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BACKGROUND OF THE DISCLOSURE
Considerable effort over the years has been devoted to discovering and
improving analytical techniques for measuring biological substances in
connection with medical and industrial applications. An example of one
such technique developed concerned polarographic electrode systems which
were used to measure various biological materials qualitatively and
quantitatively, and reference is made to my earlier U.S. Pat. No.
2,913,386 describing such a polarographic electrode system for the
measurement of oxygen and the like. Reference is also made to my U.S. Pat.
No. 3,380,905 which pertains to an improvement of the polarographic
electrode system described in the above-mentioned U.S. patent.
About twenty years ago, enzyme-coupled electrodes were reported for the
polarographic analysis of biological substances. For example, in my U.S.
Pat. Nos. 3,539,455 and 2,912,386, membrane polarographic electrode
systems and methods were described for the rapid and accurate quantitative
analysis of biological substances which theretofore could not be analyzed
directly by polarographic methods. According to the description in my U.S.
Pat. No. 3,539,455, small molecular substances, such as glucose, were
measured with a membrane polarographic electrode system. By use of
cellulose or another membrane which is permeable to small molecules, such
as glucose, but is impermeable to proteins, the membrane kept glucose
oxidase enzyme on the side of the membrane with the anode for reaction
with glucose. Therefore, for example, if a sample of blood were placed on
the membrane side opposite the electrode, with an aqueous solution of the
enzyme and oxygen on the electrode side of the membrane, the low molecular
weight materials, such as glucose, passed from the blood samples through
the membrane for enzymatic reaction adjacent the electrode. After a
certain period of time a steady state was reached when the hydrogen
peroxide concentration was directly proportional to the glucose
concentration and the cell produced a current flow as a function of the
amount of hydrogen peroxide being formed which served as an indication of
the amount of glucose present. As disclosed in my article entitled
Electrode Systems for Continuous Monitoring in Cardiovascular Surgery,
N.Y. Acad. of Sciences. 102:29-45 (1962), the Clark oxygen electrode could
be arranged so that it was sensitive to glucose by virtue of the fact that
oxygen was consumed by enzymatic reaction in proportion to glucose
content. In such arrangement, the inner membrane was impermeable to
glucose and the reaction was monitored by the drop in oxygen. My early
membrane polarographic techniques for measurement of hydrogen peroxide
were limited to the detection of small molecules which were capable of
permeating the membrane for enzymatic reaction with an enzyme being
contained on the electrode side of the membrane.
More recently, enzymatic techniques for measuring macromolecules, such as
cholesterol have been made. Generally, the enzymatic methods combined two
enzymes, cholesterol oxidase and cholesterol ester hydrolase, with
colorimetric techniques. These colorimetric methods relied on enzymatic
conversion of cholesterol or its esters to cholestenone and hydrogen
peroxide, and then on the reaction of the hydrogen peroxide with various
compounds to produce measurable chromagens and fluorogens. In my U.S. Pat.
No. 4,040,908, I described a membrane polarographic anode suitable for
measuring macromolecular substances, such as cholesterol, utilizing
enzymatic reactions as a means to measure such macromolecular substances.
Additional techniques have been developed for measuring other biological
substances in blood. For instance, ethanol is currently measured in blood
either directly or by breath sampling, by classical chemical, gas
chromatographic and enzyme methods. One of the alcohol enzyme methods, for
example, depends upon the polarographic measurement of hydrogen peroxide,
while others depend upon the consumption of oxygen. In my more recent U.S.
Pat. No. 4,458,686, I disclosed the use of a polarographic electrode as a
skin-contact analyzer to transcutaneously measure oxygen for determining
blood substances, such as glucose or alcohol as well as measurement of
alcohol going through the skin.
One of the most important biological substances is glucose. This is true
because glucose plays such a major role in the metabolism of the body in
health and disease, particularly diabetes. For instance, most of the
scientific evidence to date indicates that it is the high blood and tissue
glucose concentration per se, and not too low an insulin level or the
presence of abnormal metabolites, such as hydroxybutyric acid and the
like, which causes the organ damage in the various forms of diabetes
mellitus. This damage may be caused by glycylation of many of the tens of
thousands of proteins in the body. Such glycylation is reflected by the
glucosehemoglobin AlC level in the blood, a substance commonly measured to
give a time-integrated level for blood glucose. Since all enzymes are
proteins, the high glucose level probably impairs the catalytic functions
in every part of the body. Typical serious damage related to diabetes is
blindness, loss of limbs, cardiac and circulatory failure and death.
At present, insulin is administered either by injecting intermittently
throughout the day to control blood glucose or, in a very small population
of diabetics, by a programmable pump which injects insulin subcutaneously.
This results in considerable, potentially dangerous, fluctuation in blood
glucose depending upon the severity of the disease. In some forms of
diabetes the Beta cells which make insulin are completely destroyed and
the person becomes totally insulin dependent for survival.
In view of the above background, it would be desirable to have a device
which is capable of continuously sensing glucose in the blood of diabetic
patients so that the insulin or glucose can be more effectively
administered and regulated. Extensive efforts heretofore have been
directed toward developing an implantable glucose sensor having the
capability of controlling an insulin pump or at least to provide a
continuous signal reflecting blood glucose concentrations. However, it is
widely believed that an implanted enzyme-based glucose sensor cannot work
or, if it does work, such a sensor would last at best for only a few days,
after implantation in the blood or a body cavity. In Schichiri, M. et al:
Glycaemic Control in Pancreatectomized Dogs with a Wearable Artificial
Endocrine Pancreas, Diabetologia. 24:179-184 (1983), it was reported that
a glucose sensor was implanted and it lasted for six days after the date
of in vivo implantation. Up to this point, such success even though
limited has been considered remarkable. Nevertheless, the limited
operability of such sensors lead the scientific community to believe that
implanted glucose oxidase type glucose sensors are not practical. In
support of such belief, a penumbra of reasons are given. For instance, it
is generally thought that the enzyme, glucose oxidase, is too unstable to
remain active for any period of time in a human at human body temperature.
Furthermore, it is believed that glucose oxidase would be destroyed by
bacteria or fungi. It is further believed that the electrodes' permeable
membrane would be destroyed by tissue cells and enzymes or would become
plugged as a result of large molecules, cellular debris and white and red
blood cells collecting thereon. Additionally, it is thought that the
amount of oxygen available necessary for the enzymatic reaction would be
insufficient; or that co-enzymes would diffuse away from the enzyme
through glucose permeable membranes; or that the platinum electrode
surface would become plated, poisoned, inactivated or passivated thereby
preventing reduction of the hydrogen peroxide generated; or that tissue
response would interfere with glucose permeation through the membrane.
In summary, while there are a variety of devices and techniques available
for the measurement of biological substances, new implantable devices and
methods are needed for the measurement, administration and/or regulation
of key biological substances, such as blood glucose and insulin. It would
be especially beneficial if a satisfactory implantable device could be
provided to aid in the control and alleviation of diabetes.
SUMMARY OF THE INVENTION
In brief, the present invention seeks to alleviate the above discussed
problems and shortcomings of the present state of the art. This invention
is directed to new and improved optical, electrical or other devices for
sensing a product or reactant, such as hydrogen peroxide, derived from
enzymatic reactions between analytes and enzymes as a measure of the
amount of the analytes. This invention is predicated in part upon the
discovery that problems heretofore associated with enzyme sensors can be
overcome by providing a structure for an ample and/or steady supply of
oxygen for enzymatic reaction at the sensor surface. The device of this
invention is also uniquely suited for in vivo implantation and in
measuring analytes, such as glucose, in undiluted, whole blood. It has
been discovered that an enzyme and an electrode of this device when
implanted does in fact remain active for as long as six months or more. In
view of the present state of the art, this is remarkable. In one preferred
form of the invention, the amount or level of hydrogen peroxide is
detected as a measure of the amount or level of glucose in the animal
body. The types of animal fluid or tissue selected for implantation
include both blood and nonblood sites such as intravascular, spinal fluid,
peritoneal fluid, and extra and intracellular fluids.
In one embodiment of the present invention, the device comprises a gas
permeable membrane having opposed sides, an enzyme on one side of the
membrane for enzymatic reaction with the analyte in the presence of
molecular oxygen to generate a product, a closed container for the
molecular oxygen on the opposite side of the membrane to supply molecular
oxygen through the membrane for the enzymatic reaction, and means for
sensing the generated product or a reactant which functions as a measure
of the analyte.
The present invention contemplates providing sensing devices predicated
upon sensing a product or reactant involved in an oxygen-dependent
enzymatic reaction and having the capability to provide to the enzymatic
reaction from the container located on the side of the membrane opposite
the enzyme a steady source of oxygen either extracted from the surrounding
environment or derived independently of the surrounding environment. It is
further contemplated that such a sensing device can be uniquely designed
so that the container is adapted to generate oxygen which then can be
supplied on demand to the enzymatic reaction. In other words, the
enzymatic reaction determines the amount of oxygen necessary to convert
the analyte to a product which ultimately is sensed. It should be
appreciated, however, that the consumption of oxygen as a reactant
supplied on demand, by a servo mechanism or other device, to the enzymatic
reaction can also function as a measure for the analyte under
investigation. It is still further contemplated that, since it has been
discovered that the enzymatic reactions utilized by this invention are
oxygen limiting, such a sensing device can be constructed so that the
container provides a constant supply of oxygen for the enzymatic reaction
which is always in excess of the amount needed for the enzymatic reaction.
In a preferred embodiment, the device comprises an enclosed chamber adapted
for containing oxygen comprising a wall structure defining a hollow
interior and having an external surface wherein at least a portion of the
external surface has the enzyme associated therewith for the enzymatic
reaction, the wall structure being adapted for allowing molecular oxygen
to permeate therethrough to supply molecular oxygen for the enzymatic
reaction, an analyte permeable membrane overlying the enzyme and connected
to the chamber for allowing the analyte to permeate therethrough to supply
the analyte for the enzymatic reaction, and means for sensing the product
generated from the enzymatic reaction which functions as a measure of the
analyte.
The invention is also directed to novel methods of sensing a product or
reactant of an enzymatic reaction between an analyte and an enzyme in
presence of oxygen as a measure of the analyte in vivo or in vitro. Such a
method comprises providing a gas permeable membrane having opposed sides,
providing an enzyme on one side of the membrane for enzymatic reaction
with the analyte in presence of molecular oxygen to generate a product,
providing a closed container for the molecular oxygen on the opposite side
of the membrane to supply molecular oxygen through the membrane for the
enzymatic reaction, and sensing the generated product or reactant which
functions as a measure of the analyte.
The above features and advantages of the invention will be better
understood with reference to the accompanying drawings, detailed
description and examples. It will also be understood that the particular
devices and methods embodying the invention are exemplary only and not to
be regarded as a limitation of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Reference is made to the accompanying drawings in which is shown
illustrative embodiments of the invention from which its novel features
and advantages will be apparent.
FIG. 1 is a diagram-matic illustration of one form of an electrical device
which is illustrative of an embodiment of the invention;
FIG. 2 is a depiction of generated cyclic in vitro polarograms of changing
glucose concentrations in a liquid sample, e.g., Gomori buffer, void of
oxygen utilizing the electrical device described in Example 1;
FIG. 3a is a depiction of generated cyclic in vitro polarograms of changing
glucose concentrations in a liquid sample, e.g., Gomori buffer, utilizing
an electroenzymatic glucose sensor of the present invention; and
FIG. 3b is a depiction of cyclic in vitro polarograms of changing glucose
concentrations in undiluted fresh whole goat blood utilizing the same
sensor referred to in the description of FIG. 3a.
DETAILED DESCRIPTION OF THE INVENTION
By way of illustrating and providing a better appreciation of the present
invention, the following detailed description is given concerning the
methods of the invention.
By the term "animal tissue" as used herein, it is meant to include all
animal tissues including body fluids and blood.
As used in this specification, "closed container" is meant to refer to a
container that is impermeable to substances, such as liquids and cells,
that might interfere with the ability of the container or membrane
associated with the enzyme to supply oxygen to the enzymatic reaction. By
the term "product" generated from the enzymatic reaction, it refers herein
to the product produced from such a reaction, like hydrogen peroxide or
gluconic acid or pyruvic acid, or products generated from the "product".
The term "reactant" is meant to include a substance involved directly or
indirectly in the enzymatic reaction, such as oxygen, for instance. It
should therefore be appreciated that the biosensor and methods of this
invention can sense the products or reactants of the enzymatic reaction,
or by-products derived from the generated products, which function as a
measure of the analyte.
The term "analyte" refers herein to any material suitable for analysis with
polarographic, potentiometric, optical or other techniques. An example of
one of many other techniques is conductometric, e.g., glucose is not
electrically conductive but gluconic acid can be so detected as a measure
of the analyte. Further, hydrogen peroxide can be detected by electron
spin resonance, and so forth. Exemplary of analytes that can be detected
according to the teachings of this invention include, for instance,
glucose and lactate. The product generated, such as hydrogen peroxide, by
the enzymatic reaction between an analyte and an enzyme in the presence of
oxygen which can be sensed using known standard electrical or optical
techniques serves as a measure of the analyte under investigation. For
instance, hydrogen peroxide in an electrical system can generate an anodic
current when subjected to a sufficient voltage, or, in an optical system,
it can react with or be reduced to react with an optical substance to
generate chromagens or fluorogens which then can be detected optically.
The present invention is especially directed to devices for sensing
hydrogen peroxide generated from an enzyme reaction with glucose or
lactate in a liquid sample in the presence of oxygen as a measure of
glucose or lactate. Alternatively, oxygen may be sensed as a measure of
the analyte. The devices are especially suited for in vivo implantation
and detecting such analytes in undiluted, whole blood. The novel
biosensors are uniquely designed to provide an independent source of
oxygen for the enzymatic reaction to overcome apparent oxygen deficiencies
that otherwise arise in the liquid sample under investigation. In other
words, it has been found that problems heretofore encountered in enzymatic
electrode or optical structures are overcome by the inventive device. The
devices of the present invention have been found ideally suitable for use
or implantation in low oxygen tension areas, such as the peritoneal
cavity, and for measuring glucose therein.
Referring now to FIG. 1, it is a diagrammatic illustration of a typical
device of the present invention and illustrates sensor 5 provided with an
air space gas pocket 12 on the side of a gas permeable membrane 6a
opposite the enzyme which functions as an independent reservoir of oxygen
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