microns. A preferred embodiment of such an electrochemical cell is
illustrated in FIGS. 1, 2, and 3. The cell illustrated in FIGS. 1,
2, and 3 includes a polyester core 4 having a circular aperture 8.
Aperture 8 defines a cylindrical cell side wall 12. Adhered to one
side of core 4 is a polyester sheet 1 having a sputter coating of
palladium 2. The sheet is adhered by means of an adhesive 3 to core
4 with palladium 2 adjacent core 4 and covering aperture 8. A
second polyester sheet 7 having a second sputter coating of
palladium 6 is adhered by means of contact adhesive 5 to the other
side of core 4 and covering aperture 8. There is thereby defined a
cell having cylindrical side wall 12 closed on each end by
palladium metal 2, 6. The assembly is notched at 9 to provide for a
solution to be admitted to the cell or to be drawn in by wicking or
capillary action and to allow air to escape. The metal films 2, 6
are connected with suitable electrical connections or formations
whereby potentials may be applied and currently measured.
[0039] Such a thin layer electrochemical cell is prepared by first
forming an aperture extending through a sheet of electrically
resistive material, the aperture defining a side wall of the
electrochemical cell. Suitable electrically resistive materials,
which may be used in the sheet containing the aperture, or in other
layers in the cell, include, for example, materials such as
polyesters, polystyrenes, polycarbonates, polyolefins, polyethylene
terephthalate, mixtures thereof, and the like. In a preferred
embodiment, the aperture in the sheet is rectangular, however other
shapes, e.g., circular, may be used as well.
[0040] After the aperture is formed, a first thin electrode layer
is then mounted on one side of the sheet of electrically resistive
material, extending over the aperture and forming an end wall. The
layer may be adhered to the sheet, for example, by means of an
adhesive. Suitable adhesives include, for example, heat activated
adhesives, pressure sensitive adhesives, heat cured adhesives,
chemically cured adhesives, hot melt adhesives, hot flow adhesives,
and the like. The electrode layer is prepared by coating (e.g., by
sputter coating) a sheet of electrically resistive material with a
suitable metal, for example, palladium.
[0041] A second thin electrode layer is then mounted on the
opposite side of the electrically resistive material, also
extending over the aperture, so as to form a second end wall. In a
preferred embodiment, the electrode layers are mounted in opposing
relationship at a distance of less than about 1 millimeter,
desirably less than about 800 microns, more desirably less that
about 600, or preferably less than about 500 microns, more
preferably less than about 300 to 150 microns, more preferably less
than 150 microns, and most preferably between 25, 40, 50, 100 and
150 microns. A second aperture or ingress is then provided for
liquid to enter the cell. Such an ingress can be provided by
forming a notch along one edge of the device which extends through
the electrode layers and aperture. The electrode layers are
provided with connection means allowing the sensors to be placed in
a measuring circuit.
[0042] Chemicals for use in the cell, such as redox reagents,
lysing agents, buffers, and other substances, may be supported on
the cell electrodes or walls, on one or more independent supports
contained within cell, or may be self supporting. If the chemicals
are to be supported on the cell electrodes or walls, the chemicals
may be applied by use of application techniques well known in the
art, such as ink jet printing, screen printing, lithography,
ultrasonic spraying, slot coating, gravure printing, and the like.
Suitable independent supports may include, but are not limited to,
meshes, nonwoven sheets, fibrous fillers, macroporous membranes,
and sintered powders. The chemicals for use in the cell may be
supported on or contained within a support.
[0043] In a preferred embodiment, the materials used within the
cell as well as the materials used to construct the cell are in a
form amenable to mass production, and the cells themselves are
designed to be able to be used for a single experiment then
disposed of.
[0044] According to the present invention a disposable cell is one
that is inexpensive enough to produce that it is economically
acceptable to be used only for a single test. Secondly, that the
cell may conveniently only be used for a single test.
Inconveniently in this context means that steps such as washing
and/or reloading of reagents would need to be taken to process the
cell after a single use to render it suitable for a subsequent
use.
[0045] Economically acceptable in this context means that the
perceived value of the result of the test to the user is the same
or greater than the cost of the cell to purchase and use, the cell
purchase price being set by the cost of supplying the cell to the
user plus an appropriate mark up. For many applications, this
requires that the cells have relatively low materials costs and
simple fabrication processes. For example, the electrode materials
of the cells should be inexpensive, such as carbon, or be used in
sufficiently small amounts such that expensive materials may be
used. Screen printing carbon or silver ink is a process suitable
for forming electrodes with relatively inexpensive materials.
However, if it is desired to use electrode materials such as
platinum, palladium, gold or iridium, methods with better material
utilization, such as sputtering or evaporative vapor coating, are
more suitable as they may give extremely thin films. The substrate
materials for the disposable cells also need to be inexpensive.
Examples of such inexpensive materials are polymers such as
polyvinylchloride, polyimide, polyester and coated papers and
cardboard.
[0046] Cell assembly methods also need to be amenable to mass
production. These methods include fabricating multiple cells on
cards and separating the card into individual strips subsequent to
the main assembly steps, and web fabrication where the cells are
produced on a continuous web, which is subsequently separated into
individual strips. Card processes are most suitable when close
spatial registration of multiple features is required for the
fabrication and/or when stiff cell substrate materials are to be
used. Web processes are most suitable when the down web
registration of features is not as critical and flexible webs may
be used.
[0047] The convenient single use requirement for the disposable
cell is desirable so that users are not tempted to try to reuse the
cell and possibly obtain an inaccurate test result. The single use
requirement for the cell may be stated in user instructions
accompanying the cell. More preferably, the cell may also be
fabricated such that using the cell more than once is difficult or
not possible. This may be accomplished, for example, by including
reagents that are washed away or consumed during the first test and
so are not functional in a second test. Alternatively, the signal
of the test may be examined for indications that reagents in the
cell have already reacted, such as an abnormally high initial
signal, and the test aborted. Another method includes providing a
means for breaking electrical connections in the cell after the
first test in a cell has been completed.
[0048] The Electrodes
[0049] At least one of the electrodes in the cell is a sensing
electrode, defined as an electrode sensitive to the amount of
oxidized redox agent. In the case of a potentiometric sensor
wherein the potential of the sensing electrode is indicative of the
level of hemoglobin present, a second electrode acting as reference
electrode is present which acts to provide a reference
potential.
[0050] In the case of an amperometric sensor wherein the sensing
electrode current is indicative of the level of hemoglobin in the
sample, at least one other electrode is present which functions as
a counter electrode to complete the electrical circuit. This second
electrode may also function as a reference electrode.
Alternatively, a separate electrode may perform the function of a
reference electrode.
[0051] Materials suitable for the sensing, counter, and reference
electrodes must be compatible with the redox reagents present in
the device. Compatible materials will not react chemically with the
redox reagent or any other substance present in the cell. Examples
of such suitable materials include, but are not limited to,
platinum, palladium, carbon, indium oxide, tin oxide, mixed
indium/tin oxides, gold, silver, iridium and mixtures thereof.
These materials may be formed into electrode structures by any
suitable method, for example, by sputtering, vapor coating, screen
printing, thermal evaporation or lithography. In preferred
embodiments, the material is sputtered or screen printed to form
the electrode structures.
[0052] Non-limiting examples of materials suitable for use in the
reference electrode include metal/metal salt systems such as silver
in contact with silver chloride, silver bromide or silver iodide,
and mercury in contact mercurous chloride or mercurous sulfate. The
metal may be deposited by any suitable method and then brought into
contact with the appropriate metal salt. Suitable methods include,
for example, electrolysis in a suitable salt solution or chemical
oxidation. Such metal/metal salt systems provide better potential
control in potentiometric measurement methods than do single metal
component systems. In a preferred embodiment, the metal/metal salt
electrode systems are used as a separate reference electrode in an
amperometric sensor.
[0053] The Lysing Agent
[0054] Suitable red blood cell lysing agents include detergents,
both ionic and non-ionic, proteolytic enzymes, and lipases.
Suitable ionic detergents include, for example, sodium dodecyl
sulfate and cetyl trimethylammonium bromide. Non-limiting examples
of proteolytic enzymes include trypsin, chymotrypsin, pepsin,
papain, and Pronase E, a very active enzyme having broad
specificity. Nonionic surfactants suitable for use include, e.g.,
ethoxylated octylphenols, including the Triton X Series available
from Rohm & Haas of Philadelphia, Pa. In a preferred
embodiment, saponins, i.e., plant glycosides that foam in water,
are used as the lysing agent.
[0055] The Redox Reagent
[0056] Suitable redox reagents include those which are capable of
oxidizing hemoglobin. Examples of redox reagents suitable for use
in analyzing hemoglobin include, but are not limited, to salts of
ferricyanide, dichromate, vanadium oxides, permanganate, and
electroactive organometallic complexes. Organic redox reagents such
as dichlorophenolindophenol, and quinones are also suitable. In a
preferred embodiment, the redox reagent for analyzing hemoglobin is
ferricyanide.
[0057] The Buffer
[0058] Optionally, a buffer may be present along with the redox
reagent in dried form in the electrochemical cell. If a buffer is
used, it is present in an amount such that the resulting pH level
is suitable for adjusting the oxidizing potential of the redox
reagent to a level suitable for oxidizing hemoglobin but not other
species that it is not desired to detect. The buffer is present in
a sufficient amount so as to substantially maintain the pH of the
sample at the desired level during the test. Examples of buffers
suitable for use include phosphates, carbonates, alkali metal salts
of mellitic acid, and alkali metal salts of citric acid. The choice
of buffer will depend on the desired pH. The buffer is selected so
as not to react with the redox reagent.
[0059] Other Substances Present Within the Cell
[0060] In addition to redox reagents and buffers, other substances
may also be present within the electrochemical cell. Such
substances include, for example, viscosity enhancers and low
molecular weight polymers. Hydrophilic substances may also be
contained within the cell, such as polyethylene glycol, polyacrylic
acid, dextran, and surfactants such as those marketed by Rohm &
Haas Company of Philadelphia, Pa., under the trade name Triton.TM.
or by ICI Americas Inc. of Wilmington, Del., under the trade name
Tween.TM.. Such substances may enhance the fill rate of the cell,
provide a more stable measurement, and inhibit evaporation in small
volume samples.
[0061] Method for Measuring Hemoglobin Concentration
[0062] In measuring hemoglobin present in a whole blood sample, the
sample is introduced into the sensor cell, whereupon the sample
dissolves the dried reagents and other substances present in the
sensor cell. If the sample has not been pretreated with a lysing
agent, a lysing agent present in the sensor cell releases
hemoglobin from the red blood cells. The redox reagent then reacts
with hemoglobin present in the sample to form the reduced form of
the redox reagent. In the case of a potentiometric sensor, the
resulting ratio of oxidized to reduced form of the redox reagent
fixes the potential of the sensing electrode relative to the
reference electrode. This potential is then used as a measure of
the concentration of the hemoglobin originally in the sample.
[0063] In a preferred embodiment, the sensing cell is operated as
an amperometric sensor. According to this embodiment, the reduced
redox reagent formed by reaction with hemoglobin is
electrochemically oxidized at the sensing electrode. The current
resulting from this electrochemical reaction is then used to
measure the concentration of hemoglobin originally in the sample.
In other embodiments, the sensor is operated in potentiometric or
coulometric mode.
[0064] The cell's electrodes are used to produce an electrical
signal, i.e., a voltage or current, readable by an attached meter.
In a preferred embodiment, an interface for connecting the cell to
the meter is provided. The meter may display the measurement in a
visual, audio or other form, or may store the measurement in
electronic form.
[0065] In a preferred embodiment where ferricyanide is used as the
reagent, a typical concentration of hemoglobin, e.g., 14 g/dL,
would yield a ferrocyanide concentration of 8.2 mM upon oxidation.
The minimum detectable limit in a preferred embodiment of the
method and device is approximately 0.1 mM.
[0066] Measuring Hemoglobin Using First and Second Potential
Application Steps
[0067] In a preferred embodiment, substantially complete lysis of
all red blood cells is achieved before measurement of hemoglobin.
However, a rapid hemoglobin measurement may be obtained even if the
lysis step proceeds slowly by using a first and second potential
application step.
[0068] In such an embodiment, a sensor configuration as described
above is used, e.g., a sensor wherein the bottom electrode is
palladium, upon which the dried reagents are deposited, and wherein
the top electrode is a gold electrode. The first potential is
applied by applying a -0.3 V potential at zero time to set the gold
electrode as the working electrode. As the red blood cells begin to
lyse and the ferricyanide reacts with released hemoglobin, the
current will slowly increase. The increase in current can be used
in a number of ways: to assess the time required for full lysis and
reaction, to extrapolate the signal to longer times, or to assess
the fraction of hemoglobin reacted at a given time. The first
method gives some quality assurance, and the second and third
methods yield a shorter test.
[0069] After the first potential has been applied and current has
been measured, the potential can be reversed to +0.3 V and the
reverse transient current can be measured and analyzed using
electrochemical methods known in the art, e.g., as disclosed in
U.S. application Ser. No. 08/981385 filed Apr. 17, 1998, and U.S.
Pat. No. 5,942,102 (both incorporated herein by reference in their
entirety), to calculate the diffusion coefficient and concentration
of ferrocyanide.
[0070] The first potential application may also be used to subtract
interferents. The ferricyanide reacts more quickly with interfering
substances which are free in the plasma than with hemoglobin which
is packaged in red blood cells. The ratio of the minimum to maximum
(or extrapolated) currents resulting from the first potential
application may be used to yield the concentration of reduced
mediator measured by the second potential pulse (at +0.3 V),
thereby yielding a more accurate, corrected concentration of
hemoglobin. The simplest correction would be:
[Hb]'=[Hb]*(1-imin/imax)
[0071] wherein [Hb]' is the corrected concentration of hemoglobin,
[Hb] is the uncorrected concentration of hemoglobin, imin is the
measured current and imax is the extrapolated current resulting
from the first potential application.
[0072] The above description discloses several methods and
materials of the present invention. This invention is susceptible
to modifications in the methods and materials, as well as
alterations in the fabrication methods and equipment. Such
modifications will become apparent to those skilled in the art from
a consideration of this disclosure or practice of the invention
disclosed herein. Consequently, it is not intended that this
invention be limited to the specific embodiments disclosed herein,
but that it cover all modifications and alternatives coming within
the true scope and spirit of the invention as embodied in the
attached claims.
* * * * *