U.S. patent application number 10/866477 was filed with the patent office on 2004-11-11 for small volume in vitro analyte sensor and methods.
This patent application is currently assigned to TheraSense, Inc.. Invention is credited to Cloud, Michael S., Colman, Fredric C., Feldman, Benjamin J., Funderburk, Jeffery V., Jin, Robert Y., Krishnan, Rajesh, Liamos, Charles T., Plante, Phillip John, Vivolo, Joseph A..
Application Number | 20040225230 10/866477 |
Document ID | / |
Family ID | 23722510 |
Filed Date | 2004-11-11 |
United States Patent
Application |
20040225230 |
Kind Code |
A1 |
Liamos, Charles T. ; et
al. |
November 11, 2004 |
Small volume in vitro analyte sensor and methods
Abstract
A small volume sensor, and methods of making, for determining
the concentration of an analyte, such as glucose or lactate, in a
biological fluid such as blood or serum, using techniques such as
coulometry, amperometry, and potentiometry. The sensor includes a
working electrode and a counter electrode, and can include an
insertion monitoring trace to determine correct positioning of the
sensor in a connector. In one embodiment, the sensor determines the
concentration of the analyte by discharging an amount of charge
into the sample, determining the time needed to discharge the
charge, and determining the current used to electrolyze a portion
of the analyte using the amount of charge and the amount of
time.
Inventors: |
Liamos, Charles T.;
(Pleasanton, CA) ; Feldman, Benjamin J.; (Oakland,
CA) ; Funderburk, Jeffery V.; (Fremont, CA) ;
Krishnan, Rajesh; (Fremont, CA) ; Plante, Phillip
John; (San Francisco, CA) ; Vivolo, Joseph A.;
(San Francisco, CA) ; Jin, Robert Y.; (Castro
Valley, CA) ; Cloud, Michael S.; (Alameda, CA)
; Colman, Fredric C.; (Oakland, CA) |
Correspondence
Address: |
Attn: Mara E. Liepa
MERCHANT & GOULD P.C.
P.O. Box 2903
Minneapolis
MN
55402-0903
US
|
Assignee: |
TheraSense, Inc.
Alameda
CA
|
Family ID: |
23722510 |
Appl. No.: |
10/866477 |
Filed: |
June 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10866477 |
Jun 12, 2004 |
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10033575 |
Dec 28, 2001 |
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6749740 |
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10033575 |
Dec 28, 2001 |
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09434026 |
Nov 4, 1999 |
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6616819 |
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Current U.S.
Class: |
600/583 ;
606/181 |
Current CPC
Class: |
Y10S 439/909 20130101;
G01N 27/3272 20130101 |
Class at
Publication: |
600/583 ;
606/181 |
International
Class: |
A61B 005/00 |
Claims
We claim:
1. A sample acquisition apparatus for piercing skin, the apparatus
comprising a skin piercing lancet, a cocking mechanism operable
connected to the skin piercing lancet, the cocking mechanism
configured to move the lancet from an inactivated position to a
cocked position; a triggering mechanism operably connected to the
spin piercing lancet, the trigger mechanism configured to release
the lancet from the cocked position to a protruding position; and a
cap having a protruding ring with a surface area, the protruding
ring defining a center for passing the lancet therethrough, the
surface area configured for contact with the skin.
2. The sample acquisition apparatus according to claim 1 further
comprising a depth adjustment mechanism for adjusting the depth of
the penetration of the lancet into the skin when in the protruding
position.
3. The sample acquisition apparatus according to claim 1 wherein
the cocking mechanism requires active cocking.
4. The sample acquisition apparatus according to claim 1 wherein
the surface area is an annular surface area.
5. A method of lancing a patient's skin, the method comprising: (a)
providing a sample acquisition apparatus comprising a skin piercing
lancet and a cap having a protruding ring with a surface area, the
protruding ring defining a center for passing the lancet
therethrough, the surface area configured for contact with the
skin. (b) cocking the lancet with a cocking mechanism, thereby
moving the lancet from an inactivated position to a cocked
position; (c) after cocking the lancet, contacting the skin with
the protruding ring; and (d) triggering the lancet with a
triggering mechanism, thereby releasing the lancet from the cocked
position to a protruding position and piercing the skin with the
lancet.
6. The method according to claim 5, further comprising: (a)
controlling the depth of the lancet in the protruding position by
adjusting a depth adjustment mechanism.
7. The method according to claim 5 wherein the step of contacting
the skin with the protruding ring comprises: (a) increasing the
tension on the skin.
8. The method according to claim 7 wherein the step of increasing
the tension on the skin comprises: (a) increasing the tension on
the skin to form a bump.
9. The method according to claim 7 wherein the step of increasing
the tension on the skin comprises: (a) increasing the tension on
the skin to extend the skin.
10. The method according to claim 5, wherein the method comprises:
(a) lancing the skin on a patient's arm or thigh.
11. The method according to claim 5, wherein after piercing the
skin with the lancet, the method comprises: (a) obtaining a blood
sample having a volume no more than about 1 .mu.L.
12. The method according to claim 11, wherein the step of obtaining
a blood sample comprises: (a) obtaining a blood sample having a
volume no more than about 0.5 .mu.L.
13. The method according to claim 11, wherein the step of obtaining
a blood sample comprises: (a) obtaining a blood sample having a
volume no more than about 0.25 .mu.L.
Description
[0001] This application is a continuation of U.S. Ser. No.
10/033,575, filed Dec. 28, 2001, issued as U.S. Pat. No. 6,749,740,
which is a continuation of U.S. Ser. No. 09/434,026, filed Nov. 4,
1999, issued as U.S. Pat. No. 6,616,819, the entire disclosures of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to analytical sensors for the
detection of bioanalytes in a small volume sample, and methods of
making and using the sensors.
BACKGROUND
[0003] Analytical sensors are useful in chemistry and medicine to
determine the presence and concentration of a biological analyte.
Such sensors are needed, for example, to monitor glucose in
diabetic patients and lactate during critical care events.
[0004] Currently available technology measures bioanalytes in
relatively large sample volumes, e.g., generally requiring 3
microliters or more of blood or other biological fluid. These fluid
samples are obtained from a patient, for example, using a needle
and syringe, or by lancing a portion of the skin such as the
fingertip and "milking" the area to obtain a useful sample volume.
These procedures are inconvenient for the patient, and often
painful, particularly when frequent samples are required. Less
painful methods for obtaining a sample are known such as lancing
the arm or thigh, which have a lower nerve ending density. However,
lancing the body in the preferred regions typically produces
submicroliter samples of blood, because these regions are not
heavily supplied with near-surface capillary vessels.
[0005] It would therefore be desirable and very useful to develop a
relatively painless, easy to use blood analyte sensor, capable of
performing an accurate and sensitive analysis of the concentration
of analytes in a small volume of sample.
[0006] It would also be desirable to develop methods for
manufacturing small volume electrochemical sensors capable of
decreasing the errors that arise from the size of the sensor and
the sample.
SUMMARY OF THE INVENTION
[0007] The sensors of the present invention provide a method for
the detection and quantification of an analyte in submicroliter
samples. In general, the invention includes a method and sensor for
analysis of an analyte in a small volume of sample by, for example,
coulometry, amperometry and/or potentiometry. A sensor of the
invention preferably utilizes a non-leachable or diffusible redox
mediator. The sensor also includes a sample chamber to hold the
sample in electrolytic contact with the working electrode. In many
instances, the sensor also contains a non-leachable or diffusible
second electron transfer agent.
[0008] In a preferred embodiment, the working electrode faces a
counter electrode, forming a measurement zone within the sample
chamber, between the two electrodes, that is sized to contain no
more than about 1 .mu.L of sample, preferably no more than about
0.5 .mu.L, more preferably no more than about 0.32 .mu.L, still
more preferably no more than about 0.25 .mu.L, and most preferably
no more than about 0.1 .mu.L of sample.
[0009] In one embodiment of the invention, a sensor, configured for
insertion into an electronic meter, is provided with a working
electrode and a counter electrode, and a conductive insertion
monitor which provides electrical contact with the electronic meter
if the sensor is properly inserted into the meter. The conductive
insertion monitor is configured and arranged to close an electrical
circuit when the sensor is properly inserted into the electronic
connector.
[0010] In another embodiment of the invention, a sensor is provided
with a plurality of contacts, each contact having a contact pad,
which is a region for connection with an electronic meter. The
plurality of contacts and contact pads are disposed on a substrate
having a length and a width, and each contact pad has a contact pad
width taken parallel to the width of the substrate. The sum of the
contact pad widths is greater than the width of the substrate. In a
preferred embodiment, six electrical connections are made with six
contact pads on the sensor but in a width that is approximately the
width of four contact pads. For example, a working electrode, three
counter electrodes (e.g., one counter electrode and two indicator
electrodes), and two insertion trace connections each have a
contact pad; connection can be made to each of these six contact
pads in the same width of the contact pads of the working electrode
and three counter electrodes.
[0011] The present invention also includes an electrical connector,
for providing electrical contact between a sensor and an electrical
meter or other device. The electrical connector has a plurality of
contact structures, each which has a proximal contact end for
electrical connection to a sensor contact, and a distal end for
electrical connection to the electrical device. In one embodiment,
a plurality of first contact structures extend longitudinally
parallel from the distal to the proximal end. Additionally, one or
more second contract structures extend longitudinally next to the
first contact structures, from the distal end past the proximal end
of the first contact structures, and angle toward a longitudinal
center line of the connector. Contact to the sensor is then made
via the proximal contact ends.
[0012] Preferably, the electrical connector has at least two second
contact structures extending longitudinally past the proximal end
of the first contact structures and angling toward the longitudinal
center line of the connector. After the angled or bent portion, the
proximal contact ends of the second contact structures of one
embodiment make electrical contact with a single conductive surface
of a sensor, such as a conductive insertion monitor. In another
aspect, the first contact structures can be configured and arranged
to contact one or more working and/or counter electrodes of a
sensor, and the second contact structures are configured and
arranged to contact one or more conductive insertion monitors.
[0013] The sensors of the present invention can be configured for
side-filling or tip-filling. In addition, in some embodiments, the
sensor may be part of an integrated sample acquisition and analyte
measurement device. The integrated sample acquisition and analyte
measurement device can include the sensor and a skin piercing
member, so that the device can be used to pierce the skin of a user
to cause flow of a fluid sample, such as blood, that can then be
collected by the sensor. In at least some embodiments, the fluid
sample can be collected without moving the integrated sample
acquisition and analyte measurement device.
[0014] In one embodiment, the sensor is connected with an
electrical device, to provide a processor coupled to the sensor.
The processor is configured and arranged to determine, during
electrolysis of a sample in the sample chamber, a series of current
values. The processor determines a peak current value from the
series of current values. After the current values decrease below a
threshold fraction of the peak current values, slope values are
determined from the current values and represent a linear function
of the logarithm of current values over time. The processor
determines, from the slope values, an extrapolation slope. From the
extrapolated slope and the measured current values, the processor
determines an amount of charge needed to electrolyze the sample
and, from that amount of charge, the concentration of the analyte
in the sample.
[0015] One method of forming a sensor, as described above, includes
forming at least one working electrode on a first substrate and
forming at least one counter or counter/reference electrode on a
second substrate. A spacer layer is disposed on either the first or
second substrates. The spacer layer defines a chamber into which a
sample can be drawn and held when the sensor is completed. A redox
mediator and/or second electron transfer agent can be disposed on
the first or second substrate in a region that will be exposed
within the sample chamber when the sensor is completed. The first
and second substrates are then brought together and spaced apart by
the spacer layer with the sample chamber providing access to the at
least one working electrode and the at least one counter or
counter/reference electrode. In some embodiments, the first and
second substrates are portions of a single sheet or continuous web
of material. The invention includes particularly efficient and
reliable methods for the manufacture of these sensors.
[0016] One such efficient and reliable method includes providing an
adhesive having first and second surfaces covered with first and
second release liners and then making detailed cuts through the
first release liner and the adhesive but not through the second
release liner. These cuts define one or more sample chamber
regions. A portion of the first release liner is removed to expose
a portion of the first adhesive surface, which leaves a remaining
portion of the first release liner over the sample chamber regions.
This exposed first adhesive surface is applied to a first substrate
having one or more conductive traces disposed thereon. The second
release liner is removed together with the adhesive and the first
release liner of the sample chamber regions in order to expose the
second adhesive surface. The second adhesive surface is then
applied to a second substrate having one or more conductive traces
disposed thereon. This method forms a sensor having a sample
chamber corresponding to one of the sample chamber regions.
[0017] These and various other features which characterize the
invention are pointed out with particularity in the attached
claims. For a better understanding of the invention, its
advantages, and objectives obtained by its use, reference should be
made to the drawings and to the accompanying description, in which
there is illustrated and described preferred embodiments of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Referring now to the drawings, wherein like reference
numerals and letters indicate corresponding structure throughout
the several views:
[0019] FIG. 1 is a schematic view of a first embodiment of an
electrochemical sensor in accordance with the present invention
having a working electrode and a counter electrode facing each
other and having a sample chamber;
[0020] FIG. 2 is an exploded view of the sensor shown in FIG. 1,
the layers illustrated individually;
[0021] FIG. 3 is a schematic view of a second embodiment of an
electrochemical sensor in accordance with the principles of the
present invention having a working electrode and a counter
electrode facing each other and having an extended sample
chamber;
[0022] FIG. 4 is a top view of a third embodiment of an
electrochemical sensor in accordance with the principles of the
present invention, this sensor includes multiple working
electrodes;
[0023] FIG. 5A illustrates a top view of a first substrate with a
working electrode for use in a fourth embodiment of a sensor
according to the invention;
[0024] FIG. 5B illustrates a bottom view of a second substrate
(inverted with respect to FIG. 5A) with counter electrodes
placement over and opposite the substrate of FIG. 5A;
[0025] FIG. 5C illustrates a top view of a spacer for placement
between the first substrate of FIG. 5A and the second substrate of
FIG. 5B;
[0026] FIG. 6A illustrates a top view of a first film with a
working electrode for use in a fifth embodiment of a sensor
according to the invention;
[0027] FIG. 6B illustrates a top view of a spacer for placement on
the first film of FIG. 6A;
[0028] FIG. 6C illustrates a bottom view of a second film (inverted
with respect to FIGS. 6A and 6B) with counter electrodes placement
over the spacer of FIG. 6B and first film of FIG. 6A;
[0029] FIG. 7A illustrates a top view of a first film with a
working electrode for use in a sixth embodiment of a sensor
according to the invention;
[0030] FIG. 7B illustrates a top view of a spacer for placement on
the first film of FIG. 7A;
[0031] FIG. 7C illustrates a bottom view of a second film (inverted
with respect to FIGS. 7A and 7B) with counter electrodes placement
over the spacer of FIG. 7B and first film of FIG. 7A;
[0032] FIG. 8A illustrates a top view of a first film with a
working electrode for use in a seventh embodiment of a sensor
according to the invention;
[0033] FIG. 8B illustrates a top view of a spacer for placement on
the first film of FIG. 8A;
[0034] FIG. 8C illustrates a bottom view of a second film (inverted
with respect to FIGS. 8A and 8B) with counter electrodes placement
over the spacer of FIG. 8B and first film of FIG. 8A;
[0035] FIG. 9A illustrates a top view of a first film with a
working electrode for use in a eighth embodiment of a sensor
according to the invention;
[0036] FIG. 9B illustrates a top view of a spacer for placement on
the first film of FIG. 9A;
[0037] FIG. 9C illustrates a bottom view of a second film (inverted
with respect to FIGS. 9A and 9B) with counter electrodes placement
over the spacer of FIG. 9B and first film of FIG. 9A;
[0038] FIG. 10A illustrates a top view of a first film with a
working electrode for use in a ninth embodiment of a sensor
according to the invention;
[0039] FIG. 10B illustrates a top view of a spacer for placement on
the first film of FIG. 10A;
[0040] FIG. 10C illustrates a bottom view of a second film
(inverted with respect to FIGS. 10A and 10B) with counter
electrodes placement over the spacer of FIG. 10B and first film of
FIG. 10A;
[0041] FIG. 11A illustrates a top view of a first film with a
working electrode for use in a tenth embodiment of a sensor
according to the invention;
[0042] FIG. 11B illustrates a top view of a spacer for placement on
the first film of FIG. 11A;
[0043] FIG. 11C illustrates a bottom view of a second film
(inverted with respect to FIGS. 11A and 11B) with counter
electrodes placement over the spacer of FIG. 11B and first film of
FIG. 11A;
[0044] FIG. 12A illustrates a top view of a first film with a
working electrode for use in a eleventh embodiment of a sensor
according to the invention;
[0045] FIG. 12B illustrates a top view of a spacer for placement on
the first film of FIG. 12A;
[0046] FIG. 12C illustrates a bottom view of a second film
(inverted with respect to FIGS. 12A and 12B) with counter
electrodes placement over the spacer of FIG. 12B and first film of
FIG. 12A;
[0047] FIG. 13A illustrates a top view of one embodiment of a sheet
of sensor components, according to the invention;
[0048] FIG. 13B illustrates a top view of another embodiment of a
sheet of sensor components, according to the invention;
[0049] FIGS. 14A through 14F illustrate cross sectional views of a
sequential process of providing a sample chamber in a spacer
layer;
[0050] FIG. 14G is a bottom view of FIG. 14B;
[0051] FIG. 15A is a graphical representation of the current over
time used to calculate the concentration of an analyte;
[0052] FIG. 15B is a graphical representation of the log of current
over time used to calculate the concentration of an analyte;
[0053] FIG. 16A is a top perspective view of a sensor positioned
for insertion within an electrical connector device in accordance
with the present invention;
[0054] FIG. 16B is an exploded view of the electrical connector
device of FIG. 16A;
[0055] FIG. 17A is a top perspective view of a sensor fully
positioned within the electrical connector device of FIG. 16A;
[0056] FIG. 17B is an exploded view of the electrical connector
device of FIG. 17A;
[0057] FIG. 18A is a bottom perspective view of the electrical
connector device of FIGS. 16A and 16B;
[0058] FIG. 18B is a bottom perspective view of the electrical
connector device of FIGS. 17A and 17B;
[0059] FIGS. 19A through 19L illustrate examples of suitable
conductive pathways between contact pads;
[0060] FIG. 20 illustrates a cross-sectional view looking from
inside a connector to a sensor of the invention disposed within the
connector; and
[0061] FIG. 21 illustrates a perspective view of a ring for use
with a lancet device.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0062] As used herein, the following definitions define the stated
term:
[0063] An "air-oxidizable mediator" is a redox mediator that is
oxidized by air, preferably so that at least 90% of the mediator is
in an oxidized state upon storage in air either as a solid or as a
liquid during a period of time, for example, one month or less,
and, preferably, one week or less, and, more preferably, one day or
less.
[0064] "Amperometry" includes steady-state amperometry,
chronoamperometry, and Cottrell-type measurements.
[0065] A "biological fluid" is any body fluid in which the analyte
can be measured, for example, blood (which includes whole blood and
its cell-free components, such as, plasma and serum), interstitial
fluid, dermal fluid, sweat, tears, urine and saliva.
[0066] "Coulometry" is the determination of charge passed or
projected to pass during complete or nearly complete electrolysis
of the analyte, either directly on the electrode or through one or
more electron transfer agents. The charge is determined by
measurement of charge passed during partial or nearly complete
electrolysis of the analyte or, more often, by multiple
measurements during the electrolysis of a decaying current and
elapsed time. The decaying current results from the decline in the
concentration of the electrolyzed species caused by the
electrolysis.
[0067] A "counter electrode" refers to one or more electrodes
paired with the working electrode, through which passes an
electrochemical current equal in magnitude and opposite in sign to
the current passed through the working electrode. The term "counter
electrode" is meant to include counter electrodes which also
function as reference electrodes (i.e. a counter/reference
electrode) unless the description provides that a "counter
electrode" excludes a reference or counter/reference electrode.
[0068] An "effective diffusion coefficient" is the diffusion
coefficient characterizing transport of a substance, for example,
an analyte, an enzyme, or a redox mediator, in the volume between
the electrodes of the electrochemical cell. In at least some
instances, the cell volume may be occupied by more than one medium
(e.g., the sample fluid and a polymer film). Diffusion of a
substance through each medium may occur at a different rate. The
effective diffusion coefficient corresponds to a diffusion rate
through this multiple-media volume and is typically different than
the diffusion coefficient for the substance in a cell filled solely
with sample fluid.
[0069] An "electrochemical sensor" is a device configured to detect
the presence of and/or measure the concentration of an analyte via
electrochemical oxidation and reduction reactions. These reactions
are transduced to an electrical signal that can be correlated to an
amount or concentration of analyte.
[0070] "Electrolysis" is the electrooxidation or electroreduction
of a compound either directly at an electrode or via one or more
electron transfer agents (e.g., redox mediators and/or
enzymes).
[0071] The term "facing electrodes" refers to a configuration of
the working and counter electrodes in which the working surface of
the working electrode is disposed in approximate opposition to a
surface of the counter electrode. In at least some instances, the
distance between the working and counter electrodes is less than
the width of the working surface of the working electrode.
[0072] A compound is "immobilized" on a surface when it is
entrapped on or chemically bound to the surface.
[0073] An "indicator electrode" is an electrode that detects
partial or complete filling of a sample chamber and/or measurement
zone.
[0074] A "layer" is one or more layers.
[0075] The "measurement zone" is defined herein as a region of the
sample chamber sized to contain only that portion of the sample
that is to be interrogated during an analyte assay.
[0076] A "non-diffusible," "non-leachable," or "non-releasable"
compound is a compound which does not substantially diffuse away
from the working surface of the working electrode for the duration
of the analyte assay.
[0077] The "potential of the counter/reference electrode" is the
half cell potential of the reference electrode or counter/reference
electrode of the cell when the solution in the cell is 0.1 M NaCl
solution at pH 7.
[0078] A "redox mediator" is an electron transfer agent for
carrying electrons between the analyte and the working electrode,
either directly, or via a second electron transfer agent.
[0079] A "reference electrode" includes a reference electrode that
also functions as a counter electrode (i.e., a counter/reference
electrode) unless the description provides that a "reference
electrode" excludes a counter/reference electrode.
[0080] A "second electron transfer agent" is a molecule that
carries electrons between a redox mediator and the analyte.
[0081] A "surface in the sample chamber" is a surface of a working
electrode, counter electrode, counter/reference electrode,
reference electrode, indicator electrode, a spacer, or any other
surface bounding the sample chamber.
[0082] A "working electrode" is an electrode at which analyte is
electrooxidized or electroreduced with or without the agency of a
redox mediator.
[0083] A "working surface" is the portion of a working electrode
that is covered with non-leachable redox mediator and exposed to
the sample, or, if the redox mediator is diffusible, a "working
surface" is the portion of the working electrode that is exposed to
the sample.
[0084] The small volume, in vitro analyte sensors of the present
invention are designed to measure the concentration of an analyte
in a portion of a sample having a volume no more than about 1
.mu.L, preferably no more than about 0.5 .mu.L, more preferably no
more than about 0.32 .mu.L, still more preferably no more than
about 0.25 .mu.L, and most preferably no more than about 0.1 .mu.L
of sample.
[0085] The analyte of interest is typically provided in a solution
or biological fluid, such as blood or serum.
[0086] Referring to the Drawings in general, and in particular
FIGS. 1 and 2, a small volume, in vitro electrochemical sensor 20
of the invention generally includes a working electrode 22 on a
first substrate 32, a counter (or counter/reference) electrode 24
on a second substrate 34, and a sample chamber 26. The sample
chamber 26 is configured so that when a sample is provided in the
chamber, the sample is in electrolytic contact with both the
working electrode 22, the counter electrode 24 and any reference
electrodes or indicator electrodes that may be present. This allows
electrical current to flow between the electrodes to effect the
electrolysis (electrooxidation or electroreduction) of the analyte.
A spacer 33 is positioned between first substrate 32 and second
substrate 34 to provide a spacing between electrodes 22, 24 and to
provide sample chamber 26 in which the sample to be evaluated is
housed.
[0087] Working Electrode
[0088] The working electrode 22 may be formed from a molded carbon
fiber composite or it may include an inert non-conducting base
material, such as polyester, upon which a suitable conducting layer
is deposited. The conducting layer typically has relatively low
electrical resistance and is typically electrochemically inert over
the potential range of the sensor during operation. Suitable
conducting layers include gold, carbon, platinum, ruthenium
dioxide, palladium, and conductive epoxies, such as, for example,
ECCOCOAT CT5079-3 Carbon-Filled Conductive Epoxy Coating (available
from W.R. Grace Company, Woburn, Mass.), as well as other
non-corroding materials known to those skilled in the art. The
electrode (e.g., the conducting layer) is deposited on the surface
of the inert material by methods such as vapor deposition or
printing. Preferably, the electrode is printed onto the base
material.
[0089] The inert non-conducting base material, is also referred to
as a substrate, base, or the like. This base material is typically
an electrically non-conducting material, e.g., any insulating
material, that is not capable of carrying electric charge or
current. Examples of materials usable as the base material for
sensors of the present invention include polyesters, polyethylene
(both high density and low density), polyethylene terephthalate,
polycarbonate, vinyls, and the like. The base material can be
treated with a primer or other such coating to improve the adhesion
of the electrodes thereon.
[0090] A tab 23' can be provided on the end of the working
electrode 22 for easy connection of the electrode to external
electronics (not shown) such as a voltage source or current
measuring equipment. Contact pad 23, which is connected to working
electrode 22, such as an extension from the working electrode, can
be positioned on tab 23'.
[0091] To prevent electrochemical reactions from occurring on
portions of the working electrode not coated by the mediator, when
a non-leachable mediator is used, a dielectric or other insulating
material can be deposited on the electrode over, under, or
surrounding the region with the redox mediator. Suitable dielectric
materials include waxes and non-conducting organic polymers, such
as polyethylene. The dielectric material can also cover a portion
of the redox mediator on the electrode. The covered portion of the
redox mediator will not contact the sample, and, therefore, will
not be a part of the electrode's working surface.
[0092] Sensing Chemistry
[0093] In addition to the working electrode 22, sensing chemistry
materials are provided in the sample chamber 26 for the analysis of
the analyte. This sensing chemistry preferably includes a redox
mediator and a second electron transfer mediator, although in some
instances, one or the other may be used alone. The redox mediator
and second electron transfer agent can be independently diffusible
or non-leachable (i.e., non-diffusible) such that either or both
may be diffusible or non-leachable. Placement of sensor chemistry
components can depend on whether they are diffusible or
non-leachable. For example, non-leachable and/or diffusible
component(s) typically form a sensing layer on the working
electrode. Alternatively, one or more diffusible components can be
disposed on any surface in the sample chamber prior to the
introduction of the sample. As another example, one or more
diffusible component(s) are placed in the sample prior to
introduction of the sample into the sensor.
[0094] If the redox mediator is non-leachable, then the
non-leachable redox mediator is typically disposed on the working
electrode 22 as a sensing layer. In an embodiment having a redox
mediator and a second electron transfer agent, if the redox
mediator and second electron transfer agent are both non-leachable,
then both of the non-leachable components are disposed on the
working electrode 22 as a sensing layer.
[0095] If, for example, the second electron transfer agent is
diffusible and the redox mediator is non-leachable, then at least
the redox mediator is disposed on the working electrode 22 as a
sensing layer. The diffusible second electron transfer agent need
not be disposed on a sensing layer of the working electrode, but
can be disposed on any surface of the sample chamber, including
within the redox mediator sensing layer, or can be placed in the
sample. If the redox mediator is diffusible, then the redox
mediator can be disposed on any surface of the sample chamber or
can be placed in the sample. If both the redox mediator and second
electron transfer agent are diffusible, then the diffusible
components can be independently or jointly disposed on any surface
of the sample chamber and/or placed in the sample (i.e., each
diffusible component need not be disposed on the same surface of
the sample chamber or placed in the sample).
[0096] The redox mediator, whether it is diffusible or
non-leachable, mediates a current between the working electrode 22
and the analyte and enables the electrochemical analysis of
molecules which may not be suited for direct electrochemical
reaction on an electrode. The mediator functions as an electron
transfer agent between the electrode and the analyte.
[0097] Analytes that can be interrogated include, for example,
glucose, acetyl choline, amylase, bilirubin, cholesterol, chorionic
gonadotropin, creatine kinase (e.g., CK-MB), creatine, DNA,
fructosamine, glucose, glutamine, growth hormones, hormones,
ketones, lactate, peroxide, prostate-specific antigen, prothrombin,
RNA, thyroid stimulating hormone, and troponin. The concentration
of analytes such as drugs or medication, such as, for example,
antibiotics (e.g., gentamicin, vancomycin, and the like),
digitoxin, digoxin, drugs of abuse, theophylline, and warfarin, can
also be determined. Assays suitable for determining the
concentration of DNA and/or RNA are disclosed in U.S. patent
applications Ser. No. 09/138,888, now issued as U.S. Pat. No.
6,281,006, and Ser. No. 09/145,776 and described in PCT Application
PCT/US99/14460, all which are incorporated herein by reference.
Redox Mediators
[0098] Although any organic or organometallic redox species can be
used as a redox mediator, one type of suitable redox mediator is a
transition metal compound or complex. Examples of suitable
transition metal compounds or complexes include osmium, ruthenium,
iron, and cobalt compounds or complexes. In these complexes, the
transition metal is coordinatively bound to one or more ligands.
The ligands are typically mono-, di-, tri-, or tetradentate. The
most preferred ligands are heterocyclic nitrogen compounds, such
as, for example, pyridine and/or imidazole derivatives.
Multidentate ligands may include multiple pyridine and/or imidazole
rings. Alternatively, metallocene derivatives, such as, for
example, ferrocene, can be used. An example of one mediator is
[Os(4-(N-(6-aminohexyl)aminobipyridine)(1,1'-dimethyl-2,2'-biimidazole).s-
ub.2]Cl.sub.3.
[0099] The redox mediators can be diffusible redox mediators or
non-leachable redox mediators, such as non-leachable redox
polymers. For additional information on redox mediators, see, for
example, U.S. patent application Ser. No. 09/295,962, filed Apr.
21, 1999, now issued as U.S. Pat. No. 6,338,790, and its
continuations U.S. Pat. Nos. 6,299,757 and 6,461,496, and PCT
published application WO 98/35225, all which are incorporated
herein by reference.
Second Electron Transfer Agent
[0100] In a preferred embodiment of the invention, the sensor
includes a redox mediator and a second electron transfer agent
which is capable of transferring electrons to or from the redox
mediator and the analyte. The second electron transfer agent can be
diffusible or can be non-leachable (e.g., entrapped in or
coordinatively, covalently, or ionically bound to a redox polymer).
One example of a suitable second electron transfer agent is an
enzyme which catalyzes a reaction of the analyte. For example, a
glucose oxidase or glucose dehydrogenase, such as pyrroloquinoline
quinone glucose dehydrogenase (PQQ), is used when the analyte is
glucose. A lactate oxidase fills this role when the analyte is
lactate. Other enzymes can be used for other analytes.
[0101] Counter Electrode
[0102] Counter electrode 24, as illustrated in FIGS. 1 and 2, can
be constructed in a manner similar to working electrode 22. Counter
electrode 24 may also be a counter/reference electrode.
Alternatively, a separate reference electrode may be provided in
contact with the sample chamber. Suitable materials for the
counter/reference or reference electrode include, for example,
Ag/AgCl or Ag/AgBr printed on a non-conducting base material or
silver chloride on a silver metal base. The same materials and
methods may be used to make the counter electrode as are available
for constructing the working electrode 22, although different
materials and methods may also be used. Preferably, the counter or
counter/reference electrode is printed on an insulating base
material. A tab 25', on which contact pad 25 is disposed, can be
provided for making convenient connection to the external
electronics (not shown), such as a coulometer, potentiostat, or
other measuring device.
[0103] Optionally, a non-conductive filler material, such as a
non-conductive ink, can be formed adjacent a counter electrode, or
between multiple counter electrodes to provide a planar surface
along the path of travel of the sample fluid in the sample chamber.
The non-conductive filler material preferably creates a smooth
surface to facilitate filling of the sample chamber by capillary
action and/or for reducing the likelihood that air bubbles will
become entrapped near the counter electrodes. This non-conductive
material can be colored or colorless and may be formed on the
substrate by printing or other techniques. The non-conductive
material may be deposited prior to or subsequent to the formation
of the counter electrode. In one embodiment, a non-conductive ink
is used to fill in the area between multiple 12.3 micrometer (0.5
mil) thick counter electrodes. In another embodiment, a
non-conductive ink is used to fill the area between multiple 6.4
micrometer (0.25 mil) thick counter electrodes. Generally, no
filler ink is needed for thickness less about 6.4 micrometers, and
in some sensor designs, no filler ink is needed for 6.4 micrometer
thick counter electrodes.
[0104] Electrode Configuration
[0105] In one embodiment of the invention, working electrode 22 and
counter electrode 24 are disposed opposite to and facing each other
to form a facing electrode pair as depicted in FIGS. 1 and 2. In
this preferred configuration, the sample chamber 26 is typically
disposed between the two electrodes. For this facing electrode
configuration, it is preferred that the electrodes are separated by
a distance of no more than about 0.2 mm (e.g., at least one portion
of the working electrode is separated from one portion of the
counter electrode by no more than 200 .mu.m), preferably no more
than 100 .mu.m, and most preferably no more than 50 .mu.m.
[0106] The electrodes need not be directly opposing each other;
they may be slightly offset. Furthermore, the two electrodes need
not be the same size. Preferably, the working electrode 22 extends
the width of sensor 20 and counter electrode 24 is a portion or all
of that width. Either of working electrode 22 or counter electrode
24 may include more than one electrode, such as shown in FIG. 4 (as
counter electrodes 324, 344) and in FIG. 5B (as counter electrodes
424, 441, 442). Counter electrode 24 can also be formed with tines
in a comb shape. Other configurations of both the counter electrode
and working electrode are within the scope of the invention.
However, for this particular embodiment, the separation distance
between at least one portion of the working electrode and some
portion of the counter electrode preferably does not exceed the
limits specified hereinabove.
[0107] In another embodiment of the invention, the working and
counter electrodes are coplanar. In this case, the sample chamber
is in contact with both electrodes and is bounded on the side
opposite the electrodes by a non-conducting inert base or
substrate. Suitable materials for the inert base include
non-conducting materials such as polyester.
[0108] Other configurations of the inventive sensors are also
possible. For example, the two electrodes can be formed on surfaces
that make an angle to each other. One such configuration would have
the electrodes on surfaces that form a right angle. Another
possible configuration has the electrodes on a curved surface such
as the interior of a tube. For example, the working and counter
electrodes can be arranged so that they face each other from
opposite sides of the tube. This is another example of a facing
electrode pair. Alternatively, the electrodes can be placed near
each other on the tube wall (e.g., one on top of the other or
side-by-side). In any configuration, the two electrodes must be
configured so that they do not make direct electrical contact with
each other, to prevent shorting of the electrochemical sensor.
[0109] Referring again to FIGS. 1 and 2, the electrodes 22, 24
extend from the sample chamber 26 to the other end of the sensor 20
as electrode extensions called "traces". Each trace provides a
contact pad 23, 25 for providing electrical connection to a meter
or other device to allow for data and measurement collection, as
will be described later. Preferably, each contact pad 23, 25 is
positioned on a tab 23', 25' that extends from each non-conducting
base substrate 32, 34. In one embodiment, a tab has more than one
contact pad positioned thereon. In a second embodiment, a single
contact pad is used to provide a connection to one or more
electrodes; that is, multiple electrodes are coupled together and
are connected via one contact pad.
[0110] A spacer 33 can be used to keep the electrodes apart when
the electrodes face each other as depicted in FIGS. 1 and 2; spacer
33 is clearly seen in FIG. 2. The spacer is typically constructed
from an inert non-conducting material such as pressure-sensitive
adhesive, polyester, Mylar.TM., Kevlar.TM. or any other strong,
thin polymer film, or, alternatively, a thin polymer film such as a
Teflon.TM. film, chosen for its chemical inertness. In addition to
preventing contact between the electrodes, the spacer 33 can
function as a portion of the boundary for the sample chamber 26.
Other spacers include layers of adhesive and double-sided adhesive
tape (e.g., a carrier film with adhesive on opposing sides of the
film). Adhesive may be applied, for example by coating, onto a
polymeric material to provide spacer 33.
[0111] Sample Chamber
[0112] The sample chamber 26 is typically defined by a combination
of the electrodes 22, 24, the substrates 32, 34, and a spacer 33 as
shown in FIGS. 1 and 2. A measurement zone is contained within this
sample chamber and is the region of the sample chamber that
contains only that portion of the sample that is interrogated
during the analyte assay. In the embodiment of the invention
illustrated in FIGS. 1 and 2, sample chamber 26 is the space
between the two electrodes 22, 24 and their non-conductive base
substrates 32, 34, bounded by spacer 33. In this embodiment, the
sample chamber has a volume that is preferably no more than about 1
.mu.L, more preferably no more than about 0.32 .mu.L, and still
more preferably no more than about 0.25 .mu.L.
[0113] In the embodiment of the invention depicted in FIGS. 1 and
2, the measurement zone has a volume that is approximately equal to
the volume of the sample chamber. In a preferred embodiment the
measurement zone includes 80% of the sample chamber, 90% in a more
preferred embodiment, and about 100% in a most preferred
embodiment.
[0114] In another embodiment of the invention, shown in FIG. 3,
sensor 220 has a working electrode 222 on non-conducting substrate
base 232, a counter electrode 224 on non-conducting substrate base
234, and a spacer 233 therebetween. A contact pad 223 extends from
working electrode 222, and likewise, a contact pad 225 extends from
counter electrode 224. Sample chamber 226 (defined by base 232, 234
and spacer 233) extends the length of sensor 220 and includes much
more space than the region proximate electrodes 222, 224. In this
embodiment, the measurement zone, corresponding to the region
containing the portion of the sample which will be interrogated, is
the portion of sample chamber 226 bounded by the working surface of
the working electrode 222 and counter electrode 224. In this
embodiment, the measurement zone has a volume that is preferably no
more than about 1 .mu.L, more preferably no more than about 0.32
.mu.L, still more preferably no more than about 0.25 .mu.L, and
most preferably no more than about 0.1 .mu.L of sample.
[0115] In both of the embodiments discussed above, the thickness of
the sample chamber and of the measurement zone correspond typically
to the thickness of spacer 33, 233 (e.g., the distance between the
electrodes in FIGS. 2 and 3, or the distance between the electrodes
and the inert base in an embodiment where the electrodes are
coplanar). The spacer can be, for example, an adhesive or
double-sided adhesive tape or film. Examples of useable adhesives
include urethanes, acrylates, acrylics, latexes, rubbers, and other
known adhesive materials. Preferably, this thickness is small to
promote rapid electrolysis of the analyte, as more of the sample
will be in contact with the electrode surface for a given sample
volume. In addition, a thin sample chamber helps to reduce errors
from diffusion of analyte into the measurement zone from other
portions of the sample chamber during the analyte assay, because
diffusion time is long relative to the measurement time. Typically,
the thickness of the sample chamber is between about 50 and about
200 micrometers.
[0116] Sorbent Material
[0117] The sample chamber can be empty before the sample is placed
in the chamber, or, in some embodiments, the sample chamber can
include a sorbent material (shown in FIG. 3 as sorbent 50) to sorb
and hold a fluid sample during the measurement process. Suitable
sorbent materials include polyester, nylon, cellulose, and
cellulose derivatives such as nitrocellulose. The sorbent material
facilitates the uptake of small volume samples by a wicking action
which can complement or, preferably, replace any capillary action
of the sample chamber. In addition to or alternatively, a portion
or the entirety of the wall of the sample chamber may be coated by
a surfactant, which is intended to lower the surface tension of the
fluid sample and improve fluid flow within the sample chamber. An
example of a useable surfactant is available under the tradename
"Zonyl FSO" from Dupont of Wilmington, Del.
[0118] Methods other than the wicking action of a sorbent can be
used to transport the sample into the sample chamber or measurement
zone. Examples of such methods for transport include the
application of pressure on a sample to push it into the sample
chamber, the creation of a vacuum by a pump or other
vacuum-producing method in the sample chamber to pull the sample
into the chamber, capillary action due to interfacial tension of
the sample with the walls of a thin sample chamber, as well as the
wicking action of a sorbent material.
[0119] The entire sensor assembly is held firmly together to ensure
that the sample remains in contact with the electrodes and that the
sample chamber and measurement zone maintain the same volume. This
is an important consideration in the coulometric analysis of a
sample, where measurement of a defined sample volume is needed.
[0120] Alternative Sensor Designs
[0121] FIGS. 4 through 12 illustrate alternative sensor designs,
both tip-filling and side-filling. Referring to FIG. 4, a sensor
320 has a working electrode 322, a counter electrode 324, a second
counter electrode 344 (which may provide a fill indicator function,
as described below), and a sample chamber 326 extending along at
least a portion of the length of sensor 320 and optionally
including a sorbent 350.
[0122] FIGS. 5A through 5C illustrate three layers that when
assembled, provide a preferred embodiment of a sensor. FIG. 5A has
a working electrode 422 on substrate 432. Working electrode 422 has
a trace 453 extending from sample chamber 426 to tab 423' on which
is contact pad 423; contact pad 423 connects the sensor to a meter
or other measurement equipment. FIG. 5B, (inverted with respect to
FIG. 5A to show the electrode side up), has a counter electrode 424
on substrate 434, and also includes a first indicator electrode 441
and a second indicator electrode 442. Counter electrode 424 has a
trace 454, first indicator electrode 441 has trace 451, and second
indicator electrode 442 has trace 452, that end at contact pads
425, 443, 444, respectively, on tab 425'. Spacer 433 in FIG. 5C
defines sample chamber 426 and provides spacing between the
electrodes when the two substrates 432, 434 are positioned opposite
and facing one another.
[0123] FIGS. 6A through 6C also illustrate three layers that, when
assembled, provide a sensor. In FIG. 6A, a working electrode 502 is
formed on first substrate 500. The working electrode 502 includes a
contact pad 503 for connection with external electronics; this
contact pad 503 is connected to working electrode 502 by trace 552.
A spacer 504, shown in FIG. 6B, such as a layer of adhesive or a
double-sided tape defines a channel 506 to produce a sample chamber
for the sensor. Two counter (or counter/reference) electrodes 510,
512 are formed on a second substrate 508, as shown in FIG. 6C
(inverted with respect to FIG. 6A to show the electrode side up).
This multiple counter electrode arrangement can provide a fill
indicator function, using counter electrode 512, as described
below. Each counter electrode 510, 512 has a contact region or pad
511, 513 for connection with external electronics; these contact
pads 511, 513 are connected to counter electrodes 510, 512 by
traces 551, 553. The second substrate 508 is inverted and placed
over the first substrate 500, with the spacer 504 between, so that
the working electrode 502 and the two counter electrodes 510, 512
are facing in the region of the channel 506.
[0124] In some instances, the counter electrode 510 nearest an
entrance 514 (FIG. 6B) of the channel 506 has a surface area within
the sample chamber that is at least two times larger than the other
counter electrode 512, and may be at least five or ten times
larger. The non-leachable or diffusible redox mediator and/or
second electron transfer agent can be provided on either the first
or second substrates 500, 508 in a region corresponding to the
channel 506, as described above.
[0125] The working electrode and counter electrodes can be formed
to cover the entire channel region (except for a small space
between the two counter electrodes). In this embodiment, the sample
chamber and measurement zone are effectively the same and have the
same volume. In other embodiments, the measurement zone has, for
example, 80% or 90% of the volume of the sample chamber. It will be
understood that similar sensors could be made using one counter
electrode or three or more counter electrodes. It will also be
understood that multiple working electrodes may also be provided on
the sensor.
[0126] FIGS. 7A, 7B and 7C illustrate a side-filling sensor
arrangement. FIG. 7A shows a first substrate 520 with a working
electrode 522. FIG. 7B illustrates a spacer 524 defining a channel
526. FIG. 7C (inverted with respect to FIG. 7A and 7B) illustrate a
second substrate 528 with three counter (or counter/reference)
electrodes 530, 532, 534. This multiple counter electrode
arrangement can provide a fill indicator function, as described
below. An indentation 536, 538 or recessed or indented portion can
be formed at either edge of the opening to channel 526 to
facilitate the drawing of fluid into the sensor. This configuration
can aid in wicking or capillary filling of the channel (i.e.,
sample chamber). This configuration can also reduce the likelihood
that the user will inadvertently block the channel during
collection of the sample, which could occur by pressing the tip of
the sensor edgewise against the skin.
[0127] FIGS. 8A, 8B, and 8C illustrate another example of a
side-filling sensor arrangement. FIG. 8A illustrates a first
substrate 540 with a working electrode 542. FIG. 8B illustrates a
spacer 544 defining a channel 546. FIG. 8C (inverted with respect
to FIGS. 8A and 8B) illustrates a second substrate 548 with three
counter (or counter/reference) electrodes 550, 552, 554.
[0128] FIGS. 9A, 9B, and 9C illustrate another example of a
tip-filling sensor arrangement. FIG. 9A illustrates a first
substrate 560 with a working electrode 562. FIG. 9B illustrates a
spacer 564 defining a channel 566. FIG. 9C (inverted with respect
to FIGS. 9A and 9B) illustrates a second thin film substrate 568
with two counter (or counter/reference) electrodes 570, 572. This
multiple counter electrode arrangement can provide a fill indicator
function, as described below. A vent hole 574 (indicated as a
shaded region in FIG. 9C) is provided through the second substrate.
In the illustrated embodiment, this vent hole 574 is made through
only the substrate 568 that carries the counter electrode(s) and,
optionally, the spacer 564. In this embodiment, the vent hole can
be formed by, for example, die cutting a portion of the substrate.
This die cut can remove a portion of at least one counter
electrode, but a sufficient amount of the counter electrode should
remain for contact with the sample in the channel and for
electrical connection to a contact at the other end of the sensor.
In another embodiment, the vent hole 574 can be made through all of
the layers or through the first substrate and not the second
substrate.
[0129] Another embodiment is illustrated in FIGS. 10A, 10B, and
10C, with a different shape. This sensor includes a first substrate
579 with at least one working electrode 580, as illustrated in FIG.
10A. The sensor also includes a spacer 581 with a channel 582
formed in the spacer 581, as shown in FIG. 10B. The sensor further
includes a second substrate 583 with two counter electrodes 584,
585, as shown in FIG. 10C (inverted with respect to FIGS. 10A and
10B). A venting aperture 586 is cut typically through all of the
layers and extends from a side of the sensor. In some embodiments,
the venting aperture and the front portion 587 of the sensor are
simultaneously cut with a reproducible distance between the venting
aperture and the front portion 587 of the sensor to provide a
reproducible length for the channel 582 and the working electrode
580. This tip-filling sensor arrangement optionally includes an
indentation 588 or recessed or indented portion that can be formed
at the filling opening of the channel 582 to facilitate the drawing
of fluid into the sensor.
[0130] FIGS. 11A, 11B, and 11C illustrate another example of a
side-filling sensor arrangement. FIG. 11A illustrates a first
substrate 640 with a working electrode 642. FIG. 11B illustrates a
spacer 644 defining a channel 646. FIG. 11C (inverted with respect
to FIGS. 11A and 11B) illustrates a second substrate 648 with three
counter (or counter/reference) electrodes 650, 652, 654. This
multiple counter electrode arrangement can provide a fill indicator
function, as described below. The length of the channel 646 is
typically defined by the two parallel cuts along the sides 656, 658
of the sensors.
[0131] Because of the straight sides, these sensors (and also those
shown in FIGS. 1, 2 and 5) can be manufactured adjacent to one
another, as illustrated in FIG. 13A. Such positioning during
manufacture produces less waste material. Another optional
processing advantage of the adjacent sensor manufacturing process
is that the redox mediator and/or second electron transfer agent
can be readily disposed in the channel by striping a continuous
stream of these components along a row or column of adjacent
sensors. This can result in better efficiency and less waste of
expensive reagents such as the redox mediator and/or second
electron transfer agent, as compared to other techniques, such as
individually placing these components within the individual
channels.
[0132] FIGS. 12A, 12B, and 12C illustrate another sensor
configuration. This sensor includes a first substrate 600 with at
least one working electrode 602, as illustrated in FIG. 12A. The
sensor also includes a spacer 604 with a channel 606 formed in the
spacer 604, as shown in FIG. 12B. The sensor further includes a
second substrate 608 with two counter electrodes 610, 612, as shown
in FIG. 12C (inverted with respect to FIGS. 12A and 12B). This
multiple counter electrode arrangement may provide a fill indicator
function, as described below. The sensor can also include, for
example, an indicator, such as a slot 614 or an extension 616 from
the body of the sensor that indicates to the user which side of the
sensor should be placed adjacent to the sample. Surface printing
can also or alternatively be applied to the sensor to act as an
indicator. Some indication mechanism can be particularly important
where the sensor reading is only correct when the sample enters
from a particular side.
[0133] Multiple Electrode Sensors and Calibration Thereof
[0134] Multiple electrode sensors can be used for a variety of
reasons. For example, multiple electrodes can be used to test a
variety of analytes using a single sample. One embodiment with
multiple electrodes has one or more sample chambers, each of which
contains one or more working electrodes, with each working
electrode defining a different measurement zone. If the redox
mediator is non-leachable, one or more of the working electrodes
can have the appropriate chemical reagents, for example, an
appropriate enzyme, to test a first analyte and one or more of the
remaining working electrodes can have the appropriate chemical
reagents to test a second analyte, and so on. For example, a
multiple electrode sensor might include a working electrode having
glucose oxidase disposed thereon to determine glucose concentration
and another working electrode can have lactate oxidase disposed
thereon to determine lactate concentration.
[0135] Multiple electrodes could also be used to improve the
precision of the resulting readings. The measurements from each of
the working electrodes (all of which are detecting the same
analyte) can be averaged or otherwise combined together to obtain a
more precise or reliable reading. In some cases, measurements could
be rejected if the difference between the value and the average
exceeds a threshold limit. This threshold limit could be, for
example, determined based on a statistical parameter, such as the
standard deviation of the averaged measurements. The average could
then be recalculated while omitting the rejected values. In
addition to using multiple electrode sensors to increase precision,
multiple measurements could be made at each electrode and averaged
together to increase precision. This technique could also be used
with a single electrode sensor to increase precision.
[0136] One example of a multiple electrode sensor that can be used
to accurately determine the volume of the measurement zones of the
electrode pairs and that is also useful in reducing noise is
presented herein. In this example, one of the working electrodes is
prepared with a non-leachable redox mediator and a non-leachable
second electron transfer agent (e.g., an enzyme). Another working
electrode includes non-leachable redox mediator, but no second
electron transfer agent on the electrode. An optional third working
electrode has no redox mediator and no second electron transfer
agent bound to the electrode. A similar configuration can be
constructed using diffusible redox mediator and/or diffusible
second electron transfer agent although diffusible components are
not limited to being disposed on the working electrode. Preferably,
the distance between the working electrodes is sufficient that
redox mediator and/or enzyme do not substantially diffuse between
electrodes within the measurement period (e.g., in the time period
from introduction of the same sample into the sample chamber to the
end of the measurement).
[0137] The sensor error caused by the redox mediator being in a
non-uniform oxidation state prior to the introduction of the sample
can be measured by concurrently electrolyzing the sample in the
measurement zones that are proximate working and counter
electrodes. At the first working electrode, the analyte is
electrolyzed to provide the sample signal. At the second working
electrode, the analyte is not electrolyzed because of the absence
of the second electron transfer agent (assuming that a second
electron transfer agent is necessary). However, a charge will pass
(and a current will flow) due to the electrolysis of the redox
mediator that was in a mixed oxidation state (i.e., some redox
centers in the reduced state and some in the oxidized state) prior
to the introduction of the sample and/or the shuttling of a
diffusible redox mediator between the working electrode and the
counter electrode. The small charge passed using this second
working electrode can be subtracted from the charge passed using
the first electrode pair to substantially remove the error due to
the oxidation state of the redox mediator and/or to remove the
background current caused by a diffusible redox mediator. This
procedure also reduces the error associated with other electrolyzed
interferents, such as ascorbate, urate, and acetaminophen, as well
as errors associated with capacitive charging and faradaic
currents.
[0138] The thickness of the sample chamber can be determined by
measuring the capacitance, preferably in the absence of any fluid,
between an electrode and its corresponding counter electrode. The
capacitance of an electrode pair depends on the surface area of the
electrodes, the interelectrode spacing, and the dielectric constant
of the material between the plates. The dielectric constant of air
is unity which typically means that the capacitance of this
electrode configuration is a few picofarads (or about 100-1000
picofarads if there is fluid between the electrode and counter
electrode given that the dielectric constant for most biological
fluids is approximately 75). Thus, since the surface area of the
electrodes are known, measurement of the capacitance of the
electrode pair allows for the determination of the thickness of the
measurement zone to within about 1-5%.
[0139] Other electrode configurations can also use these techniques
(i.e., capacitance measurements and coulometric measurements in the
absence of a critical component) to reduce background noise and
error due to interferents and imprecise knowledge of the volume of
the interrogated sample. Protocols involving one or more working
electrode(s) and counter electrode(s) and one or more of the
measurements described above can be developed and are within the
scope of the invention. For example, only one electrode pair is
needed for the capacitance measurements, however, additional
electrodes can be used for convenience.
[0140] Fill Indicator
[0141] When using a sample chamber that is filled with 1 .mu.L or
less of fluid, it is often desirable to be able to determine when
the sample chamber is filled. FIGS. 6A through 6C illustrate a
sensor having a fill indicator structure. In particular, FIG. 6A
illustrates a first substrate 500 upon which a working electrode
502 is printed. A spacer 504 (FIG. 6B), such as, for example, a
layer of adhesive or a double-sided tape, is formed over the first
substrate 500 and working electrode 502 with a channel 506 formed
in the layer to provide a sample chamber. A second substrate 508 is
printed with two counter electrodes 510, 512, as shown in FIG. 6C
(inverted with respect to FIGS. 6A and 6B to show the electrode
side up). Preferably, the counter electrode 510 nearest an entrance
514 of the channel 506 has a surface area within the sample chamber
that is at least two times larger than the other counter electrode
512, and preferably at least five or ten times larger.
[0142] The sensor can be indicated as filled, or substantially
filled, by observing a signal between the second counter electrode
512 and the working electrode 502 as the sensor fills with fluid.
When fluid reaches the second counter electrode 512, the signal
from that counter electrode will change. Suitable signals for
observing include, for example, voltage, current, resistance,
impedance, or capacitance between the second counter electrode 512
and the working electrode 502. Alternatively, the sensor can be
observed after filling to determine if a value of the signal (e.g.,
voltage, current, resistance, impedance, or capacitance) has been
reached indicating that the sample chamber is filled.
[0143] In alternative embodiments, the counter electrode or working
electrode can be divided into two or more parts and the signals
from the respective parts observed to determine whether the sensor
has been filled. In one example, the working electrode is in a
facing relationship with the counter electrode and the indicator
electrode. In another example, the counter electrode, working
electrode, and indicator electrode are not in a facing
relationship, but are, for example, side-by-side. Typically, the
indicator electrode is further downstream from a sample inlet port
than the working electrode and counter electrode.
[0144] For side-fill sensors, such as those illustrated in FIGS. 5,
7, 8 and 11, an indicator electrode can be disposed on each side of
the primary counter electrode. This permits the user to fill the
sample chamber from either the left or right side with an indicator
electrode disposed further upstream. This three-electrode
configuration is not necessary. Side-fill sensors can also have a
single indicator electrode and, preferably, some indication as to
which side should be placed in contact with the sample fluid.
[0145] Alternately or additionally, two indicator electrodes, used
in combination with one counter/reference electrode, detect when
the sample chamber begins to fill and when the sample chamber has
been filled to prevent partial filling of the sample chamber. The
two indicator electrodes are optionally held at a different
potential than the counter/reference electrode. The start and
completion of filling of the sample chamber is indicated by the
flow of current between the indicator and counter/reference
electrodes.
[0146] In other instances, the potential of each of the
counter/reference electrodes may be the same. When the potential at
all three counter/reference electrodes is the same, for example, 0
volts, then as the measurement zone begins to fill, the fluid
allows for electrical current flow between a working electrode and
the first counter/reference electrode, causing a current at the
first counter/reference electrode due to the reaction of the
analyte with the enzyme and the mediator. When the fluid reaches
the third counter/reference electrode, another current may be
measured similar to the first counter/reference electrode
indicating that the measurement zone is full. When the measurement
zone is full, the three counter/reference electrodes are optionally
shorted together or their signals can be added or otherwise
combined.
[0147] The indicator electrode can also be used to improve the
precision of the analyte measurements. The indicator electrode may
operate as a working electrode or as a counter electrode or
counter/reference electrode. In the embodiment of FIGS. 6A through
6C, the indicator electrode 512 can act as a second counter or
counter/reference electrode with respect to the working electrode
502. Measurements from the indicator electrode/working electrode
can be combined (for example, added or averaged) with those from
the first counter/reference electrode/working electrode to obtain
more accurate measurements. In one embodiment, the indicator
electrode operates as a second working electrode with the counter
electrode or counter/reference electrode. In another embodiment,
the indicator electrode operates as a second working electrode with
a second counter electrode or counter/reference electrode. In still
another embodiment, the indicator electrode operates as a second
counter electrode or counter/reference electrode with a second
working electrode.
[0148] The sensor or a sensor reader can include a sign (e.g., a
visual sign or auditory signal) that is activated in response to
the indicator electrode to alert the user that the measurement zone
has been filled. The sensor or a sensor reader can be configured to
initiate a reading when the indicator electrode indicates that the
measurement zone has been filled with or without alerting the user.
The reading can be initiated, for example, by applying a potential
between the working electrode and the counter electrode and
beginning to monitor the signals generated at the working
electrode.
[0149] General Method for Manufacturing Sensors
[0150] Referring now to FIGS. 13A and 13B, one example of a method
for making thin film sensors is described with respect to the
sensor arrangement displayed in FIGS. 5A through 5C, although this
method can be used to make a variety of other sensor arrangements,
including those described before. When the three layers of FIGS. 5A
through 5C are assembled, a sensor 420 is formed.
[0151] In FIGS. 13A and 13B, a substrate 1000, such as a plastic
substrate, is moving in the direction indicated by the arrow. The
substrate 1000 can be an individual sheet or a continuous roll on a
web. Multiple sensors 420 can be formed on a substrate 1000 as
sections 1022 that have working electrodes 422 (FIG. 5A) thereon
and sections 1024 that have counter electrode 424 and indicator
electrodes 441, 442 (FIG. 5B). These working, counter and indicator
electrodes are electrically connected to their corresponding traces
and contact pads. Typically, working electrode sections 1022 are
produced on one half of substrate 1000 and counter electrode
sections 1024 are produce on the other half of substrate 1000. In
some embodiments, the substrate 1000 can be scored and folded to
bring the sections 1022, 1024 together to form the sensor. In some
embodiments, as illustrated in FIG. 13A, the individual working
electrode sections 1022 can be formed next to or adjacent each
other on the substrate 1000, to reduce waste material. Similarly,
individual counter electrode sections 1024 can be formed next to or
adjacent each other. In other embodiments, the individual working
electrode sections 1022 (and, similarly, the counter electrode
sections 1024) can be spaced apart, as illustrated in FIG. 13B. The
remainder of the process is described for the manufacture of
multiple sensors, but can be readily modified to form individual
sensors.
[0152] Carbon or other electrode material (e.g., metal, such as
gold or platinum) is formed on the substrate 1000 to provide a
working electrode 422 for each sensor. The carbon or other
electrode material can be deposited by a variety of methods
including printing a carbon or metal ink, vapor deposition, and
other methods. The printing may be done by screen printing, gravure
roll printing, transfer printing, and other known printing methods.
Trace 453 and contact pad 423 are preferably applied together with
working electrode 422, but may be applied in a subsequent step.
[0153] Similar to the working electrode 422, the counter
electrode(s) 424, 441, 442 (shown in FIG. 5B) are formed on the
substrate 1000. The counter electrodes are formed by providing
carbon or other conductive electrode material onto the substrate.
In one embodiment, the material used for the counter electrode(s)
is a Ag/AgCl ink. The material of the counter electrode(s) may be
deposited by a variety of methods including printing or vapor
deposition. The printing may be done by screen printing, gravure
roll printing, transfer printing, and other known printing methods.
Traces 454, 451, 452 and contact pads 425, 443, 444 are preferably
applied together with counter electrodes 424, 441, 442, but maybe
applied in a subsequent step.
[0154] Preferably, multiple sensors 420 are manufactured
simultaneously; that is, the working electrodes, including their
traces and contact pads, for a plurality of sensors are produced
(e.g., printed) on a polymer sheet or web, and simultaneously or
subsequently, the counter electrodes, and their traces and contact
pads, for a plurality of sensors are produced (e.g., printed). The
working electrode(s) and counter electrode(s) can be formed on
separate substrates that are later positioned opposite one another
so that the electrodes face each other. Alternately, to simplify
registration of the substrates, the working electrodes can be
formed on a first half of a substrate sheet of web and the counter
electrodes are formed on a second half of the substrate sheet or
web so that the sheet or web can be folded to superimpose the
working and counter electrodes in a facing arrangement.
[0155] To provide a sample chamber 426, a spacer 433 is formed over
at least one of the substrate/working electrode and
substrate/counter electrode(s). The spacer 433 is preferably an
adhesive spacer, such as a single layer of adhesive or a
double-sided adhesive tape (e.g., a polymer carrier film with
adhesive disposed on opposing surfaces). Suitable spacer materials
include adhesives such as urethanes, acrylates, acrylics, latexes,
rubbers and the like.
[0156] A channel, which will result in the sample chamber, is
provided in the spacer 433, either by cutting out a portion of the
adhesive spacer or placing two adhesive pieces in close proximity
but having a gap therebetween. The adhesive can be printed or
otherwise disposed on the substrate according to a pattern which
defines the channel region. The adhesive spacer can be optionally
provided with one or more release liners prior to its incorporation
into the sensor. The adhesive can be cut (e.g., die-cut or slit) to
remove the portion of the adhesive corresponding to the channel
prior to disposing the spacer on the substrate.
[0157] In one method of the invention, illustrated in FIGS. 14A
through 14G, the adhesive includes a first and a second release
liner. Prior to applying the adhesive to a first substrate/working
electrode or substrate/counter electrode(s), the adhesive is
"kiss-cut" through the first release liner and at least a portion
of the underlying adhesive, preferably the entire adhesive but not
through the second release liner, to form one or more sections, one
of which will eventually result in a sample chamber. The first
release liner is removed from the adhesive over its length but is
retained over the sample chamber section. The exposed adhesive is
then applied to the first substrate/working electrode or
substrate/counter electrode(s). On removal of the second release
liner, the adhesive positioned within the sample chamber sections
is also removed, leaving a channel or opening in the adhesive
spacer. The second substrate/working electrode or substrate/counter
electrode(s) is then applied to the adhesive to form the layered
sensor structure.
[0158] FIGS. 14A through 14G describe in detail the processing of
an adhesive layer by a "kiss-cut". An adhesive layer that will
eventually become spacer 433 is preferably obtained from an
adhesive structure including an adhesive layer 1030 between a first
release liner 1020 and a second release liner 1040. A blade (e.g.,
a knife or die blade) cuts through the first release liner 1020 and
at least a portion of the adhesive layer 1030, but not through the
second release liner 1040, as shown in FIG. 14B and 14G, to form
release liner sections 1020a, 1020b, and adhesive sections 1030a,
1030b. The space occupied by 1030b is the area that will result in
the sample chamber of the sensor and can be referred to as "sample
chamber region". A cut configuration such as shown in FIG. 14G
allows removal of one release liner section 1030a, leaving behind
release liner section 1030b. The liner could be cut to provide
individual strips extending the entire length of the liner; this
would then need removal of each individual liner strip.
[0159] The release liner section 1030a is removed to provide the
adhesive configuration shown in FIGS. 14C. The exposed adhesive is
positioned over and securely adhered to the substrate 1050, as
illustrated in FIG. 14D. The adhesive sections 1030a without the
release liner will adhere to the substrate 1050. As shown in FIG.
14E, the second release liner 1040 is removed, pulling the cut and
non-adhered section of adhesive 1030b and the first liner 1020b
with it; this results in adhesive areas 1030a providing spacer
layer sections 433a, 433b, with sample chamber 426 therebetween, as
illustrated in FIG. 14F. The second substrate is then positioned
over the adhesive layer and laminated to the first substrate via
the spacer 433. The thickness of the spacer 433 typically
determines the spacing between the working and counter electrodes,
and thus impacts the volume of the sample chamber 426. Preferably,
the thickness does not vary more than .+-.5% over the individual
sensor and/or among individual sensors in a batch. Use of the
"kiss-cut" method for manufacturing the layered sensor structure
preserves the integrity of the sample chamber walls during
manufacturing, thereby providing for more reliable and reproducible
sample chamber volumes.
[0160] The non-leachable or diffusible redox mediator and/or second
electron transfer agent are disposed onto the substrate in at least
the sample chamber regions. If either or both of these components
is non-leachable, that component or components is preferably
disposed on the working electrode. If either or both of these
components is diffusible, that component or components can be
disposed on any surface of the substrate in the channel region. The
redox mediator and/or second electrode transfer agent can be
disposed independently or together on the substrate prior to or
after disposition of the spacer. The redox mediator and/or second
electrode transfer agent may be disposed by a variety of methods
including, for example, screen printing, ink jet printing,
spraying, painting, striping along a row or column of aligned
and/or adjacent electrodes, and the like. Other components can be
deposited separately or together with the redox mediator and/or
second electrode transfer agent; these components can include, for
example, surfactants, polymers, polymer films, preservatives,
binders, buffers, and cross-linkers.
[0161] After disposing the spacer, redox mediator, second electron
transfer agent, sensing layers, and the like, the first and second
substrates (having the working and counter electrodes thereon) are
positioned opposite each other to form the sensor. The faces of the
substrate are joined by the adhesive of the spacer. Preferably,
after bringing the faces together, individual sensors are cut out
from the web of sensors using a variety of methods including, for
example, die cutting, slitting, or otherwise cutting away the
excess substrate material and separating the individual sensors. In
some embodiments, a combination of cutting or slitting methods is
used. As another alternative, the individual sensor components can
first be cut out of the substrates and then brought together to
form the sensor by adhesively joining the two components, such as
by using the spacer adhesive.
[0162] The sides of the sensor can be straight to allow the sensor
to be cut out from the remainder of the substrate and/or from other
sensors by slitting the substrate in parallel directions using, for
example, a gang arbor blade system. The edges of the sensor can
define edges of the sample chamber and/or measurement zone. By
accurately controlling the distance between cuts, variability in
sample chamber volume can often be reduced. In some instances,
these cuts are preferably parallel to each other, as parallel cuts
are typically the easiest to reproduce.
[0163] Sensor Connection to Electrical Device
[0164] Referring generally now to FIGS. 16A and 16B, 17A and 17B,
and 18A and 18B, the assembled sensor of FIGS. 5A through 5C (as
will be referred to as sensor 1420) is typically coupled to a meter
or other electrical device by electrical connector 1500 which is
configured to couple with and contact the end of the sensor 1420 at
contact pads 423, 425, 443, 444. The sensor meter typically
includes a potentiostat or other component to provide a potential
and/or current for the electrodes of the sensor. The sensor reader
also typically includes a processor (e.g., a microprocessor or
hardware) for determining analyte concentration from the sensor
signals. The sensor meter also includes a display or a port for
coupling a display to the sensor. The display displays the sensor
signals and/or results determined from the sensor signals
including, for example, analyte concentration, rate of change of
analyte concentration, and/or the exceeding of a threshold analyte
concentration (indicating, for example, hypo- or
hyperglycemia).
[0165] One example of a suitable connector is shown in FIGS. 16A
and 16B, 17A and 17B, and 18A and 18B. Connector 1500 (which is
used to connect a sensor to a meter or other electrical device) is
generally a two part structure, having top portion 1510 and bottom
portion 1520 (see FIG. 16B). Positioned between and secured by top
portion 1510 and bottom portion 1520 are contact leads 1423, 1425,
1443, 1444 that provide electrical connection between the sensor
1420 and a meter. These leads 1423, 1425, 1443, 1444 have proximal
ends to physically contact pads 423, 425, 443, 444 (in FIGS. 5A and
5B, respectively) and distal ends to connect to any attached meter.
The end of the sensor 1420 which has the contact pads can be slid
into or mated with the connector 1500 by placing the sensor 1420
into slide area 1530, which provides a support for and retains
sensor 1420. It is typically important that the contact structures
of the connector 1500 make electrical contact with the correct pads
of the sensor so that the working electrode and counter
electrode(s) are correctly coupled to the meter.
[0166] One optional feature is an insertion monitor 1450, that is
illustrated in FIGS. 16A and 16B, which are top views of the sensor
prior to insertion into connector 1500. This conductive insertion
monitor is positioned on the non-conductive base substrate and has
a contact pad for electrical contact with a connector. The
insertion monitor is configured and arranged to close an electrical
circuit between two contact structures 1451 and 1452 when the
sensor is properly inserted into the connector. Proper insertion
into the connector 1500 means that the sensor strip 1420 is
inserted right side up, that the correct end of the strip is
inserted into the connector, and that the strip is inserted far
enough into the connector that reliable electrical connections are
made between the electrodes' contact pads on the strip and the
corresponding contact structures of the connector. Preferably, no
closed circuit is made unless all electrode pads have properly
contacted the contact structures of connector 1500. The insertion
monitor may have shapes other than a stripe across the width of the
sensor; for example, other designs include an individual dot, a
grid pattern, or may include stylistic features, such as words or
letters.
[0167] Because this insertion monitor 1450 is not at the end with
the contact regions for the electrodes, the insertion monitor 1450
does not require additional width space on the sensor. The width of
the contact pads 443, 425, 444, 423 is defined as the width on
which a lead could be placed that would result in an electrical
connection; typically, the contact width is the width of the
exposed contact area. In one embodiment, six contact lead
structures on the connector (i.e., 1451, 1443, 1425, 1444, 1423,
1452) can contact sensor 1420 in the same width as the four contact
pads (i.e., 443, 425, 444, 423). This concept of having contact
points on the sensor that occupy more width than the width of the
sensor may be used for any number of contact points; this may be
used with or without an insertion monitor.
[0168] As a particular example, four leads 1443, 1425, 1444, 1423
make contact with contact pads 443, 425, 444, 423. If each lead
and/or contact pad is one millimeter wide, a sensor of at least 4
mm wide is needed to make contact. Additional leads, such as those
for insertion monitor 1450 (i.e., contact leads 1451, 1452), can
make contact by having leads 1451, 1452 extend along the side of
leads 1443 and 1423 and then angle in toward the center of the
strip 1420 after the point where leads 1443, 1425, 1444, 1423
contact strip 1420. The insertion monitor stripe leads 1451, 1452
cross side edges of sensor 1420 to make contact with the sensor,
thus not requiring additional sensor width.
[0169] The contact structures are parallel and non-overlapping and
they run longitudinally from the distal end to the proximal end.
The lead structures 1443, 1425, 1444, 1423 terminate at their
proximal end, but lead structures 1451, 1452 continue
longitudinally past the proximal end of lead structures 1443, 1425,
1444, 1423. Once past the proximal end, lead structures 1452, 1452
angle in toward the center of the sensor strip.
[0170] The insertion monitor can also be used to encode information
on a test strip. The encoded information can be, for example,
calibration information for that manufacturing lot or for that
specific strip. Such calibration information may relate to the
sensitivity of the strip or to the y-intercept of its calibration
curve. In one embodiment, the insertion monitor comprises two or
more contact pads for connection to a meter. The two or more
contact pads are connected to each other by a conductive material,
such as a conductive ink. The resistance of conductive path between
the two or more contact pads is related to the encoded information.
As an example of discrete calibration values, resistance values in
a given range can correspond to one calibration setting, and
resistance values in a different range can correspond to a
different calibration setting. As an example of continuous
calibration values, the calibration setting may be a continuous
function of the resistance. Examples of suitable conductive
pathways between contact pads are shown in FIGS. 19A through
19L.
[0171] In FIGS. 19A-19F, multiple conductive pathways with
different resistances are printed on the strip. The resistance of
the pathway is varied by cutting or scoring some or all of the
conductive pathways so that they do not carry charge. In FIGS.
19G-19L, the resistance is controlled by the width or length of the
conductive path. While it is preferred to provide this encoded
information on the insertion monitor, it should be recognized that
the insertion monitor function and the encoding of information can
also be implemented separately using separate conductive traces on
the strip.
[0172] In another embodiment to ensure proper insertion of a sensor
into a meter, the meter may include a raised area or bump that
prevents or hinders the insertion of the sensor in an improper
direction, as shown in FIG. 20. To ensure proper insertion of the
sensor 1100 into a connector 1110, the connector 1110 may include a
raised area 1140 that prevents or hinders the insertion of the
sensor in an improper direction. For example, the width, w2, of the
contact region of the second substrate 1130 may be wider than the
width, w1, of the contact region of the first substrate 1120. In
this instance, the raised area 1140 is positioned to allow sensor
1100 to be slid into the connector so that the first substrate 1120
is next to the surface 1150 from which the raised area 1140
protrudes, but would prevent or hinder having the second substrate
1130 next to the surface 1150 from which the raised area 1140
protrudes. Objects other than a raised area can also be used to
guide the user in correct introduction of the sensor into the
meter.
[0173] Integrated Sample Acquisition and Analyte Measurement
Device
[0174] An analyte measurement device constructed according to the
principles of the present invention typically includes a sensor, as
described hereinabove, combined with a sample acquisition apparatus
to provide an integrated sampling and measurement device. The
sample acquisition apparatus typically includes, for example, a
skin piercing member, such as a lancet, that can be injected into a
patient's skin to cause blood flow. In a preferred embodiment, the
integrated sample acquisition and analyte measurement device
comprises a lancing instrument that holds a lancet and measurement
strip. The lancing instrument preferably requires active cocking.
By requiring the user to cock the device prior to use, the risk of
inadvertently triggering the lancet is minimized. Preferably, the
lancing instrument will also permit the user to adjust the depth of
penetration of the lancet into the skin. Such devices are already
commercially available from companies such as Boehringer Mannheim
and Palco. This feature allows users to adjust the lancing device
for differences in skin thickness, skin durability, and pain
sensitivity across different sites on the body and across different
users.
[0175] Typically, a larger sample of body fluid such as blood or
interstitial fluid is expressed when pressure is applied around a
site where a hole has been created the skin. A sample acquisition
device having a protruding ring, such as illustrated in FIG. 21,
may be used. Ring 1200 circumferentially surrounds the lancing site
to create increased pressure and extract a larger sample out of the
wound. A lancet would protrude from the center 1220 of ring 1200.
Ring 1200 has a surface area 1210 that presses against the skin,
thus increasing the tension on the skin and often providing a bump
of skin. Skin that is more taught, and even extended, provides a
large volume of fluid from the wound. The increased surface area
1210 and tighter skin provided by ring 1200, decreases the amount
of pain experienced by the patient during the lancing. Further, the
tendency to bruise is typically decreased.
[0176] In one embodiment, the lancing instrument and the meter are
integrated into a single device. To operate the device the user
need only insert a disposable cartridge containing a measurement
sensor and lancing device into the integrated device, cock the
lancing instrument, press it against the skin to activate it, and
read the result of the measurement. Such an integrated lancing
instrument and test reader simplifies the testing procedure for the
user and minimizes the handling of body fluids.
[0177] Operation of the Sensor and Meter
[0178] An electrochemical sensor of the invention can be operated
with or without applying a potential across the electrodes. In one
embodiment, the electrochemical reaction occurs spontaneously and a
potential need not be applied between the working and counter
electrodes. In another embodiment, a potential, which may or may
not remain constant, is applied between the working and counter
electrodes. The magnitude of the required potential depends on the
redox mediator used. The potential at which the electrode poises
itself, or where it is poised by applying an external bias, and
where the analyte is electrolyzed, is typically such that the
electrochemical reaction is driven to or near completion, but it
is, preferably, not oxidizing enough to result in significant
electrochemical reaction of interferents, such as urate, ascorbate,
and acetaminophen, that can affect the signal measured. For
non-leachable redox mediators, the potential is typically between
about -350 mV and about +400 mV versus the standard calomel
electrode (SCE). Preferably, the potential of the redox mediator is
more negative than +100 mV, more preferably the potential is more
negative than 0 mV, and most preferably the potential is about -150
mV versus SCE.
[0179] When an external potential is applied, it can be applied
either before or after the sample has been placed in the sample
chamber. If the measurement zone includes only a portion of the
sample chamber then the potential is preferably applied after the
sample has come to rest in the sample chamber to prevent
electrolysis of sample passing through the measurement zone as the
sample chamber is filling. Alternatively, in the case where the
measurement zone includes most or all of the sample chamber, the
potential can be applied before or during the filling of the sample
chamber without affecting the accuracy of the assay. When the
potential is applied and the sample is in the measurement zone, an
electrical current will flow between the working electrode and the
counter electrode. The current is a result, at least in part, of
the electrolysis of the analyte in the sample. This electrochemical
reaction occurs via the redox mediator and the optional second
electron transfer agent. For many biomolecules, B, the process is
described by the following reaction equations: 1
nA(red).fwdarw.nA(ox)+ne (2)
[0180] Biochemical B is oxidized to C by redox mediator A in the
presence of an appropriate enzyme. Then the redox mediator A is
oxidized at the electrode. Electrons are collected by the electrode
and the resulting current is measured. The measured current may
also include a background current resulting in a measured
background charge, due, at least in part, to the shuttling of a
diffusible redox mediator between the working electrode and the
counter electrode. This background current can be minimized or
accounted for, as described in PCT Application PCT/US99/23425,
incorporated herein by reference.
[0181] A variety of techniques can be used to determine the
concentration of an analyte in a sample. These techniques include,
for example, coulometry, amperometry, and potentiometry. To
determine the concentration of analyte (e.g., glucose) in the
sample by coulometry, the charge passing or projected to pass
between the working electrode(s) and counter electrode(s) during
electrolysis of the analyte is determined. Knowledge of the charge
and the volume of the sample chamber permit the calculation of the
concentration of the electrolyzed analyte in the sample. This
charge can be determined by several methods.
[0182] For example, the charge can be measured directly. This can
be accomplished using a coulometer and known coulometric
techniques. Typically, the charge is measured during the complete
or nearly complete electrolysis of the analyte.
[0183] As another example, the charge can be determined from
measurements of the electrolysis current, i.sub.t, over time, t. A
series of currents (i.sub.x, i.sub.x+1, i.sub.x+2, . . . ) is
measured for a series of times (t.sub.x, t.sub.x+1, t.sub.x+2, . .
. ). The current can then be integrated (e.g., numerically
integrated using known numerical methods) to give the charge. In
some instances, current can be measured during the entire
electrolysis. In other instances, current can be extrapolated after
a period of time and the charge determined using a combination of
the measured currents and extrapolated currents.
[0184] Extrapolation of current utilizes a projected relationship
between current and time during at least a portion of the
electrolysis. Typically, when the system is diffusion limited, the
natural logarithm of the current is linearly related to the time.
This typically occurs after a period of time during which the
sensor "equilibrates". FIG. 15A is an example of a graph of current
versus time for electrolysis of an analyte, such as glucose, in one
of the previously described sensors; FIG. 15B is a graph of the
natural logarithm of current versus time for the same data. After
the sample completely fills the sample chamber, the current
increases to a peak current value, designated as "P" in FIGS. 15A
and 15B, during which time the sensor is equilibrating. The peak
current typically occurs during a period of time in which the
system is kinetically, rather than diffusionally, limited.
Typically, the current then begins to decrease, although, in some
instances, further increases may occur before the current becomes
diffusion limited. Eventually, the current values enter a region,
designated as "L" in FIG. 15B, where there is a linear relationship
between the natural logarithm of current and time. The remaining
current needed to electrolyze a remaining portion of the analyte
can then be extrapolated using estimation methods, such as
nonlinear or, preferably, linear estimation methods (e.g., linear
least squares techniques). The region where extrapolation occurs is
designated as "E" in FIG. 15B and the extrapolation is indicated as
a solid line.
[0185] For this discussion, the current values being referred to
are the absolute value of the actual current, thus, the sign of the
current measurement is ignored. For example, the peak current is
the largest deviation from zero current, whether positive or
negative.
[0186] During the period when the system is diffusion limited the
relationship between the natural logarithm of the current and time
is linear. The remaining charge required to completely electrolyze
the analyte remaining in the sample can be reliably estimated from
the slope of this line. Care must be taken to ensure that the
system is in the diffusion limited range before performing the
estimation. One method of ensuring that the system is in the
diffusion limited regime includes observing the current values
until the peak current, i.sub.peak, ("P") is achieved. Current
values continue to be observed until the current drops below a
threshold value, i.sub.thresh, that is typically a fraction of the
peak current. For example, the threshold value can be one half, one
third, or one quarter of the peak current (e.g.,
i.sub.thresh=j*i.sub.pea- k where j is, for example, 0.5, 0.33, or
0.25). The threshold value is chosen based on the characteristics
of the sensor so that one has a high degree of confidence that the
system will be diffusion limited at this time.
[0187] Many other methods may be used to ensure that the system is
in a diffusion limited regime. For example, one could observe the
current values after i.sub.peak until an inflection point is
reached and the second derivative of the natural log of the current
turns positive. At this point the system is typically in a
diffusion limited regime.
[0188] After achieving the threshold value, slope values are
determined using natural logarithms of two or more of the measured
current values (e.g.,
m.sub.x=(log(i.sub.x)-log(i.sub.x-y))/(t.sub.x-t.sub.x-y) where
m.sub.x is the slope at time t.sub.x, i.sub.x is the current at
time t.sub.x, and i.sub.x-y is the current at time t.sub.x-y before
t.sub.x). Although the slope can be determined using natural
logarithms of any two current values (e.g., current values measured
one directly after the other), preferably the slope is determined
using natural logarithms of multiple consecutive current values or
current values that are spaced apart by at least about ten (i.e.,
y=10), and, preferably, at least about thirty (i.e., y=30), current
values. This procedure can reduce the effect of random noise in the
system. Optionally, the consistency of multiple slope values (e.g.,
three or five or ten slope values) can be observed for
verification. For example, the system can look for ten
monotonically decreasing slope values before accepting the slope
value for use in the estimation.
[0189] The determined slope value is used to extrapolate the charge
required to completely electrolyze the analyte remaining in the
sample. That is, the current is extrapolated to zero current. Known
extrapolation techniques (e.g., linear extrapolation techniques)
can be used. The total charge required to electrolyze the analyte
is determined by adding the measured charge and extrapolated
charge.
[0190] Current values (or other measured values) can also be used
to monitor the operation of the sensor and meter. For example,
ratios of current values can be checked to determine if there has
been some failure of the measurement (e.g., the sensor has been
pulled out of the measuring device, etc.). The acceptable ratio
range will typically depend on the type and components of the
sensor, the rate at which measurements are made, and the acceptable
noise level. As an example, an acceptable ratio range could be, for
example, 2:1 to 1:2 for immediately adjacent measurements.
[0191] The discussion above has focused on the extrapolation of
charge required to complete electrolysis of an analyte in a sample
using the slope of the natural logarithm of current versus time
under diffusion limited conditions. Those skilled in the art will
recognize that this slope is related to a Effective Diffusion
Coefficient, and that other linear or non-linear mathematical
functions related to the Effective Diffusion Coefficient can also
be used to extrapolate the charge.
[0192] The extrapolation of current measurements and the
determination of charge passed or projected to pass, as well as the
concentration of the analyte, and other functions can be performed
by a processor with or without a storage medium, in which the
desired procedures are performed by software, hardware, or a
combination thereof. According to another embodiment, these same
procedures are accomplished using discrete or semi-programmable
hardware configured, for example, using a hardware descriptive
language, such as Verilog. In yet another embodiment, the
procedures are performed using a processor having at least one
look-up table arrangement with data stored therein to represent the
complete result or partial results of the above equations based on
a given set of input data.
[0193] Current measurements can be made by a variety of methods.
For example, the current measurements can be made by measuring the
current directly using any known techniques and devices.
[0194] Another method of measuring current includes discharging a
known amount of charge into the sample (by electrolysis of the
analyte) and measuring the time required for the discharge. Current
is then calculated as the quotient of charge and discharge time. As
an example, a capacitor can be charged by circuitry in the meter
and then coupled to the working or counter electrode(s) to
discharge by electrolysis of the analyte. The time to discharge to
a threshold level can be measured using, for example, a clock
circuit that is part of the processing circuitry of the meter. The
use of a clock circuit permits very accurate measurements of time.
This can be advantageous over designs that directly measure current
or charge and must convert these analog measurements to processable
digital representations using the more expensive A/D
(analog-to-digital) converters.
[0195] The accuracy of the charge and time measurement, which
provides the current values, affects the accuracy of the final
analyte concentration measurement. Although it is desired to use a
high-quality and highly accurate measuring device, the capacitor
used to store the charge can be a high-quality, expensive capacitor
that has a precisely known capacitance. Alternatively, an accurate
charge measurement can be obtained by using a calibrating resistor
in parallel with the capacitor. During a calibration period, the
capacitor discharges through the resistor and the current through
the resistor or the time constant of the discharge can be
determined. This can then be used to determine the charge stored in
the capacitor. A high-quality, very accurate resistor is typically
less expensive than a similarly accurate capacitor, and will
provide the same amount of accuracy to the charge measurement.
[0196] The charge, Q, no matter how determined, is then used to
calculate the concentration of the analyte (C.sub.A) by the
following equation (when the redox mediator is non-leachable):
C.sub.A=Q/nFV (3a)
[0197] where n is the number of electron equivalents required to
electrolyze the analyte, F is Faraday's constant (approximately
96,500 coulombs per equivalent), and V is the volume of sample in
the measurement zone. When using a diffusible mediator, the
concentration of the analyte can be obtained from the following
equation:
C.sub.A=(Q.sub.tot-Q.sub.back)/nFV (3b)
[0198] where Q.sub.tot is the total charge transferred during the
measurement and Q.sub.back is the amount of charge transferred that
was not due to the analyte, e.g., charge transferred by the
shuttling of the diffusible mediator between the working electrode
and the counter electrode. In at least some instances, the sensor
is constructed so that the background charge is at most 5 times the
size of the charge generated by electrolysis of an amount of
analyte. Preferably, the background signal is at most 200%, 100%,
50%, 25%, 10%, or 5% of the charge generated by electrolysis of the
analyte.
[0199] One example of a method for determining the ratio of
background signal to signal generated by electrolysis of the
analyte is described as follows. If the shuttling of the redox
mediator is not disabled by the applied potential, the charge that
results from the shuttling may be represented by the following
formula:
Q.sub.back=(A F D.sub.M C.sub.M/d)(t n.sub.M)
[0200] where A is the area of the working electrode; F is Faraday's
constant (96,500 coulombs/equivalent); D.sub.M is the effective
diffusion coefficient of the redox mediator; C.sub.M is the
concentration of the redox mediator in the measurement zone; d is
the distance separating facing electrodes; t is the amount of time
for the measurement; and n.sub.M is the number of electrons gained
or lost by the redox mediator.
[0201] Additionally, the charge of the analyte, when the analyte is
electrooxidized to about 90% completion in the measurement period,
may be represented by the following formula:
Q.sub.G=A d(0.90)C.sub.G n.sub.G F
[0202] where A is the area of the working electrode; d is the
distance separating facing electrodes; C.sub.G is the concentration
of glucose; n is the number of electrons needed to electrolyze the
analyte (e.g., 2 electrons per glucose molecule); and F is
Faraday's constant. For glucose, when C.sub.G is 5 mM (or
5.times.10.sup.-6 moles/cm.sup.3), t is 60 seconds, n.sub.G is 2,
and n.sub.M is 1, the ratio of charge from the redox mediator to
the charge from electrooxidation of the analyte may be represented
by the following formula:
[0203] Q.sub.Back/Q.sub.G=(D.sub.M C.sub.M/d.sup.2)(t n.sub.M/(0.9
n.sub.G C.sub.G))=(D.sub.M
C.sub.M/d.sup.2).times.(6.7.times.10.sup.6)
[0204] For example, if the ratio of Q.sub.Back/Q.sub.G is 5, then
(D.sub.M C.sub.M)/d.sup.2 is 7.5.times.10.sup.-7 moles/(cm.sup.3
sec). Also for example, if the ratio of Q.sub.Back/Q.sub.G is 1,
then (D.sub.M C.sub.M)/d.sup.2 is 1.5.times.10.sup.-7
moles/(cm.sup.3 sec). Still another example, if the ratio is 0. 1,
then (D.sub.M C.sub.M)/d.sup.2 is 1.5.times.10.sup.-8
moles/(cm.sup.3 sec). Thus, depending on the ratio desired, a
sensor may be configured to have the desired ratio by choosing
D.sub.M, C.sub.M, and d accordingly.
[0205] The ratio can be affected, for example, by reducing the
concentration of the redox mediator (i.e., C.sub.M may be reduced).
Alternatively, or additionally, the diffusion of the redox mediator
may be reduced. Other sensor configurations are also suitable for
controlling the ratio of background signal to signal generated by
the analyte and will be described below.
[0206] The background charge, Q.sub.back, can be accounted for in a
variety of ways. Q.sub.back can be made small, for example, by
using only limited amounts of diffusible redox mediator; by
providing a membrane over the counter electrode that limits
diffusion of the redox mediator to the counter electrode; or by
having a relatively small potential difference between the working
electrode and the counter electrode. Other examples of sensor
configurations and methods suitable for reducing Q.sub.back include
having a redox mediator reaction rate at the working electrode that
is significantly faster than that at the counter electrode;
immobilizing the redox mediator on the working electrode; having
the redox mediator become immobilized on the counter or
counter/reference electrode upon its reaction at the counter or
counter/reference electrode; or slowing the diffusion of the redox
mediator.
[0207] For coulometric measurements, at least 20% of the analyte is
electrolyzed, preferably at least 50%, more preferably at least
80%, and even more preferably at least 90% of the analyte is
electrolyzed. In one embodiment of the invention, the analyte is
completely or nearly completely electrolyzed. The charge can then
be calculated from current measurements made during the
electrochemical reaction, and the concentration of the analyte is
determined using equation (3a) or (3b). The completion of the
electrochemical reaction is typically signaled when the current
reaches a steady-state value. This indicates that all or nearly all
of the analyte has been electrolyzed. For this type of measurement,
at least 90% of the analyte is typically electrolyzed, preferably,
at least 95% of the analyte is electrolyzed and, more preferably,
at least 99% of the analyte is electrolyzed.
[0208] For coulometry, it is typically desirable that the analyte
be electrolyzed quickly. The speed of the electrochemical reaction
depends on several factors, including the potential that is applied
between the electrodes and the kinetics of reactions (1) and (2).
Other significant factors include the size of the measurement zone.
In general, the larger the potential, the larger the current
through the cell and therefore, the faster the reaction will
typically occur. However, if the potential is too large, other
electrochemical reactions may introduce significant error in the
measurement. Typically, the potential between the electrodes as
well as the specific redox mediator and optional second electron
transfer agent are chosen so that the analyte will be almost
completely electrolyzed in less than 5 minutes, based on the
expected concentration of the analyte in the sample. Preferably,
the analyte will be almost completely electrolyzed within about 2
minutes and, more preferably, within about 1 minute.
[0209] Although coulometry has the disadvantage of requiring the
volume of the measured sample be known, coulometry is a preferred
technique for the analysis of the small sample because it has the
advantages of, for example, no temperature dependence for the
measurement, no enzyme activity dependence for the measurement, no
redox-mediator activity dependence for the measurement, and no
error in the measurement from depletion of analyte in the sample.
As already described above, coulometry is a method for determining
the amount of charge passed or projected to pass during complete or
nearly complete electrolysis of the analyte. One coulometric
technique involves electrolyzing the analyte on a working electrode
and measuring the resulting current between the working electrode
and a counter electrode at two or more times during the
electrolysis. The electrolysis is complete when the current reaches
a steady state. The charge used to electrolyze the sample is then
calculated by integrating the measured currents over time and
accounting for any background signal. Because the charge is
directly related to the amount of analyte in the sample there is no
temperature dependence of the measurement. In addition, the
activity of the enzyme does not affect the value of the
measurement, but only the time required to obtain the measurement
(i.e., less active enzyme requires a longer time to achieve
complete electrolysis of the sample) so that decay of the enzyme
over time will not render the analyte concentration determination
inaccurate. And finally, the depletion of the analyte in the sample
by electrolysis is not a source of error, but rather the objective
of the technique. (However, the analyte need not be completely
electrolyzed if the electrolysis curve is extrapolated from the
partial electrolysis curve based on well-known electrochemical
principles.)
[0210] It may be desirable in some instances to utilize
non-coulometric assays, such as amperometric or potentiometric
measurement techniques. "Amperometry" and "chronopotentiometry"
refer to taking a potentiometric measurement at one or more points
in time. These measurement techniques are useful when the volume of
the measured sample is unknown; the volume of the sample in the
measurement zone of a small volume sensor (i.e., no more than one
microliter) may be difficult to accurately reproduce if the
manufacturing tolerances of one or more dimensions of the
measurement zone have significant variances. For information
regarding using non-coulometric measuring, such as amperometric and
potentiometric techniques, see, for example, U.S. application Ser.
No. 09/295,962, filed Apr. 21, 1999, now issued as U.S. Pat. No.
6,338,790, and its continuations U.S. Pat. Nos. 6,299,757 and
6,461,496, all incorporated herein by reference in its
entirety.
[0211] Heating of Sample
[0212] The sample can be heated to increase the rate of diffusion,
oxidation, or reduction of the analyte. This heating can be
accomplished by a variety of techniques including placing the
sensor in a heated environment or applying a heating unit to the
sensor.
[0213] Another technique includes providing a thermal heating
element, such as, for example, a wire or an ink that is capable of
converting electrical energy into heat energy, on the sensor. This
wire or ink can be applied, for example, on the opposite side of a
base material, such as a polymer film, from one or more of the
working, counter, reference, or counter/reference electrodes, or
applied around the periphery of the working, counter, reference, or
counter/reference electrodes. In some instances, the sample is
heated up to 5 to 20.degree. C. above an initial temperature. In
other instances, the temperature of the sample may not be known but
a constant amount of power or current is applied to the wire or
ink.
GENERAL EXAMPLES
[0214] The invention will be further characterized by the following
general examples. These examples are not meant to limit the scope
of the invention which has been fully set forth in the foregoing
description. Variations within the concepts of the invention are
apparent to those skilled in the art.
[0215] One method for manufacturing the sensor shown in FIGS. 5A
through 5C, having a working electrode, multiple counter/reference
electrodes, and a side filled sample chamber, is described below.
According to the method described herein, a plurality of sensors
are produced and then separated.
[0216] A first non-conducting substrate, preferably polyester, is
provided. A conductive carbon ink, such as that available from
Ercon of Wareham, Mass. under the trade designation "Graphite
#G4491", is screen printed onto a first half of the first polymeric
substrate in the pattern shown in FIG. 5A and dried, to provide the
working electrode 422, trace 453 and contact pad 423. Once the
substrate web is cut to the final size, each working electrode 422
will extend across the width of the sensor and have a trace 453
that extends to the far end of the sensor. As an example, a sensor
can be made with a width of about 6.1 mm (0.240 inch). Typical
sensor widths are about 2 mm to 20 mm, preferably about 4 mm to 10
mm, but wider or narrower sensors can be used as desired.
[0217] A sensing layer, as described in U.S. patent application
Ser. No. 09/295,962, now U.S. Pat. No. 6,338,790, comprising a
diffusible mediator and diffusible glucose dehydrogenase enzyme, is
applied to the working electrode in a sample chamber region. The
sensing layer can be provided on the electrode prior to or
subsequent to providing the sample chamber. On the other half of
the polyester substrate, a conductive silver/silver chloride ink,
such as that commercially available from Ercon under the trade
designation "Silver/Silver Chloride #R414", is screen printed onto
a second half of the first polymeric substrate in the pattern shown
in FIG. 5B and dried. In the embodiment shown in FIG. 5B, the
Ag/AgCl ink provides a first indicator or reference electrode 441,
a counter electrode 424, and a second indicator electrode 442.
Counter electrode may extend across the entire sensor surface, or
may extend only a portion thereof. As an example, the width of a
counter electrode is about 1 mm to 20 mm, preferably about 2 to 10
mm. In one sensor configuration, the counter electrode 424 has a
width of about 3.71 mm (0.146 inch). The indicator electrodes can
have a width, for example, of about 0.1 mm to 5 mm, preferably
about 0.25 mm to about 1 mm.
[0218] In one sensor configuration, working electrode, such as
working electrode 422 shown in FIG. 5A, extends the width of the
sensor, which is about 6.1 mm (0.240 inch). A counter electrode,
such as counter electrode 424, has a width of about 3.71 mm (0.146
inch). Indicator electrodes, such as indicator electrodes 441, 442
in FIG. 5B, each can have a width of about 0.51 mm (0.020 inch). A
spacing of 0.30 mm (0.012 inch) is present between these indicator
electrodes and the counter electrode. The indicator electrodes may
be each set back from the eventual side edge of the sensor strip,
for example, by approximately 0.38 mm (0.015 inch), so that the
electrodes 441, 442 are not damaged, for example, smeared,
streaked, chipped or the like, by the process of slitting the
substrate into individual sensors.
[0219] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it will
be apparent to one of ordinarily skill in the art that many
variations and modifications may be made while remaining within the
spirit and scope of the invention.
[0220] All publications and patent applications in this
specification are indicative of the level of ordinary skill in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually incorporated by reference.
* * * * *