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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to monitoring glucose levels, and more particularly
relates to an implantable glucose sensor and a method for using same for
detection or quantitation of an elevated glucose level in a body fluid.
2. Background Description
Over five million Americans have diagnosed diabetes and another five
million are estimated to have undiagnosed diabetes. Diabetes is a chronic
metabolic disorder manifested by degenerative disease of the blood
vessels, kidneys, retina and nervous system and is characterized by the
body's abnormal metabolism of carbohydrates, proteins and fats.
Carbohydrates are normally digested to glucose in the gut, the glucose
being absorbed into the circulatory system and carried to most cells of
the body where, it is utilized as the principal source of nutrition. In
one form .of diabetes, the glucose cannot enter the liver, muscle and fat
cells in normal amounts for storage or energy use and as a consequence
builds up in the blood and urine. Abnormally high blood glucose levels may
lead to the accumulation of toxic ketone metabolites often leading to coma
and death.
Glucose is normally present in the blood stream at a level of about 0.8 to
1.0 mg/ml and is maintained within this narrow range by a continuous
moment to moment sensing and correction of the glucose concentration by
hormones released from the pancreas. If glucose concentration in the blood
stream rises above the normal range, insulin is released and causes
metabolism of glucose which lowers the concentration. If the glucose
concentration falls below the normal range, glucogen is released to raise
it to normal. The pathological condition of diabetes is primarily due to a
long term hyperglycemia resulting from reduced insulin production or
release.
Many diabetics control their disease merely by diet and weight control.
Others require drug treatment, generally insulin or an oral hypoglycemic
agent, to control blood glucose levels. Oral administration of insulin is
not practical because it is destroyed by proteolytic enzymes in the
gastro-intestinal tract. Injected insulin provides only partial control of
the degenerative effects of diabetes, apparently because periodic
injections do not closely correspond to changing metabolic requirements
consequent to fluctuating blood glucose levels. For this reason, a variety
of methods have been proposed for rapid and accurate assessment of blood
glucose levels.
Glucose measurement systems known in the art are generally based on the
oxidation of glucose by oxygen in the presence of glucose oxidase. U.S.
Pat. Nos. 4,452,887 to Kitajina et al., and 4,390,621 and 4,460,684 to
Bauer exemplify a chromogenic system in which hydrogen peroxide formed
during the oxidation oxidizes a substrate in the presence of peroxidase to
produce a color which is measured.
Conversion of the chemical energy of the oxidation reaction to electrical
energy, which is measured at electrodes, is the subject of U.S. Pat. Nos.
4,392,933 to Nakamura et al., 4,436,094 to Cerami, 4,431,004 to Bessman et
al. and 4,317,817 to Busby.
Cerami, in U.S. Pat. No. 4,330,299, discloses an indicator element, such as
a dye, as part of a complex containing a carbohydrate or a lectin. The
indicator remains undetected until released from the complex by glucose in
direct proportion to the glucose concentration.
Boehringer Mannheim Diagnostics (Indianapolis, IN) recently marketed an in
vitro enzyme-based blood glucose monitoring system, (Accu-Chek.TM.
Chemstrip bG.TM.), which may be read colorimetrically or
photo-electronically.
Fiber optic probes for determination of oxygen pressure in a body fluid
have been described. Peterson et al., in U.S. Pat. No. 4,476,870, disclose
an implantable device for measurement of partial oxygen pressure in a
blood stream based on oxygen quenching of fluorescence.
U.S. Pat. No. 4,399,099 to Buckles discloses a dual fiber optic device
useful in a method for measuring glucose concentration. Oxygen permeable
sheaths containing an oxygen quenchable fluorescent dye surround optical
fibers, one of the sheaths containing glucose oxidase. The enzyme oxidizes
glucose and thereby lowers oxygen concentration which is detected by
reduced quenching of the fluorescence emission from the dye.
Prior art methods and devices disclosed to date for glucose measurement all
suffer from deficiencies such as insufficient accuracy, speed or use of
methodology or equipment which is impractical for an implantable device.
There remains a definite need for a simple and accurate method for glucose
monitoring using a small, light and compact apparatus. It is toward
fulfillment of this need that the present invention is directed.
SUMMARY OF THE INVENTION
One aspect of the present invention is a method to detect, either in vivo
or in vitro, a glucose level in a body fluid which differs from a
reference level. A fluorescent dye, the fluorescence emission of which is
sensitive to oxygen quenching so that the emission is maximum in the
absence of oxygen, is conjugated to active glucose oxidase. The dye
conjugated to active glucose oxidase is hereinafter called the test dye.
The dye-enzyme conjugate is immobilized in contact with a body fluid, and
glucose in the fluid is oxidized at the active enzyme with consumption of
oxygen. The oxygen concentration at the dye is thereby reduced in inverse
proportion to the extent of oxidation and therefore also to the glucose
concentration. Application of excitation light to the dye causes
fluorescence emission, which is measured. The magnitude of the emission is
inversely proportional to oxygen concentration at the dye and therefore
directly proportional to glucose concentration in the fluid.
The dye may also be conjugated to inactivated glucose oxidase, hereinafter
called the control dye. This conjugate is also immobilized in contact with
the body fluid. Glucose in the fluid is not oxidized by the inactive
enzyme and the oxygen concentration at the control dye therefore remains
unchanged. Quenching therefore does not occur, and the magnitude of
fluorescence emission from the control dye remains constant, irrespective
of changing glucose concentration, and provides a base line control for
comparison with the magnitude of emission from the test dye which does
fluctuate in proportion to glucose concentration. If emission from the
test dye is greater than that from the control dye, an elevated glucose
concentration in the fluid is indicated.
The method of the invention may also be used to quantitate the glucose
concentration in the body fluid. In this embodiment of the invention, the
magnitude of the fluorescence emission from the test dye may be compared
with the magnitude of emission measured when the fluid contains a
predetermined quantity of glucose. Emission from a plurality of fluids
containing predetermined quantities of glucose may be measured to prepare
a standard curve which relates glucose concentration in the fluid to the
magnitude of fluorescence emission.
Another aspect of the invention is a glucose-monitoring apparatus. The two
conjugates having active and inactive enzymes, described above, are coated
onto the surfaces of separate optical fibers adapted for insertion into
the fluid to be tested. The apparatus includes a suitable source of
excitation light and a suitable fluorescence emission detector. The
excitation light passes through the fibers, excites the dyes and induces
fluorescence emission which passes back through the fibers where it is
detected by the detector.
The preferred apparatus has four fibers, two for passage of excitation
light from the light source to the dye-conjugates and two for passage of
fluorescence emission from the dye conjugates to the detector. The most
preferred apparatus has two pairs of concentric fibers and uses a light
emitting diode (LED) as light source and a photocell as detector. One pair
of fibers is coated with active enzyme-dye conjugate and is further coated
with a glucose permeable membrane. The dye in this conjugate serves as the
test dye. The other pair is also coated with active enzyme-dye conjugate,
but is further coated with an oxygen permeable membrane which precludes
passage of glucose so that the enzyme is effectively rendered inactive and
its dye serves as the control dye. One fiber in each pair introduces
excitation light to the conjugate and the other fiber in each pair
conducts fluorescence emission from the conjugate to the detector.
Thus, in accordance with the invention, an elevated glucose level in a body
fluid may be detected or quantitated, in vivo or in vitro, by a method
using a glucose monitoring apparatus. The apparatus employs glucose
oxidase covalently conjugated to a fluorescent dye whereby the dye and the
enzyme are in close proximity so that the local oxygen concentration at
the site of the enzyme reaction can be determined with exceptional
accuracy. The apparatus includes an optical fiber which may be very thin
and flexible thereby providing advantages for comfort and safety when
inserted into the body fluid through the skin. The LED and photocell of
the preferred apparatus are small and light and may easily be assembled
into a simple and inexpensive unit to be either implanted or worn
externally on the surface of the body, and may, if desired, be used in
conjunction with any insulin delivery system. Because of these and other
features, the apparatus of the invention may easily and safely be used on
an outpatient basis.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view of an apparatus of the invention using two
optical fibers;
FIG. 2 is a vertical sectional view of the apparatus of FIG. 1 taken along
line 2--2 thereof;
FIG. 3 is a perspective view of an apparatus of the invention using four
optical fibers;
FIG. 4 is a perspective view of an apparatus of the invention using
concentric optical fibers;
FIG. 5 is a horizontal section view of the apparatus of FIG. 4 taken along
line 5--5 thereof; and
FIG. 6 is a perspective view of an apparatus of the invention, similar to
the apparatus of FIG. 4, using enzyme conjugates coated with membranes.
DETAILED DESCRIPTION OF THE INVENTION
While this invention is satisfied by embodiments in many different forms,
there will herein be described in detail preferred embodiments of the
invention, with the understanding that the present disclosure is to be
considered as exemplary of the principles of the invention and is not
intended to limit the invention to the embodiments illustrated and
described. The scope of the invention will be measured by the appended
claims and their equivalents.
The method of the present invention for continuous monitoring of glucose in
a body fluid is based on the well-known oxidative conversion of glucose to
gluconic acid catalyzed by glucose oxidase. When glucose is oxidized, the
consumption of oxygen causes a decrease in the local oxygen concentration
at the active site of the enzyme. This decrease is proportional to glucose
concentration and may be detected by fluorescence emission from a dye
conjugated to the enzyme. Glucose oxidase is a well-known and
well-characterized enzyme and is commercially available, for example, from
Sigma Chemical Co., St. Louis, Missouri.
The dye to be conjugated to the enzyme may be any fluorescent dye sensitive
to quenching of its fluorescence emission by oxygen. Such a dye fluoresces
with maximum intensity in the absence of oxygen, and the intensity of its
fluorescence emission is decreased in inverse proportion to the oxygen
concentration in the immediate vicinity of the dye. Such dyes preferably
are hydrophobic fluorescent dyes having strong absorbance in the visible
part of the spectrum. Exemplary of, but not limited to, such dyes are
those listed in Peterson et al. (op. cit.), preferably perylene
dibutyrate, most preferably fluoranthrene.
Conjugation of the dye to the enzyme may be carried out by any conventional
procedure as, for example, by covalently coupling active functional groups
on the dye and enzyme. The functional groups may be bonded directly, as in
amide bond formation between amino and carboxyl groups, or they may be
coupled through linking groups which couple, for example, amino, hydroxyl
or sulfhydryl groups on one component to a carboxyl group on the other
component. Suitable linking groups may be, for example, but not limited
to, a methylene chain of from one to six carbon atoms. If desired, the
technique of affinity labeling may be used to conjugate the dye near the
active site of the enzyme. The ratio of dye molecules to enzyme molecules
in the conjugate is not critical, but preferably is as high as possible in
order that the emission signal be as intense as possible. The coupling of
enzymes and dyes, including affinity labeling, is well-known in the art
and further details in this respect are not necessary for a complete
understanding of the invention.
The dye-enzyme conjugate is immobilized on a solid support introduced into
the body fluid in such a way that the enzyme contacts glucose in the
fluid. Excitation light is applied to the dye and fluorescence emission is
detected therefrom. Any support material may be used which substantially
does not interact with the fluid or interfere with the oxidation reaction
or the fluorescence detection system. Exemplary of such supports are glass
and plastics, such as polyethylene, polystyrene, polyvinyl chloride and
polytetrafluoroethylene.
A particularly preferred support is an optical fiber which, in addition to
providing the support for immobilization of the conjugate, also serves as
the means for introduction of excitation light to the dye and conduction
of fluorescence emission from the dye. Optical fibers act as pipelines for
passage of light. They are made of a transparent material, such as glass,
and are designed in such a way that very little light can leak out through
their sidewalls. A thorough discussion of optical fibers is given by D. M.
Considine et al. in Encyclopedia of Chemistry, Van Nostrand Reinhold,
(1984) p 645.
The dye-enzyme conjugate may be coated onto a segment of an optical fiber
to be contacted with the body fluid. Alternatively, the conjugate may be
coated onto a solid support as described above, and the optical fiber
brought into intimate contact with the conjugate on the support in such a
way that light passed through the fiber is absorbed by the dye.
Fluorescence emission from the dye passes back through the fiber and its
intensity is measured on a detector.
As mentioned above, fluorescence intensity from the test dye is directly
proportional to glucose concentration in the fluid. In order to determine
whether the intensity of the emission from the test dye indicates an
elevated glucose level in the fluid, a base line level of fluorescence
emission may be determined, preferably simultaneously, from the control
dye. A second optical fiber may be coated with dye only, the quantity of
dye being substantially the same as conjugated to the enzyme. Passage of
excitation light through this second optical fiber excites the dye to emit
fluorescence which is independent of glucose concentration and thus a
measure of ambient oxygen concentration. If the intensity of the emission
from the test dye is greater than that from the control dye, an elevated
glucose level in the fluid is indicated.
In a preferred embodiment of the method of the invention, the base line
level of fluorescence emission from the control dye may be obtained with a
second dye-enzyme conjugate. The second conjugate may be prepared in the
same way as the first conjugate, except inactive glucose oxidase is used.
The enzyme may be rendered inactive, i.e., incapable of catalyzing
oxidation of glucose, either prior to or subsequent to coupling to the
dye. Methods to inactivate enzymes are routine, well-known to those
skilled in the art, and do not constitute a part of this invention.
Most preferably, the inactive enzyme-dye conjugate may be prepared by
coating active enzyme-dye conjugate with a membrane. In this embodiment of
the method of the invention, active enzyme-dye conjugate immobilized on a
first fiber is coated with a selective membrane permeable to molecules the
size of glucose and smaller. When introduced into the body fluid, this
membrane allows glucose to pass through and contact the conjugate where it
is oxidized. Dye in this conjugate is thus the test dye and fluorescence
emission therefrom measures glucose concentration. Conjugate on a second
fiber is coated with a selective membrane permeable only to molecules the
size of oxygen and smaller. Since glucose molecules are larger than oxygen
molecules, glucose in the body fluid cannot reach this conjugate to be
oxidized, so that its enzyme has, in effect, been inactivated. Oxygen,
however, can reach the conjugate and thus provide measurement of ambient
oxygen concentration. The dye in this conjugate is thus the control dye,
and comparison of the intensities of fluorescence emission from the two
dyes indicates, as described above, whether the fluid contains an elevated
glucose level.
The method of the present invention may be adapted to quantitate the
glucose concentration in a body fluid; In this embodiment of the
invention, the intensity of fluorescence emission from the test dye is
determined and compared to the intensity of emission determined when the
method of the invention is applied to a body fluid having a predetermined
glucose concentration. For this embodiment, the invention contemplates a
standard curve which relates fluorescence emission intensity, as
determined with the device of the invention, to glucose concentration. In
accordance with this embodiment of the method, glucose concentration, for
example in a diabetic's blood stream, may be ascertained merely by finding
the test dye fluorescence intensity on the standard curve and reading the
corresponding glucose concentration.
Having now described the method of the invention, various embodiments of
the blood glucose monitoring apparatus of the invention will be described
with the aid of the figures. FIG. 1 shows glucose monitor 10 having
optical fibers 12 and 14, each having a sidewall portion 16 and 17,
respectively, and a bottom portion 18 and 19, respectively. Bottom portion
18 of optical fiber 12 has a coating of conjugate 20 of active glucose
oxidase conjugated to fluorescent dye (the test dye). Bottom portion 18 of
optical fiber 14 has a coating of conjugate 21 of inactive glucose oxidase
conjugated to fluorescent dye (the control dye). Alternatively, reference
numeral 21, representing the control dye, may be unconjugated fluorescent
dye, i.e., the enzyme is omitted. Arrows 22 diagrammatically illustrate
excitation light passing down fibers 12 and 14 from a light source (not
shown) where it is absorbed by the dyes and emitted therefrom as
fluorescence emission 24. Emission 24 returns up fibers 12 and 14 and is
measured by a detector (not shown).
FIG. 2 is a vertical sectional view of the apparatus of FIG. 1 after
insertion into body fluid 26, illustrated in the Figure as a blood stream.
Optical fibers 12 and 14 are shown surrounded by cladding material 27,
which separates the fibers and prevents leakage of light through the
sidewall portions 16 and 17. Any cladding material conventional in optical
fiber technology, such as plastic or glass having a refractive index lower
than that of the light-transmissive core of the fiber, may be used.
An embodiment of the apparatus in which two fibers are used for each of the
test dyes and the control dye is shown in FIG. 3. In FIGS. 3-6, elements
identical to elements described in FIGS. 1 and 2 are given the same
reference numbers and elements similar are given the same base reference
number followed by a different suffix (letter).
In FIG. 3, a solid support, shown in the form of a disc 28, is coated on
its upper surface 30 with active glucose oxidase-fluorescent dye conjugate
20. Disc 28a is coated on its upper surface 30a with inactive glucose
oxidase-fluorescent dye conjugate 21. Discs 28 and 28a preferably are made
of a porous plastic material such as polystyrene foam through which the
body fluid may freely pass. Optical fibers 12a,12b,14a and 14b are
attached to discs 28 and 28a so that their bottom portions 18 and 19 are
in intimate contact with conjugate coatings 20 and 21, respectively.
Excitation light 22 from the light source passes down fibers 12a and 14a
and contacts conjugates 20 and 21 in contact with body fluid 26 in porous
discs 28 and 28a where it is absorbed by the fluorescent dye and emitted
as fluorescence emission 24. Emission 24 passes up through fibers 12b and
14b to the detector (not shown).
FIG. 4 gives a perspective view of an embodiment of the apparatus using
concentric fibers. Active enzyme-dye conjugate 20 and inactive enzyme-dye
conjugate 21 are coated onto upper surfaces 30 and 30a of porous solid
supports 28 and 28a, respectively. Optical fibers 12a and 14a have
dimensions which allow them to fit inside of hollow optical fibers 32 and
2a. Fibers 12a and 14a and 32 and 32a are separated by layers of cladding
material 27, as shown in horizontal sectional FIG. 5.
FIG. 6 shows an embodiment of the apparatus in which the conjugates are
coated with membranes. Concentric optical fibers 12a and 32 are coated on
their bottom surfaces 30 with active enzyme-dye conjugate 20. The coating
of conjugate 20a is then itself coated with membrane 34 which is permeable
to glucose and molecules smaller than glucose so that its dye serves as
the test dye. Concentric optical fibers 14a and 32a are likewise coated on
their bottom surfaces 30a with active conjugate 20, which is further
coated with membrane 36 permeable only to molecules the size of oxygen and
smaller so that its dye serves as the control dye.
It is understood that support discs 28 and 28a, depicted in FIG. 4, may be
included in the apparatuses of FIGS. 1 or 6. Likewise, membranes 34 and 36
of FIG. 6 may be included in any of the other embodiments described. The
invention is contemplated to encompass these and any other modifications
of the apparatus, which provide glucose monitoring in accordance with the
principles of the method of the invention herein described.
The following examples are provided to further describe the invention, but
are in no way to be considered as limitative.
In summary, the invention provides a system including a method and an
apparatus for monitoring of glucose in a body fluid, preferably on a
continuous basis. The method is based on oxidation of glucose by glucose
oxidase, the extent of oxidation being proportional to glucose
concentration. The oxidation reaction depletes oxygen at the active site
of the enzyme, and the reduced oxygen concentration is detected and
measured by changes in fluorescence intensity proportional to the oxygen
concentration. The system may be used either in vitro or in vivo and is
particularly suitable for blood glucose determinations. When used in vivo
for monitoring glucose concentration in a diabetic's blood stream, the
system may be used in conjunction with any insulin delivery system, and is
easily adapted for out patient use.
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