U.S. patent application number 11/710329 was filed with the patent office on 2007-08-30 for flux limiting membrane for intravenous amperometric biosensor.
Invention is credited to Kenneth M. Curry.
Application Number | 20070202562 11/710329 |
Document ID | / |
Family ID | 38268740 |
Filed Date | 2007-08-30 |
United States Patent
Application |
20070202562 |
Kind Code |
A1 |
Curry; Kenneth M. |
August 30, 2007 |
Flux limiting membrane for intravenous amperometric biosensor
Abstract
A flux limiting layer for an intravenous amperometric biosensor
is formed on a substrate to limit a diffusion rate of an analyte
from blood to an enzyme electrode. The layer may be formed from
ethylene vinylacetate (EVA) dissolved in a solvent such as
paraxylene, spray-coated to cover a portion of the electrode, and
cured to seal the electrode to the substrate. In a glucose sensor
having glucose oxidase disposed on the electrode, thickness and
concentration of the EVA layer are optimized to promote a linear
output of electrode current as a function of blood glucose
concentration.
Inventors: |
Curry; Kenneth M.;
(Oceanside, CA) |
Correspondence
Address: |
EDWARDS LIFESCIENCES CORPORATION
LEGAL DEPARTMENT, ONE EDWARDS WAY
IRVINE
CA
92614
US
|
Family ID: |
38268740 |
Appl. No.: |
11/710329 |
Filed: |
February 23, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60777139 |
Feb 27, 2006 |
|
|
|
Current U.S.
Class: |
435/14 ;
600/316 |
Current CPC
Class: |
A61B 5/14532 20130101;
A61B 5/14865 20130101 |
Class at
Publication: |
435/14 ;
600/316 |
International
Class: |
C12Q 1/54 20060101
C12Q001/54; A61B 5/00 20060101 A61B005/00 |
Claims
1. A flux limiting membrane for an intravenous amperometric
biosensor, comprising: an ethylene vinylacetate (EVA) polymer at
least partially coating a reagent disposed on an electrode of the
biosensor to limit a rate at which a reactant from blood diffuses
to the reagent.
2. The flux limiting membrane of claim 1, wherein the reactant
comprises glucose.
3. The flux limiting membrane of claim 1, wherein the EVA polymer
is deposited from a solution comprising EVA dissolved in a
solvent.
4. The flux limiting membrane of claim 3, wherein the solution
comprises between about 0.5 wt % and about 6.0 wt % of an EVA
composition.
5. The flux limiting membrane of claim 4, wherein the EVA
composition has a vinyl acetate content between about 9 wt % and
about 50 wt %.
6. The flux limiting membrane of claim 3, wherein the solvent is
selected from the group comprising cyclohexanone, paraxylene, and
tetrahydrofuran.
7. The flux limiting membrane of claim 1, wherein the EVA polymer
comprises an average diffusion layer thickness between about 0.5
microns and about 10 microns.
8. The flux limiting membrane of claim 1, further comprising a
biocompatibility layer.
9. The flux limiting membrane of claim 8, wherein the
biocompatibility layer comprises heparin.
10. The flux limiting membrane of claim 1, further comprising
poly(methylmethacrylate-co-butyl methacrylate) blended with the EVA
polymer.
11. The flux limiting membrane of claim 1, wherein the EVA polymer
is cross-linked with diglycidil ether.
12. The flux-limiting membrane of claim 1, wherein the EVA polymer
is cross-linked with a diisocyanate.
13. An intravenous amperometric biosensor, comprising: a substrate;
an electrode bonded to the substrate; a reagent disposed on the
electrode; and an ethylene vinylacetate (EVA) flux limiting
membrane at least partially coating the reagent to limit a rate at
which a reactant from blood diffuses to the reagent.
14. The biosensor of claim 13, wherein the reagent comprises
glucose oxidase and the reactant comprises glucose.
15. The biosensor of claim 13, wherein the EVA flux limiting
membrane adheres to a portion of the electrode.
16. The biosensor of claim 13, wherein the EVA flux limiting
membrane seals the electrode to the substrate.
17. The biosensor of claim 13, wherein the EVA flux limiting
membrane is deposited from a solution comprising EVA dissolved in a
solvent.
18. The biosensor of claim 17, wherein the solution comprises
between about 0.5 wt % and about 6.0 wt % of an EVA
composition.
19. The biosensor of claim 18, wherein the EVA composition has a
vinyl acetate content between about 9 wt % and about 40 wt %.
20. The biosensor of claim 17, wherein the solvent is selected from
the group comprising cyclohexanone, paraxylene, and
tetrahydrofuran.
21. The biosensor of claim 13, wherein the EVA flux limiting
membrane comprises an average diffusion layer thickness between
about 0.5 microns and about 10 microns.
22. The biosensor of claim 13, further comprising
poly(methylmethacrylate-co-butyl methacrylate) blended with the EVA
flux limiting membrane.
23. The biosensor of claim 13, wherein the EVA flux limiting
membrane is cross-linked with diglycidil ether.
24. The biosensor of claim 13, wherein the EVA flux limiting
membrane is cross-linked with a diisocyanate.
25. On a biosensor having an enzyme electrode disposed on a
substrate, a method for forming a flux limiting membrane on the
enzyme electrode, comprising: dissolving ethylene vinylacetate
(EVA) in a solvent; applying a layer of the dissolved EVA to an
area of the substrate that includes at least a portion of the
enzyme electrode; and curing the applied layer.
26. The method of claim 25, wherein the solvent is selected from
the group comprising cyclohexanone, paraxylene, and
tetrahydrofuran.
27. The method of claim 25, wherein the dissolving step further
comprises dissolving between about 0.5 wt % and about 6.0 wt % of
an EVA composition in the solvent.
28. The method of claim 27, wherein the EVA composition has a vinyl
acetate content between about 9 wt % and about 40 wt %.
29. The method of claim 25, wherein the applying step comprises
spray-coating the EVA solution onto the area of the substrate.
30. The method of claim 25, wherein the applying step further
comprises creating a layer of the EVA solution having a thickness
between about 0.5 microns and about 10 microns.
Description
CLAIM OF PRIORITY UNDER 35 U.S.C. .sctn.119
[0001] The present Application for Patent claims priority to
Provisional Application No. 60/777,139 filed Feb. 27, 2006, and
assigned to the assignee hereof and hereby expressly incorporated
by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to amperometric biosensors for
measuring blood chemistry. In particular, the invention relates to
an intravenous sensor for measuring a biological parameter such as
blood glucose concentration.
BACKGROUND
[0003] Amperometric biosensors are known in the medical industry
for analyzing blood chemistry. Early biosensors, also known as
enzyme electrodes, were first proposed by Clark and Lyons and
implemented by Updike and Hicks. Enzyme electrodes typically
include an oxidase enzyme, such as glucose oxidase, that is
immobilized behind a dialysis membrane at the surface of an
electrode. In the presence of blood, the membrane selectively
passes an analyte of interest, e.g. glucose, to the oxidase enzyme
where it undergoes oxidation or reduction, e.g. the reduction of
oxygen to hydrogen peroxide. Amperometric biosensors function by
producing an electric current when a potential sufficient to
sustain the reaction is applied between two electrodes in the
presence of the reactants. For example, in the reaction of glucose
and glucose oxidase, the hydrogen peroxide reaction product may be
subsequently oxidized by electron transfer to an electrode. The
resulting flow of electrical current in the electrode is indicative
of the concentration of the analyte of interest.
[0004] Applications for amperometric biosensors include measuring
analytes in blood-borne gases, electrolyte levels in blood and in
particular, blood glucose concentration. For measuring glucose,
subcutaneous methods have been proposed. For example, see Renard,
"Implantable Glucose Sensors for Diabetes Monitoring," Minim
Invasive Ther Allied Technol, Vol. 13, No. 2, pp. 78-86 (2004).
While these minimally invasive glucose monitoring systems properly
display trends in plasma glucose concentration, they do not track
glucose accurately enough to be used for intensive insulin therapy,
for example, where inaccuracy at conditions of hypoglycemia could
pose a very high risk to the patient. In addition, sensors based
upon the enzyme glucose oxidase need to have access to adequate
oxygen to provide a linear glucose response. Sensor systems
optimized for subcutaneous tissue would not necessarily function
well in venous blood, where oxygen tension can be 20 mm Hg or
less.
[0005] At the present time, the most reliable way to obtain a
highly accurate blood glucose measurement in an ICU patient is by a
direct time-point method, which involves drawing a blood sample and
sending it off for laboratory analysis. This is a time-consuming
method that is often incapable of producing needed results in a
timely manner. Despite ongoing research in this field, many
improvements in glucose monitoring are still needed.
[0006] One of the difficulties impeding the development of an
intravenous amperometric sensor is that the sensor must be small
enough to be suspended within a blood vessel, but robust enough to
immobilize an enzyme so that a reaction may be sustained for a
sufficient length of time. An intravenous sensor must also be
biocompatible, such that it does not release any toxins into a
patient, and when implanted, e.g. through a catheter in a femoral
vein, discourages clotting of blood at the membrane surface that
would prevent plasma from diffusing to the enzyme layer.
SUMMARY
[0007] The invention discloses a biocompatible flux limiting
membrane for an amperometric biosensor designed for intravenous use
and continuous analyte monitoring. The flux limiting membrane may
be formed on a sensor electrode that is at least partially coated
with a reagent selected to react with a substance found in blood.
The flux limiting membrane limits a rate at which the substance
diffuses through the flux limiting membrane to react with the
reagent. The flux limiting membrane may include an ethylene
vinylacetate (EVA) polymer selected for its biocompatibility,
adhesion, physical, and diffusion properties. In one embodiment,
the membrane may include one or more cured layers of EVA that are
applied by spraying a solution having a percentage of EVA dissolved
in paraxylene.
[0008] An intravenous amperometric biosensor may be formed using
the EVA membrane as a flux limiting layer to at least partially
cover the surface of an enzyme electrode. The biosensor may be
formed on a flex circuit substrate having reference, counter, and
working electrodes mounted thereon, wherein one working electrode
may be the enzyme-bearing electrode. In one embodiment, the
biosensor may be a glucose sensor, the working electrode may be at
least partially coated with glucose oxidase, and an EVA membrane
may be formed on the working electrode to provide a flux limiting
barrier that selectively allows diffusion of oxygen and glucose
from blood to the glucose oxidase. Adhesive properties of EVA
mechanically seal the glucose oxidase to the electrode and the
electrode to the substrate to improve mechanical integrity during
intravenous insertion. The composition of the EVA membrane may be
optimized such that, when the biosensor is located intravenously
with the working electrode energized, the current output of the
working electrode is a linear function of blood glucose
concentration.
[0009] A related method is also disclosed for forming a flux
limiting layer on an enzyme electrode that is bonded to a substrate
of an amperometric biosensor. The method may include dissolving EVA
in a solvent such as paraxylene, applying a layer of the dissolved
EVA to an area of the substrate that includes at least a portion of
the enzyme electrode, and curing the applied layer. The EVA may be
dissolved in paraxylene to facilitate application by spray-coating,
and the thickness and concentration of the EVA membrane may be
optimized to promote a linear output of electrode current as a
function of blood glucose concentration.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The features, objects, and advantages of the invention will
become more apparent from the detailed description set forth below
when taken in conjunction with the drawings, wherein:
[0011] FIG. 1 shows an amperometric biosensor in the form of a flex
circuit having a working electrode coated with a flux limiting
membrane according to an embodiment of the invention.
[0012] FIG. 2 is a magnified side cross-sectional view of the
working electrode portion of the biosensor of FIG. 1, shown prior
to application of a flux limiting membrane according to an
embodiment of the invention.
[0013] FIG. 3 is a magnified cross-sectional view of the working
electrode portion of the biosensor of FIG. 1, shown after
application of the flux limiting membrane according to an
embodiment of the invention.
[0014] FIG. 4 is a process flow chart illustrating steps for
forming a flux limiting membrane on a biosensor substrate according
to an embodiment of the invention.
[0015] FIG. 5 is a graph of current output vs. glucose
concentration for biosensors formed with flux limiting membranes
according to an embodiment of the invention.
[0016] FIG. 6 is a graph of glucose assay results of the current
output over time covering multiple step changes in glucose
concentration, for a biosensor formed with flux limiting membranes
according to an embodiment of the invention.
[0017] FIG. 7 shows results of an acute in vivo swine test for
response of a glucose sensor having a flux limiting membrane
according to an embodiment of the invention.
DETAILED DESCRIPTION
[0018] The invention discloses an adhesive biocompatible polymer
for forming a flux-limiting membrane on an enzyme-bearing electrode
in an intravenous amperometric biosensor. When the sensor is
installed in a patient to measure blood chemistry, the membrane
improves sensor accuracy by allowing oxygen to pass from the blood
to the sensor while limiting the passage of larger molecules. The
biocompatibility of the membrane limits the number of toxins that
may be introduced into the bloodstream, and the adhesive properties
enhance the mechanical integrity of the sensor during installation
and operation.
[0019] In one embodiment, the membrane may be used on a glucose
sensor for limiting the amount of glucose flux from blood to the
sensor electrode. The biocompatible polymer that forms the membrane
may contain an optimized content of dissolved ethylene vinylacetate
(EVA). A solvent such as paraxylene may be used to dissolve the EVA
into a solution suitable for application to the electrode by
spray-coating or other means.
[0020] EVA is selected for its many properties that are
advantageous for forming a flux limiting membrane. EVA is a
biocompatible, linear polymer. Formed as a membrane layer on a
biosensor electrode, it can provide a general hydrophobic property
to accentuate oxygen transport, but with sufficient hydrophilic
segments to still allow for limited glucose transport. This is
important in intravenous applications, where glucose is in
preponderance in the blood plasma in comparison to free oxygen
molecules. Thus, an EVA membrane may provide a desired diffusion
barrier that passes an abundance of oxygen while restricting the
passage of glucose. In addition, an EVA polymer as applied herein
may provide a mechanically strong adhesive for coating an
enzyme-bearing electrode on a flex circuit substrate suitable for
long-term continuous intravenous use. Also, films of EVA are very
elastomeric, which is important in a situation where the electrode
may need to navigate a tortuous path, for example, into venous
anatomy. Moreover, the concentration of EVA and the thickness of
the applied layers may be controlled in the manufacturing process
to promote a linear output of electrode current as a function of
blood glucose concentration.
[0021] One application for a flux limiting membrane is in a
thin-film amperometric biosensor formed on a flex circuit. Flex
circuits have been applied in medical devices as microelectrode
substrates for in vivo applications. For example, one flex circuit
design uses a laminate of a conductive foil (e.g., copper) on a
flexible dielectric substrate (e.g., polyamide). The flex circuit
may be formed on the conductive foil using masking and
photolithography techniques. Flex circuits are desirable due to
their low manufacturing cost, ease in design integration, and
physical flexibility during transport in applications such as
central venous catheter (CVC) insertion.
[0022] FIG. 1 is an amperometric biosensor 11 in the form of a flex
circuit that incorporates a flux limiting membrane according to an
embodiment of the invention. The biosensor or sensor 11 may be
formed on a substrate 13 (e.g., a flex substrate). One or more
electrodes 15, 17 and 19 may be attached or bonded to a surface of
the substrate 13. The biosensor 11 is shown with a reference
electrode 15, a counter electrode 17, and a working electrode 19.
In another embodiment, one or more additional working electrodes
may be included on the substrate 13. Electrical wires 21 may
transmit power to the electrodes for sustaining an oxidation or
reduction reaction, and may also carry signal currents to a
detection circuit (not shown) indicative of a parameter being
measured. The parameter being measured may be any analyte of
interest that occurs in, or may be derived from, blood chemistry.
In one embodiment, the analyte of interest may be hydrogen
peroxide, formed from reaction of glucose with glucose oxidase,
thus having a concentration that is proportional to blood glucose
concentration.
[0023] The magnified cross-sectional side view of FIG. 2 shows a
distal portion of the substrate 13 in the vicinity of the working
electrode 19. The working electrode 19 may be at least partially
coated with a reagent or enzyme layer 23 that is selected to
chemically react when the sensor is exposed to certain reactants
found in the bloodstream. For example, in an embodiment for a
glucose biosensor, enzyme layer 23 may contain glucose oxidase,
such as may be derived from Aspergillus niger (EC 1.1.3.4), type II
or type VII.
[0024] To promote a reaction of the enzyme with blood, the enzyme
layer 23 may be formed within a matrix that is active on its
surface. This may be achieved, for example, by adding or
cross-linking the enzyme to an active hydrogel. The hydrogel layer
may be water absorbent, and swell to provide active transport of a
reactant in the blood (e.g. glucose) from the blood to the enzyme.
Intermolecular bonds may be formed throughout the hydrogel layer to
create adhesion and a density of matrix to allow for even
dispersion of the reagent across the hydrogel surface and
throughout the hydrogel layer. Reaction products may then be
communicated to the electrode layer.
[0025] FIG. 3 shows a magnified cross sectional side view of the
working electrode site on the sensor substrate 13. A flux limiting
membrane 25 is added onto the enzyme layer 23, such that the
membrane 25 at least partially covers the enzyme layer 23. With the
sensor 11 installed in an intravenous location, the flux limiting
membrane 25 may selectively allow diffusion, from blood to the
enzyme layer 23, a blood component that reacts with the enzyme. In
a glucose sensor embodiment, the flux limiting membrane 25 passes
an abundance of oxygen, and selectively limits glucose, to the
enzyme layer 23. In addition, a flux limiting membrane 25 that has
adhesive properties may mechanically seal the enzyme layer 23 to
the working electrode 19, and may also seal the working electrode
19 to the sensor substrate 13. It is herein disclosed that a flux
limiting membrane formed from an EVA polymer may serve as a flux
limiter at the top of the electrode, but also serve as a sealant or
encapsulant at the enzyme/electrode boundary and at the
electrode/substrate boundary. An additional biocompatible layer
(not shown), including a biocompatible anti-thrombotic substance
such as heparin, may be added onto the flux limiting membrane
25.
[0026] The sensor 11 works on an amperometric measurement
principle, where the working electrode 19 is held at a positive
potential relative to the counter electrode 17. The positive
potential is sufficient to sustain an oxidation reaction of
hydrogen peroxide, which is the result of a glucose reaction with
the glucose oxidase. Thus, the working electrode 19 functions as an
anode, and collects electrons produced at the surface of the
working electrode 19 that result from the oxidation reaction. The
collected electrons flow into the working electrode 19 as an
electrical current. In one embodiment with the working electrode
coated with glucose oxidase, the oxidation of glucose produces a
hydrogen peroxide molecule for every molecule of glucose, when the
working electrode 19 is held at a potential between about +450 mV
and about +650 mV. The hydrogen peroxide produced oxidizes at the
surface of the working electrode 19 according to the equation:
H.sub.2O.sub.2.fwdarw.2H.sup.++O.sub.2+2e.sup.-
[0027] The equation indicates that two electrons are produced for
every hydrogen peroxide molecule oxidized. Thus, under certain
conditions, the amount of electrical current may be proportional to
the hydrogen peroxide concentration. Since one hydrogen peroxide
molecule is produced for every glucose molecule oxidized at the
working electrode, a linear relationship may exist between the
blood glucose concentration and the resulting electrical current.
The reader may refer to the following article for additional
information on electronic sensing theory for amperometric glucose
biosensors: J. Wang, "Glucose Biosensors: 40 Years of Advances and
Challenges," Electroanaylsis, Vol. 13, No. 12, pp. 983-988
(2001).
[0028] To achieve the linear relationship or substantially linear
relationship, the working electrode 19 is designed to promote the
desired chemical reactions. In the amperometric sensor 11, the
chemistry may be controlled by applying one or more membranes, or
layers, of varying composition on the surface of a flex circuit
substrate. In one embodiment, the substrate 13 may be a polyimide
material, the enzyme layer 23 may be a cross-linked hydrogel, and
the flux limiting layer 25 may be an EVA polymer according to an
embodiment of the invention. EVA is selected, inter alia, for its
adhesive and biocompatible qualities in polymeric implant devices
for controlling drug delivery rates.
[0029] The substrate 13 provides an insulated structure for
mounting the electrodes and membrane layers. In one embodiment, the
substrate 13 may be between about 0.050 and 0.060 inches wide and
between about 1.0 and 2.0 inches long. The thickness of the
membrane layers may vary between about 0.5 microns and about 10
microns. In one embodiment, one or more of the flux limiting
membrane layers may have a thickness in the about 0.5 micron to
about 10 micron range.
[0030] The electrical wires 21 may be coupled or soldered to
conductive traces formed on the substrate 13 using flex circuit
technology. For example, the traces may be gold-plated copper. In
one embodiment, the sensor 11 may be designed so that the flex
circuit terminates to a tab that mates to a multi-pin connector,
such as a 3-pin, 1 mm pitch ZIF Molex connector. Such a connection
facilitates excitation of the working electrode and measurement of
electrical current signals, for example, using a potentiostat or
other controller.
[0031] The electrodes 15, 17 and 19 may be applied to the substrate
13 using a thick film process and commercially available inks. In
one embodiment, the reference electrode 15 may be a silver/silver
chloride type deposited or formed on the substrate 13. The
reference electrode 15 establishes a fixed potential from which the
potential of the counter electrode 17 and the working electrode 19
may be established. The reference potential is Nernstian. For the
silver/silver chloride electrode, the reference potential is
maintained by the following half-reaction:
Ag.sup.0.fwdarw.Ag.sup.++e.sup.-
[0032] The counter electrode 17 may be constructed from conductive
materials such as platinum or graphite. These materials may be
formulated as an ink for application to the substrate 13 using a
thick film process and cured accordingly. The counter electrode 17
provides a working area for conducting the majority of electrons
produced from the oxidation chemistry back to the blood solution.
Otherwise, all the current would likely pass through the reference
electrode 15, and may reduce its service life. In one embodiment,
the counter electrode 17 may be formed with a surface area greater
than that of the working electrode 19.
[0033] The working electrode 19 may be formed using
platinum/graphite materials similar to those used for forming the
counter electrode 17. In other embodiments, the working electrode
19 may be formed from other conductive materials. Its operation has
been described thus far as promoting anodic oxidation of hydrogen
peroxide at its surface. Other embodiments are possible, for
example, the working electrode 19 may be held at a negative
potential. In this case, the electrical current produced at the
working electrode 19 may result from reduction of oxygen.
[0034] In one embodiment, the biosensor 11 may be installed within
a probe or catheter for intravenous insertion into a patient, for
example, via a CVC. The biosensor 11 may function as an
amperometric sensor while immersed in a patient's bloodstream by
the addition of an enzyme-bearing hydrogel layer 23 to a surface of
the working electrode 19. The hydrogel layer 23 may be sealed to
the working electrode 19, and the working electrode 19 may be
sealed to the substrate 13, by using the flux limiting layer 25.
That is, in addition to its diffusion function, the flux limiting
layer 25 also serves to bond the hydrogel and electrode firmly to
the substrate 13.
[0035] Based on experimental trials, the substance of which is
disclosed in sections that follow, a method has been developed and
is herein disclosed as a series of process steps forming a flux
limiting EVA membrane on a biosensor electrode. FIG. 4 illustrates
one such embodiment of a method 400.
[0036] Method 400 includes step 402, in which EVA is dissolved in a
solvent. The EVA may be provided from a source having a composition
anywhere from about 9 wt % vinyl acetate (EVA-9) to about 40 wt %
vinyl acetate (EVA-40). The solvent should be chosen for its
ability to dissolve EVA, to promote adhesion to the biosensor
substrate and enzyme electrode, and to form a solution that may be
effectively applied (e.g. spray-coated). Solvents such as
cyclohexanone, paraxylene, and tetrahydrofuran may be suitable for
this purpose. In this step, the solution may include about 0.5 wt %
to about 6.0 wt % of the EVA composition. In addition, the solvent
is sufficiently volatile to evaporate without undue agitation to
prevent issues with the underlying enzyme, but not so volatile as
to create problems with the spray process.
[0037] Step 404 involves applying a layer of the EVA solution to an
area of the biosensor substrate to at least partially coat the
enzyme electrode. In one embodiment, step 404 may include
completely coating the enzyme electrode and sealing the electrode
to the biosensor substrate. Step 404 may be performed, for example,
by spraying the EVA solution onto the enzyme electrode area of the
substrate 13 to form a layer having a uniform or near uniform
thickness. Additional layers may be added in this step to achieve a
desired membrane thickness. Layers of EVA solution formed in this
step may also be applied by brushing, immersion, or similar
technique.
[0038] In step 406, the applied EVA layer or layers are cured to
form the flux limiting membrane. This step may be performed by
drying in ambient air, by curing in a low-temperature oven (between
about 30 and about 40 degrees C.), or alternatively, by annealing
at a temperature between about 50 and 65 degrees C. (preferably,
slightly below 65 degrees C.), which is the softening temperature
region of EVA. An additional step may be added to method 400, in
which the cured flux limiting membrane is coated with a
biocompatibility layer composed of a biocompatible material such as
heparin.
[0039] Using method 400, a population of eight prototype sensors
was fabricated with flux limiting layers and each sensor was tested
for glucose response. The prototype sensors were denoted G1, G2,
G3, G5, G6, G7, G8 and G9. Each sensor was fabricated on a flex
circuit configured with a platinum/graphite working electrode and a
silver/silver chloride reference electrode. A recess was formed in
the working electrode and filled with a glucose oxidase enzyme
layer. A 2.0 wt % solution of EVA-40 was prepared by dissolving in
tetrahydrofuran (THF). The working electrode area of each sensor
was then coated with a flux limiting layer by dipping the electrode
in the EVA solution one or more times and allowing the coating to
cure. Curing was performed either by air-drying at ambient
temperature or by oven-curing at about 59 degrees C. for ten
minutes. The table below indicates how each prototype sensor was
made, where the value under L indicates the number of times
(1.times. or 3.times.) each sensor was dipped in the EVA solution
to coat the working electrode, and the value under T indicates
whether the sensor was cured in ambient air (0) or in a 59-degree
C. oven for ten minutes (59/10).
TABLE-US-00001 SENSOR L T G1 3.times. 0 G2 1.times. 59/10 G3
1.times. 0 G5 3.times. 0 G6 1.times. 59/10 G7 1.times. 0 G8
3.times. 59/10 G9 3.times. 59/10
[0040] FIG. 5 shows the results of a glucose assay performed on
each prototype sensor to determine the linearity of response. Each
sensor was exposed to a solution of known glucose concentration,
and its working electrode excited at a potential of about 650 mV.
The resulting electrical current output in amperes in the working
electrode was then measured and plotted versus glucose
concentration in mg/dL. For each sensor, the current output was
measured at four values of glucose concentration: 0.00, 50.00,
100.00, and 150.00 mg/dL.
[0041] A linear regression technique was applied to the test data
to derive a slope for a theoretical line through each group of four
data points, and to determine the linearity of response. The
coefficient of multiple determination, r.sup.2, was computed for
each sensor to determine the adequacy of the linear regression
model, where
r.sup.2=SSR/SST
and where SSR represents the sum of squares due to regression, and
SST represents the total sum of squares. The results are tabulated
below:
TABLE-US-00002 SENSOR SLOPE r.sup.2 G1 1.24E-10 0.8563 G2 3.42E-10
0.9994 G3 3.37E-10 0.9998 G5 2.95E-10 0.9813 G6 3.44E-10 0.9981 G7
2.11E-10 0.9581 G8 2.21E-10 0.9480 G9 2.28E-10 0.9701
[0042] The results indicate excellent linearity, with r.sup.2
values for eight prototype sensors varying between 0.8563 and
0.9998, including three sensors having r.sup.2 values greater than
0.9990. Exact values for the average thickness of the flux limiting
layer were difficult to determine due to inconsistencies in
membrane thickness achieved using the dipping technique.
[0043] The graph of FIG. 6 shows a plot of the results of the
glucose assay on sensor G6 as a function of current output over
time. For simplicity, FIG. 6 includes only the curve for sensor G6
as being representative of the behavior of all eight sensors. The
time period shown covers the three step changes in glucose
concentration that correspond to the 50, 100, and 150 mg/dL
concentrations. These step changes occurred at about 310, 372, and
432 seconds, respectively. As shown in the plot, after an initial
transient spike in the current signal coincident with each step
change, the response at each concentration quickly levels off to a
steady state response. The linearity of this response over time for
different glucose concentrations indicates that the flux limiting
membrane is able to pass a proper oxygen-to-glucose ratio for a
wide range of blood glucose concentrations. Similar qualitative
behavior was observed in the other prototype sensors.
[0044] Additional tests were performed on sensor populations using
a method for spray-coating EVA polymer dissolved in paraxylene to
achieve superior control over membrane thickness. Based on studies
conducted by the present inventor, spray-coating EVA in paraxylene
has been shown to provide a consistent and uniform membrane layer
that adheres to the polyimide substrate of the sensor. Paraxylene
was selected as a solvent for its effectiveness in spray-coating
applications. Paraxylene has been commonly used in electronics, for
example, in low pressure vapor deposition processes forming a thin
conformal coating on printed circuit boards. Paraxylene has a
boiling point in a range that allows for effective evaporation for
spraying, but prevents overly rapid evaporation that could cause
clogging of the spray nozzle. Once deposited, the evaporation time
of paraxylene allows for reasonably short drying times. In
addition, paraxylene has certain adhesion promoting properties that
facilitate bonding to the sensor substrate that is based upon a
polymer. As a spray, it lends itself for application to the
substrate of a very small flex circuit on which, in one embodiment,
the working electrode may be effectively mounted for intravenous
use.
[0045] Using 0.5 wt % to 6.0 wt % EVA-40 dissolved in paraxylene,
an EVA solution was created and sprayed on working electrodes, and
cured, to form the desired flux limiting membrane. Glucose assays
were performed in the manner previously described. The results
indicated that good linearity may be achieved by forming the flux
limiting membrane from about 4 or 5 layers of sprayed-on EVA
solution, where each layer is about 1 micron in thickness. Final
membrane thickness may be process-dependent, since the spray method
may deposit layers of varying porosities, i.e. surface areas having
different flatness qualities or average depth of interstices. Thus,
the number of layers needed for good linearity depends on the EVA
formulation used, and on the process used for applying the spray.
It has been found, however, that linear sensors may be manufactured
by producing an average membrane thickness, a.k.a. diffusion layer
thickness, between about 0.5 microns and about 10 microns. A
preferred range for thickness may be between about 4 microns and
about 6 microns, so that a sufficient amount of material may be
deposited to withstand the mechanical stress of an intravenous
insertion.
[0046] In the foregoing experiments, an EVA composition of 40 wt %
(EVA-40) was used to create the solution that formed the flux
limiting membranes. Membranes according to the invention, however,
are not limited to this composition. Membranes may be formed using
any EVA composition, for example, EVA having vinyl acetate
compositions ranging from between about 9 wt % and about 50 wt %.
As vinyl acetate is varied within the polyethylene content,
solubility may also change (i.e. become less soluble at lower EVA
compositions) and may not spray as effectively. A preferred range
of EVA composition may be between about 25 wt % and about 40 wt %
to promote good solubility and adhesion properties. The EVA may
also be cross-linked to other polymers, such as
poly(methylmethacrylate-co-butyl methacrylate), to create a
different diffusion coefficient for glucose. The EVA may also be
cross-linked with other compounds such as diglycidil ether or a
diisocyanate, for example, to allow lower compositions of EVA to be
used or to achieve better spray-coating performance.
[0047] Spray coating a flux limiting membrane may be especially
effective in improving quality control for mass-production of
biosensors. For example, a production lot of about 50 to 100
biosensors may be formed from a common flex circuit substrate.
During fabrication, a step may be performed for spray-coating an
EVA solution on the common substrate. After curing, the substrate
may be cut or sliced into multiple, uniform strips to allow the
flux limiting membrane of each biosensor to have approximately the
same thickness.
[0048] To test the biocompatibility and mechanical strength of a
flux limiting membrane, an acute study was performed as an in vivo
test on a swine. A prototype sensor was manufactured according to
method 400 and then installed intravenously by mounting it in one
lumen of a catheter that was inserted in a jugular vein of the
swine using an over-the-wire technique. Glucose was periodically
injected into the swine at a different entry site over a 6 hour
period. The current output of the sensor was monitored during this
period. Glucose concentration was also measured at periodic
intervals over the same duration, by drawing blood samples and
determining glucose concentration using a YSI 2300 glucose
analyzer. The blood samples were drawn from a different lumen of
the same catheter.
[0049] FIG. 7 shows the results of the swine test. With the
exception of two transient periods between time 700-720 minutes and
860-880 minutes, the results show that in the early stages of the
test, at time periods 600 through 700 minutes, the sensor readings
agreed very well with the results of the reference standard. After
the non-agreement interval at 860 minutes, it was discovered that
the electrode was pressed against a vessel wall. Upon repositioning
the catheter, the response was once again in good agreement with
the YSI 2300 analyzer. Near the conclusion of the test period, at
times 900 through 950 minutes, the readings still agreed, without
any significant deviations. The consistent performance over a
prolonged test period indicates no detachment of the flux limiting
membrane, and no protein build-up, blood clotting, or other
biofouling that would degrade the performance of the sensor over
time. The transient portions may be explained as excursions where
some non-linearity or other instability is experienced during
abnormally high glucose levels or positioning difficulty with the
catheter.
[0050] The foregoing disclosure and experimental test results
demonstrate the efficacy of using EVA polymer to form a flux
limiting membrane. In the particular case of a glucose sensor, EVA
has the correct adhesion properties, biocompatibility properties,
solubility suitable for spray-coating, and hydrophobic/hydrophilic
properties to create a flux differential between glucose and oxygen
to enable a sensor to exhibit a linear glucose response. In sum,
EVA has been found to possess the proper material, chemical,
performance, and manufacturing properties for forming a flux
limiting membrane on an intravenous amperometric biosensor.
[0051] The invention has been disclosed in an illustrative manner.
Accordingly, the terminology employed throughout should be read in
an exemplary rather than a limiting manner. Although minor
modifications of the invention will occur to those well versed in
the art, it shall be understood that what is intended to be
circumscribed within the scope of the patent warranted hereon are
all such embodiments that reasonably fall within the scope of the
advancement to the art hereby contributed, and that that scope
shall not be restricted, except in light of the appended claims and
their equivalents.
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