 |
Claims  |
|
|
We claim:
1. An apparatus suitable for use in measuring blood glucose concentration
in a living being using glucose which is present at the buccal mucosal
surface of said being, said apparatus comprising:
membrane means for containing glucose oxidase enzyme;
means for positioning the said membrane means against said buccal mucosal
surface;
means for providing oxygen for said membrane means, said provided oxygen
used for peroxidative oxidation of said glucose when said membrane means
is positioned against said mucosal surface, said oxidation yielding
hydrogen peroxide as a product; and
means for measuring in said membrane means changes in the concentration of
at least one compound selected from the group including said provided
oxygen and said hydrogen peroxide product.
2. The apparatus of claim 1, wherein said measuring means comprises
electrode means for generating a signal proportional to changes in the
concentration of said at least one compound.
3. The apparatus of claim 2, wherein said electrode means includes wire
means for transmitting said signal.
4. The apparatus of claim 3, wherein said positioning means comprises a
handle member, a holder member connected to said handle member and cap
member connected to said holder member, said membrane means being
stretchingly fit across the face of said cap member.
5. The apparatus of claim 4, further including a first wire pathway
disposed in said handle member and a second wire pathway disposed in said
holder member, and wherein said wire means is housed within said first and
second wire pathways.
6. The apparatus of claim 5, wherein said holder member includes a pillar
extending opposite from said handle member when said holder member is
connected to said handle member, said pillar including said second wire
pathway.
7. The apparatus of claim 6, wherein said cap member includes a central
cutout, and wherein said pillar of said holder member projects through
said cutout when said cap member is connected to said holder member.
8. The apparatus of claim 7, wherein said pillar extends beyond said cap
member when said cap member is connected to said holder member, said
pillar including a face which abuts against said membrane means
stretchingly fit over said cap member, whereby said wire means contacts
said membrane means at said face for transmitting said signal.
9. The apparatus of claim 2, wherein said electrode means comprises an
anode member and a cathode member.
10. The apparatus of claim 9, wherein said electrode means further
comprises an electrical grounding member.
11. The apparatus of claim 10, wherein said positioning means comprises a
handle member, a stem member connected to said handle member, a base
member connected to said stem member and a probe member supported by said
base member, said membrane means being coveringly fit over said probe
member.
12. The apparatus of claim 11, wherein positioning means further includes a
ridge member extending laterally from said stem member.
13. The apparatus of claim 11, wherein said probe member includes said
anode member and said cathode member.
14. The apparatus of claim 13, wherein said anode member is centrally
positioned on the face of said probe member and projects beyond said probe
member so as to contact said membrane means.
15. The apparatus of claim 14, wherein said cathode member is positioned
along the peripheral region of said probe member.
16. The apparatus of claim 1, wherein said membrane means comprises an
outer layer, a central layer and an inner layer, said outer layer being in
contact with said buccal mucosal surface when said membrane means is
positioned against said surface.
17. The apparatus of claim 16, wherein said outer layer comprises
cellulose.
18. The apparatus of claim 16, wherein said outer layer comprises
polycarbonate.
19. The apparatus of claim 16, wherein said central layer includes said
glucose oxidase enzyme.
20. The apparatus of claim 16, wherein said inner layer comprises cellulose
acetate.
21. The apparatus of claim 1, wherein said oxygen providing means comprises
means for supplying excess oxygen to said membrane means.
22. The apparatus of claim 21, wherein said oxygen supplying means
comprises means for conducting atmospheric air to said membrane means.
23. The apparatus of claim 22, wherein said positioning means comprises a
handle member, a holder member connected to said handle member and a cap
member connected to said holder member, said membrane means being
stretchingly fit across the face of said cap member.
24. The apparatus of claim 23, wherein said conducting means includes a
first pathway disposed in said handle member, a second pathway
communicating with said first pathway and disposed in said holder member,
and a third pathway communicating with said second pathway and disposed in
said cap member.
25. The apparatus of claim 24, wherein said handle member includes means
for detachably receiving said holder member.
26. The apparatus of claim 25, wherein said receiving means comprises a
side arm.
27. The apparatus of claim 26, wherein said holder member comprises a plug
portion received in said side arm, a disc portion and a central pillar
extending from said disc portion opposite said plug portion.
28. The apparatus of claim 27, wherein said plug portion includes a ridge
and said side arm includes a groove for mating engagement with said ridge.
29. The apparatus of claim 27, wherein said cap member includes a central
cutout, and wherein said pillar of said holder member projects through
said cutout when said cap member is connected to said holder member.
30. The apparatus of claim 29, wherein the diameter of said cutout is
greater than the diameter of said pillar to form a chamber therebetween,
said third air pathway comprising said chamber.
31. The apparatus of claim 24, wherein said second pathway disposed in said
holder member comprises a plurality of air orifices.
32. The apparatus of claim 21, wherein said oxygen supplying means
comprises means for conducting oxygen in the air of the mouth of said
living being to said membrane means.
33. The apparatus of claim 32, wherein said positioning means comprises a
probe member, said membrane means being stretchingly fit across the face
of said probe member.
34. The apparatus of claim 33, wherein said membrane means is saturated
with an electrolyte solution, said saturated membrane means comprising
said oxygen conducting means.
35. A method of measuring blood glucose concentration in a living being
using glucose which is present at a mucosal surface located in an orifice
of said being, said method comprising:
positioning a membrane member against said mucosal surface, said membrane
member including glucose oxidase enzyme;
providing oxygen to said membrane member to enable peroxidative oxidation
of said glucose, said oxidation producing hydrogen peroxide as a product;
measuring the change in concentration of at least one compound selected
from the group including said provided oxygen and said hydrogen peroxide
product; and
calibrating said measurement in order to determine blood glucose
concentration.
36. The method of claim 35, wherein said measuring step comprises measuring
the amount of said hydrogen peroxide product.
37. The method of claim 35, wherein said positioning step comprises
positioning said membrane member against a buccal mucosal surface.
38. The method of claim 35, wherein said providing oxygen step comprises
supplying excess oxygen to said membrane member.
39. The method of claim 38, wherein said providing oxygen step comprises
conducting atmospheric air to said membrane member.
40. The method of claim 38, wherein said providing step comprises
conducting oxygen in the air of the mucosal orifice to said membrane
member.
41. An apparatus suitable for use in measuring blood glucose concentration
in a living being using glucose which is present at a mucosal surface of
said being, said apparatus comprising:
membrane means for containing glucose oxidase enzyme;
means for positioning said membrane means against said mucosal surface;
means for providing oxygen for said membrane means, said provided oxygen
used for peroxidative oxidation of said glucose when said membrane means
is positioned against said mucosal surface, said oxidation yielding
hydrogen peroxide as a product; and
means for measuring in said membrane means changes in the concentration of
at least one compound selected from the group including said provided
oxygen and said hydrogen peroxide product. |
|
 |
|
 |
Claims |
|
|
|
Description |
|
|
BACKGROUND OF THE INVENTION
This invention relates to a method and apparatus for measuring blood
glucose concentration using glucose which is present at the buccal mucosal
surface, and more particularly to an apparatus and method for measuring
blood glucose concentration by means of the peroxidative enzyme glucose
oxidase.
In general, an enzymatic electrode is often used to measure the level of a
blood substance. A typical enzymatic electrode combines enzymatic and
polarographic technologies. The enzyme is usually bound, physically or
chemically, to an inert support.
Many enzymatic electrodes utilize enzymes which catalyze the peroxidative
oxidation of a substrate and generate hydrogen peroxide. The specificity
of the electrode is determined by the substrate specificity of the enzyme.
Two types of analytical measurements are possible, each one using the
polarographic technique. Specifically, either the consumption of free
oxygen or the production of hydrogen peroxide may be measured. Both serve
as stoichiometric indicators of substrate oxidation. When the enzyme
electrode is used after it has been calibrated using known concentrations
of the substrate, the electrode becomes a tool for the quantitative
analysis of unknown concentrations of that same substrate.
Enzymatic peroxidation requires two substrates: oxygen, which is usually
present in excess, and the unknown, for which the enzyme is specific and
which is usually present in a limited amount. In operation, the enzyme is
placed in an oxidized state before reacting with a substrate. Then, after
enzyme-substrate interaction, the enzyme is reduced. This reduced enzyme
is re-oxidized in order to produce hydrogen peroxide. If oxygen were not
present, the catalytic enzyme cycle would be inhibited.
In view of the above, it is desirable to provide an enzyme electrode
apparatus for the analysis of glucose in an animal or human using the
enzyme glucose oxidase. Such a device would be very useful in a program of
diabetes management, relieving the diabetic of the need to obtain blood
samples for glucose analysis.
In order to determine blood glucose concentration for diagnostic and other
purposes, the present state of the art requires that blood samples be
obtained. In a patient, samples are usually obtained either by pricking
the finger or by intravenous withdrawal. However, these invasive methods
are not satisfactory since tissue damage and patient discomfort often
result.
A glucose oxidase-containing enzyme electrode apparatus has been previously
constructed which is suitable for intravenous implanation. Such an
apparatus, however, is not fully satisfactory since fibrous tissue growth
and immune system rejection may inhibit proper functioning of the
apparatus over long periods of time.
As a non-invasive alternative, the measurement of blood glucose
concentration at a body surface may be used. This is achieved without the
necessity of obtaining a blood sample. Body surface areas that may be
suitable include the skin and the mucous membranes.
It is generally known that glucose diffuses from subdermal capillaries onto
the surface of hydrated skin. This was shown by experiments which
consisted of placing drops of buffer solution (which contained enzymes and
coenzymes used in the enzymatic analysis of glucose) onto the skin, and
then measuring the increase in fluorescence as NADP was reduced to NADPH.
Significantly, although dry skin is an effective barrier to the diffusion
of hydrophilic molecules such as glucose, that barrier is reduced by the
removal of keratinizized layers, thereby leaving only the dermis. However,
the buccal mucosa and the dermis have nearly identical diffusion
characteristics for many hydrophilic molecular species, including glucose.
Accordingly, it is an object of the present invention to provide a method
for measuring blood glucose concentration using glucose which is present
at the buccal mucosal surface of a living being.
It is another object of the present invention to provide a method for
measuring blood glucose concentration by means of the peroxidative enzyme
glucose oxidase.
Still a further object of the present invention is to provide a method for
measuring blood glucose concentration which is non-invasive.
Yet another object of the present invention is to provide a method of
measuring blood glucose concentration using an excess oxygen supply.
It is still a further object of the present invention to provide a method
of measuring blood glucose concentration by means of an electrode
apparatus.
Still other objects of the invention will in part, be obvious, and will, in
part, be apparent from the following description.
SUMMARY OF THE INVENTION
Generally speaking, in accordance with the invention, a method of measuring
blood glucose concentration using glucose which is present at a mucosal
surface of a living being is provided. The method includes the steps of
positioning a glucose oxidase membrane on the mucosal surface, providing
oxygen to the surface in order to peroxidatively oxidize the glucose and
produce hydrogen peroxide as a product, electrically measuring the amount
of hydrogen peroxide produced, and calibrating the measured amount of
hydrogen peroxide in order to determine blood glucose concentration.
The method can be achieved by using an electrode apparatus which supports a
glucose oxidase membrane at one end and which supplies oxygen to the
region of the membrane, either from the atmosphere or from the air in the
orifice containing the mucosal surface. The membrane is placed against the
mucosal surface of the living being in order to initiate a peroxidative
oxidation reaction. Then, by means of electrodes in communication with the
membrane, the amount of hydrogen peroxide produced during peroxidative
oxidation is electrically measured and calibrated in order to determine
blood glucose concentration.
In the preferred embodiments, glucose concentration is measured at the
buccal mucosal surface.
The invention accordingly comprises the several steps and the relation of
one or more of such steps with respect to each of the others, and the
apparatus embodying the features of construction, combination of elements
and arrangement of parts which are adapted to effect such steps, all as
exemplified in the following detailed disclosure, and the scope of the
invention will be indicated in the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
For a fuller understanding of the invention, reference is made to the
following description taken in connection with the accompanying drawings,
in which:
FIG. 1 is a schematic view, partially in phantom, showing an apparatus in
accordance with the invention inserted within the mouth of the user;
FIG. 2 is an exploded perspective view showing the apparatus of FIG. 1 in
accordance with the invention;
FIG. 3 is a cross-sectional view taken along line 3--3 of FIG. 1;
FIG. 4 is a cross-sectional view of the apparatus of FIG. 1 in accordance
with the invention and illustrates the oxygen pathway and electrode wire
pathway disposed therein;
FIG. 5 is an enlarged cross-sectional view of a portion of the apparatus
shown in FIG. 4 and shows in detail the 3-layer membrane and electrode of
the apparatus;
FIG. 6 is a perspective view showing an alternate embodiment of the
electrode holder of the apparatus;
FIG. 7 is a perspective view showing the membrane attached to the cap
member of the apparatus;
FIG. 8 is a perspective view of an alternate embodiment of the apparatus in
accordance with the invention;
FIG. 9 is a side view in partial cross-section, showing the electrode
apparatus of FIG. 8 positioned within the mouth of the user and held in
place by the user's teeth;
FIG. 10 is a cross-sectional view taken along line 10--10 of FIG. 9,
showing the probe tip of the electrode apparatus in contact with the
buccal mucosa;
FIG. 11 is a frontal view taken along line 11--11 of FIG. 10; and
FIG. 12 is an enlarged cross-sectional view taken along line 12--12 of FIG.
11.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
In order to measure diffused glucose at the buccal mucosal surface by means
of peroxidative oxidation, an adequate oxygen supply is needed. However,
the oxygen concentration found in the buccal mucosal is normally
inadequate to support peroxidative oxidation. In accordance with the
invention, the oxygen deficiency is overcome by providing an electrode
apparatus in which the components are arranged so that oxygen is
transmitted to a membrane/electrode interface. Consequently, when placed
in an oxygen-deficient environment, such as on the buccal mucosal surface,
an oxygen concentration gradient is formed in order to drive oxygen into
and through the membrane which covers the face of the electrode.
Referring first to FIGS. 2 and 4, an electrode apparatus 11 in accordance
with the invention is shown. Apparatus 11 includes a handle 13, an
electrode holder 15, a cap member 17, a membrane member 19 and an O-ring
21.
Handle 13 of apparatus 11 is substantially tubular in shape and has a top
member 41, a bottom 33, and four sides 35a-d. As shown in FIGS. 2 and 3,
handle 13 also includes ridge members 37 extending laterally from side 35c
adjacent top member 41. During use of the apparatus, ridge members 37 abut
the lingual surface of the teeth to hold apparatus 11 securely in position
in the mouth.
Handle 13 also includes a cylindrical housing side arm 23 on side 35a
proximate top member 41. Side arm 23 receives electrode holder 15 during
assembly of apparatus 11, as best shown in FIG. 2.
Referring once again to FIGS. 2 and 4, handle 13 is formed with an
electrode wire pathway 25 and an air pathway 27 longitudinally disposed
therein. Electrode wire pathway 25 communicates with the outside by means
of an electrode wire opening 29 formed in bottom member 33. Electrode wire
pathway 25 houses a plurality of electrode wires. In the preferred
embodiment, as shown in FIG. 4, two wires 43 are illustrated, one of which
is made of platinum and the other of which is made of silver. Each wire 43
is provided with a conventional insulating material 45 thereabout. An
additional insulating material 46, which is preferably a Teflon shrink
tubing, but which alternatively may be an epoxy resin, may be used within
wire pathway 25.
Air pathway 27 communicates with the outside by means of an air opening 31
formed in bottom member 33. The other end of air pathway 27 is in
communication with an air space 39 formed below top member 41 of handle
13.
As best illustrated in FIG. 2 and FIG. 4, electrode holder 15 of apparatus
11 is detachably receivable in side arm 23 of handle 13 and includes a
plug portion 51, a disc portion 49 integrally formed therewith and a
central pillar 47 extending from disc portion 49 opposite plug portion 51.
Pillar 47 includes a face 48 and a cylindrical wall 50, both of which are
preferably fabricated of or coated with a non-wettable material, such as
Teflon. In order that holder 15 can be readily received in and retained by
side arm 23, plug portion 51 of holder 15 is preferably formed with a
circumferential ridge 55 which matingly engages circumferential groove 53
disposed along the inside of side arm 23.
Holder 15 is formed with centrally positioned orifices 57 for receiving
electrode wires 43. Orifices 57 extend through pillar 47, including face
48 thereof. During assembly, electrode wires 43 are inserted into wire
opening 29 of bottom member 33, through wire pathway 25 of handle 13,
outside arm 29 and into orifices 57.
Holder 15 is also formed with a plurality of circumferentially positioned
air orifices 59. Orifices 59 extend through plug portion 51 and disc
portion 49 of holder 15, but do not extend through and are
circumferentially positioned with respect to pillar 47, preferably, as
shown best in FIG. 2, in a semi-circular arrangement. This construction is
advantageous since it enables uniform diffusion of oxygen to the membrane
surface when the apparatus of the invention is operatively positioned on
the buccal mucosal of a living being. Orifices 59 communicate with air
space 39 of handle 13 so that air which enters air space 39 from air
channel 27 can pass therethrough.
As shown in FIGS. 2 and 4, cap member 17 of apparatus 11 includes a central
cut-out 65, an annular face 70, a circumferential groove 68 and a
protruding annular lip 20. Lip 20 includes a circumferential rib 22 which
engages the rim of disc portion 49 when cap member 17 is attached to
holder 15. Cut-out 65 includes an internal wall 66 which is preferably
fabricated of or coated with a non-wettable material, such as Teflon. Cap
member 17 is attached to and matingly engaged with holder 15 such that
central pillar 47 of holder 15 projects through cut-out 65, as FIG. 4
illustrates.
Once cap member 17 is attached to holder 15, an annular interior space 63
is formed therebetween which communicates with air orifices 59 of holder
15. Moreover, since cut-out 65 has a diameter larger than the diameter of
pillar 47, a circumferential air chamber 67 is formed around pillar 47
which communicates with interior space 63. By this construction, air in
air space 39 will pass through air orifices 59 in holder 15, then enter
interior space 63 and finally pass into circumferential air chamber 67.
Referring now to FIG. 7, membrane member 19 of apparatus 11 is stretchingly
fit across cap member 17. This is achieved by positioning the perimeter of
membrane member 19 along circumferential grove 68 of cap member 17 and
then fitting elastic O-ring 21 thereabout. Moreover, since pillar 47
projects beyond cap member 17, forcing membrane member 19 to assume a
somewhat convex shape, an annular aqueous reservoir 69' is formed between
membrane member 19 and cap member 17. Because membrane member 19 must be
maintained in a physiological isotonic solution, or alternatively in a
preservative liquid, when the apparatus of the invention is not being
used, reservoir 69' becomes filled with that liquid and remains filled
with that liquid when the device is operatively positioned on the buccal
mucosal surface of the user. Accordingly, air which enters the apparatus
through the various air pathways previously described will ultimately pass
into aqueous reservoir 69' and contact membrane member 19. As a result, a
sufficient oxygen supply is provided for the peroxidative oxidation
reaction in membrane member 19.
Referring again to FIGS. 4 and 7, after attaching membrane member 19 to cap
member 17, face 48 of pillar 47 abuts membrane member 19 such that
electrode wires 43 contact membrane member 19. Moreover, since face 48 is
rounded, membrane member 19 is retained snugly and smoothly against face
48.
Referring now to FIG. 5, membrane member 19 preferably comprises three
layers: an outermost protective layer 67, a central glucose oxidase layer
69, and an inner limiting membrane layer 71. Outermost layer 67 extends
along the entire surface of cap member 17, and is preferably made of
cellulose. Layer 67 serves to stabilize membrane member 19 since it is the
thickest and strongest of the three layers. Layer 67 also serves to
exclude all substances having a molecular weight greater than 3500, while
offering little resistance to molecules of a much lower molecular weight.
Central layer 69 is not coextensive with outermost layer 67, but extends
only along face 48 of pillar 47. Layer 69 contains covalently-immobilized
glucose oxidase and may be made of any material which has free reactive
groups suitable for attaching the glucose oxidase molecules. Preferably, a
natural membranous material which has free --NH.sub.2 groups, such as
bovine mesentery (obtainable from a slaughter house or butcher shop), is
used. In preparing the central layer 69, the membranous material is first
incubated with glucose oxidase and the coupling reagent glutaraldehyde
(which both attaches the enzyme to the membrane and crosslinks residual
membrane --NH.sub.2 groups). Layer 69 is then extensively washed and
dried. Thereafter, layer 69 is rehydrated before use with the invention.
Layer 69 occupies an area overlying electrode wires 43 at their tips. In
the preferred embodiment, glucose diffuses through outer membrane layer 67
from the buccal mucosal surface, while oxygen diffuses across inner layer
71, which is positioned between layer 69 and pillar 47 of holder 15.
Glucose oxidase catalyzes the following reaction:
glucose+O.sub.2 -------- H.sub.2 O.sub.2 +gluconate
Inner membrane layer 71 is made preferably of cellulose acetate by means of
a cast film, and prevents the passage of molecules having a molecular
weight greater than 100. Hydrogen peroxide, which is a product of the
glucose oxidase catalyzed reaction, diffuses through membrane layer 71 to
electrode wires 43, where it is detected polarographically, yielding a
current proportional to concentration as a result of the following
reaction:
H.sub.2 O.sub.2 -------- O.sub.2 +2H.sup.+ +2e.sup.-
Turning now to FIGS. 1, 3, and 5, apparatus 11 is illustratively shown in
FIG. 1 operatively positioned in the mouth of a human user. Cap member 17,
which has membrane member 19 disposed thereover, is placed on the rinsed
buccal mucosal surface. As shown schematically in FIG. 5, glucose
molecules G diffuse from a subdermal capillary 73 through interstitial
tissue 75 and mucosal tissue 76 to membrane member 17 along a
concentration gradient. The presence of excess oxygen in the region of
membrane member 17 enables the peroxidative oxidation reaction to take
place with glucose as the limiting factor. Accordingly, the concentration
of hydrogen peroxide produced bears a direct stoichiometric relationship
to the concentration of glucose which was initially present.
Electrode wires 43, which extend through the device and contact membrane
member 19, transmit an electrical signal when apparatus 11 is operatively
placed on the buccal mucosal surface. Specifically, the presence of
hydrogen peroxide, the product of peroxidative oxidation, is detected
polarographically by electrode wires 43. This is achieved by the platinum
electrode wire detecting the negative potential (electron migration)
produced by the reaction. The strength of the signal generated is
proportional to the concentration of hydrogen peroxide produced and the
wires transmit the electrical signal to an external device (a readout
means) which amplifies the signal and displays and records changes in
concentration as a function of time. Significantly, since membrane member
19 is pliable, it adheres tightly to face 48 of pillar 47 so that no gap
is formed between face 48 and membrane member 19 which would otherwise
inhibit peroxide diffusion towards wires 43.
Reference is now made to FIG. 6, which illustrates a second embodiment of
the electrode holder, generally designated at 81, in accordance with the
invention. Holder 81 includes a disc portion 79 and a plug portion 83
integrally formed therewith. Holder 81 also includes a pillar 87 which
projects opposite plug portion 83. In this embodiment, holder 81 includes
a plurality (four) of holes 85 disposed at equally spaced intervals about
the circumference. This construction is also advantageous since it enables
uniform diffusion of oxygen to the membrane surface.
In general, the handle of the apparatus has the following functions:
1. It serves as a conduit for the transmission of air to the interior of
the holder.
2. It contains the wiring through which electrode polarization is
transmitted and from which a current, proportional in intensity to the
glucose concentration at the mucosal surface, is sent to amplifiers and
then to a display device. The electronic functions may be contained within
the handle or may be located in a separate unit connected by wires to the
handle.
3. It stabilizes the electrode apparatus when in position against the
buccal mucosal surface. Stability is maintained by a ridge, running
lengthwise on the top and bottom of the handle, which is placed tightly
against the lingual margin of the teeth. This stabilization eliminates
non-glucose related signals, which may be caused by the physical movement
of the device after electrode-buccal contact has been made, or by the
intrusion of saliva.
The electrode holder serves the following functions:
1. It permits the flow of oxygen from the electrode housing into the space
between the electrode holder and the cap member.
2. It provides a watertight, insulated housing for the connecting wiring
and working electrode surfaces which are used to detect the hydrogen
peroxide product of the enzymatic reaction.
The oxygen-transmitting glucose electrode apparatus in accordance with the
invention yields steady-state signals wherein the amount of glucose
measured at the buccal mucosal surface equals the amount of glucose in the
capillaries minus the amount taken up by the cells in transit from the
capillaries to the buccal mucosal surface. At a steady state, the glucose
concentration at the buccal mucosal surface equals that of the
interstitial fluid which bathes the living cells. Therefore, these signals
in turn can be correlated with blood glucose under both preprandial and
glucose tolerance test conditions.
During operation, between 5 and 10 minutes is required for the
glucose-related signal to attain a steady-state level. At this
steady-state level, the glucose concentration equilibrium between the
membrane and the buccal mucosal surface is achieved. Maintaining a
constant contact, however, between the electrode face and the mucosal
surface for that time period is difficult and requires training.
Accordingly, since the relationship between the initial rate of an
enzyme-catalyzed reaction, at a given temperature, and the concentration
of substrate is well known, that relationship may be substituted for a
steady state analysis. Thus, the initial rate of change of the electrode
signal is preferably used to indicate buccal glucose concentration.
The electrode of the inventive apparatus may be calibrated at room
temperature (25 degrees Celsius), using known concentrations of glucose.
Specifically, when the apparatus of the invention is placed operatively on
the buccal surface, the face of the electrode may be warmed by contact
with the body. Since the rate of an enzyme-catalyzed reaction is
temperature-dependent, measurements of temperature are made (such as by a
thermistor), both in vitro and in vivo, to allow calibration and analysis
at known temperatures. This allows the user to calibrate the electrode at
any temperature within 20-40 degrees Celsius (68-104 degrees Fahrenheit),
and then to measure buccal mucosal glucose concentration at any other
temperature within this range, multiplying the measured amperage by a
constant in order to compensate for the effect of temperature. The rate at
which the electrode-membrane interface comes to thermal equilibrium after
being placed on the buccal mucosal may also be measured.
In accordance with the invention, an output signal is produced which
increases in proportion to the amount of glucose present at the mucosa.
Both the rate at which the signal increases and the steady state which is
ultimately obtained are directly proportional to the concentration of
glucose in the capillaries.
As soon as the membrane is applied to the buccal mucosa, a concentration
gradient for oxygen is formed (this gradient can range between
approximately 8.6 mM in the air and about 0.2 mM at the interface).
Consequently, oxygen from the atmosphere is conducted through the oxygen
pathway of the apparatus to the membrane retained against the buccal
mucosal surface. The electrode system thus uses the atmosphere as a
reservoir to supply oxygen for the peroxidative enzymatic reaction at the
membrane-electrode interface.
The principles embodied in the present invention extend beyond glucose
analysis to all applications involving the use of oxygen-requiring enzyme
electrodes in oxygen-deficient environments.
After the membrane is placed over the electrodes, each electrode is
calibrated by first immersing the membrane in a physiological buffer
solution and then adding aliquots of Beta-D-glucose. The current from the
electrodes increases in proportion to increasing glucose concentration.
The presence of glucose at the buccal surface is analyzed after each
subject first rinses his mouth thoroughly with distilled water to cleanse
the mouth of surface glucose, including salivary glucose. Thereafter, an
abrasive material (such as a piece of gauze) is used to wipe the surface
to which the membrane is to be applied in order to remove mucosal residue
and other contaminants. Placement of the membrane onto the buccal mucosal
yields a current which increases to a plateau level over a time interval
of about 5 minutes.
Referring now to FIGS. 8 and 9, an electrode apparatus 101 in accordance
with a second embodiment of the invention is shown. Apparatus 101 includes
a handle 107, a stem 105 connected to handle 107, a base member 109, and
an electrode or probe member generally designated at 102 supported by base
member 109. Apparatus 101 also includes a ridge member 103 extending
laterally from base member 109 and stem 105. During use of apparatus 101
in the mouth of the user, as best shown in FIG. 9, ridge member 103 is
securely gripped between the user's upper molar 121 and lower molar 123 in
order to hold apparatus 101 securely in position in the mouth.
Apparatus 101 is formed with an electrode wire pathway 125 longitudinally
extending through handle 107 and stem 105. Electrode wire pathway 125
communicates with the outside by means of an electrode wire opening (not
shown) formed at one end of handle 107. Electrode wire pathway 125
receives and houses an electrical cable 112 containing a plurality of
electrical wires 127. Electrical wires 127 extend through apparatus 125
and can transmit an electrical signal when apparatus 101 is operatively
placed on the buccal mucosal surface of a patient's mouth.
Turning now to FIG. 10, electrode member 102, which is supported by base
member 109, is shown in contact with the buccal mucosa 129 of a patient's
mouth. Electrode member 102 includes a face region 117 and an anode 111,
preferably made from platinum, centrally positioned on face 117 and
protruding slightly therefrom. Electrode member 102 also includes a
substantially annular cathode 115 preferably made of silver and disposed
substantially along the peripheral region of face 117. Anode 111 and
cathode 115 are electrically connected to electrical wires 127 inside
electrode member 102 (not shown).
A membrane member 119 of apparatus 101 is stretchingly fit across face 117
of electrode member 102. This is achieved by positioning the perimeter of
membrane member 119 along circumferential groove 124 of electrode member
102 and then fitting an elastic O-ring 113 thereabout. Since anode 111
protrudes beyond face 117, thereby forcing membrane member 119 to assume a
somewhat convex shape, an annular gap 128 is formed between membrane
member 119 and face 117, as best shown in FIGS. 10 and 12. Moreover,
annular cathode 115 protrudes somewhat beyond the periphery of face 119
and also contacts membrane member 119.
Optionally, electrode member 102 includes a reference electrode 120 which
is positioned beneath O-ring 113, as illustrated in FIG. 10. Reference
electrode 120 is preferably made of platinum and has the function of
increasing electrical stability; electrode member 120 is normally kept at
0 volts.
Turning now to FIGS. 11 and 12, and still referring also to FIG. 10,
membrane member 119 is shown operatively positioned over face 117 of
electrode member 102 and abutting anode 111 and cathode 115. Membrane
member 119 is preferably made from three layers: an outermost protective
layer 135, a central glucose oxidase layer 133 and an inner membrane layer
131. Outermost layer 135 extends along the entire surface of electrode
member 102 and is made of polycarbonate. Inner membrane layer 131, which
overlies both anode 111 and cathode 115, is made of cellulose acetate.
This material is chosen since it is selectively permeable and will enable
diffusion of H.sub.2 O.sub.2 more readily than other substances having a
greater molecular weight than H.sub.2 O.sub.2.
Central layer 133 contains covalently-immobilized glucose oxidase and may
be made of any material which has free reactive groups suitable for
attaching glucose oxidase molecules, similar to central layer 69 of the
first embodiment.
Before placing apparatus 101 in the mouth of a patient, three-layer
membrane member 119 is saturated with an aqueous electrolyte or buffer
solution. The buffer solution then becomes saturated with atmospheric
oxygen. For optimal performance, annular gap 128 formed between membrane
member 119 and face 117 is filled with the buffer solution.
After the apparatus is placed in the patient's mouth, the central region of
membrane member 119 contacts bucccal mucosa 129, as shown in FIG. 10.
However, the peripheral region of membrane member 119 does not contact the
buccal mucosa, but remains in communication with the air in the patient's
mouth. Because of the glucose oxidase reaction which occurs in the region
of membrane member contact with the buccal mucosa, the oxygen level in the
buffer solution which has saturated membrane member 119 decreases. As a
result, oxygen will diffuse from the air in the patient's mouth through
the peripheral region of membrane member 119 and toward the central region
of membrane member 119, as arrows A in FIGS. 10 and 11 illustrate.
Consequently, an oxygen gradient is formed in membrane member 119, and can
be characterized as the difference between the oxygen concentration in the
saturated buffer solution (approximately 0.21 mM) at the periphery and
that at the buccal mucosa (approximately 0.08 mM to 0.13 mM). This enables
a sufficient quantity of oxygen to be supplied to the buccal mucosal
region in order to peroxidatively oxidize all of the glucose present
thereat, and thereby produce hydrogen peroxide as a product.
In operation, electrode wires 127, which extend through apparatus 101, and
which are electrically connected to cathode 115 and anode 111, as well as
to reference electrode 120, transmit an electrical signal when electrode
member 102 is operatively positioned on the buccal mucosal surface. More
particularly, the presence of hydrogen peroxide, the product of
peroxidative oxidation, is detected polarographically by cathode 115 and
anode 111, and is transmitted along electrode wires 127 to an external
device (a readout means) which amplifies the signal and displays and
records changes in concentration as a function of time. Electrical
detection of hydrogen peroxide is similar to detection in the first
embodiment of the electrode apparatus.
Electrode apparatus 101, however, has certain advantages over the first
embodiment. Specifically, its sensitivity is at least five times greater,
the noise level is reduced at least ten times and the signal to noise
ratio is increased at least fifty times. This is because of the following
structural and functional changes:
1. a substantial increase in electrode surface area;
2. the inclusion of a third electrode surface --the reference electrode;
3. supplying oxygen from the air within a patient's mouth to the mucosal
surface;
4. a unique three-layer membrane member which includes glucose oxidase in
the central layer and which substantially inhibits the diffusion of
various undesirable substances to the surface of the anode;
5. increased stabilization of the position of the electrode and membrane
against the buccal mucosal; and
6 a reduction in the size and weight of the electrode apparatus.
Although the method in accordance with the invention preferably includes
measuring hydrogen peroxide production in order to determine glucose
concentration, the method can alternatively include measuring oxygen loss
in order to determine glucose concentration. This may be achieved by
reversing the polarity of the electrodes and measuring the decrease in
current as molecular oxygen is reduced to peroxide.
Even though the method described herein is for measuring blood glucose
concentration using g | | |
