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This invention relates to a means and method for monitoring blood glucose
levels and to elements employed in the practice of same.
Referring to diabetes, as illustrative of the need for rapid and frequent
analysis of blood glucose levels, diabetes is characterized by elevated
blood glucose. The severity of this disturbance, and the extent to which
diet and insulin treatment are successful in maintaining blood glucose in
the normal range is believed to determine onset and severity of the
devastating renal, retinal and cardiovascular manifestations of the
disease.
In diabetic patients dependent on insulin injections, total absence of
endogenous insulin, antibody against insulin, or less understood types of
"insulin resistance", control of glucose can be particularly difficult.
In these patients, checking for spill of glucose into the urine and spot
blood glucose determinations on an out-patient basis may not provide
sufficient information to bring blood glucose back under control. Thus
hospitalization for more intense study is required.
Furthermore, determining glucose spill into the urine can sometimes lead to
an erroneous judgment about the patient's current insulin requirement.
This is because some diabetics spill glucose into the urine from the
effects of too much, rather than too little, insulin. The cause of this
seeming paradox is recurrent hypoglycenic insulin reactions. Each of these
reactions is transient and may or may not cause symptoms, but is followed
by a massive rebound hyperglycemia, mediated through adrenalin and
glucagon release and less well understood mechanism. To evaluate and
successfully treat these patients, the physician must obtain frequent
blood glucose determinations.
Frequent blood glucose determination is also desirable when a diabetic
patient is acutely ill, undergoing surgery, childbirth, or suffering from
severe ketoacidosis. Occasionally, non-diabetic patients such as the
acutely ill patient treated with a pharmacologic dose of corticosteroid,
or the patient with recurrent fainting spells who is suspect of having
functional hypoglycemia needs to have frequent serial blood glucose
determinations made.
In summary, there is a need for an instrument, preferably a portable
instrument, suitable for continuous glucose monitoring. Numerous attempts
have been made to provide this capability but, to the present, no
instrument has emerged which is sufficiently free from problems for
acceptance into broad clinical use. Problems encountered have included
poor precision of the glucose detector, clotting and drift in the blood
sampling system, and non-linearity of the signal output. Such instruments
tend to be complicated and have required frequent and sometimes complex
calibration.
It is an object of this invention to provide an instrument and method for
frequent and rapid analysis of blood glucose, which is relatively free of
the problems heretofore encountered, which is portable to enable use for a
bedside instrument for continuous monitoring of blood glucose levels in a
patient, which can be automated for maintaining a predetermined analysis
program, and which is relatively free of clotting and/or drift in the
system for blood sampling and linear in the signal output, and it is a
related object to produce and to provide elements for use in the
successful operation of the blood glucose analyzer for monitoring blood
glucose levels.
These and other objects of this invention will hereinafter appear and for
purposes of illustration, but not of limitation, embodiments of the
invention are shown in the accompanying drawings in which:
FIG. 1 is a flow diagram of a blood glucose analyzer embodying the features
of this invention;
FIG. 2 is a flow diagram similar to that of FIG. 1 with modifications in
the analyzer;
FIG. 3 is a graph of the determination for blood glucose levels derived
from the example in the application; and
FIG. 4 is a schematic sectional elevational view of an electrode embodying
the features of this invention.
In accordance with the practice of this invention, use is made of a glucose
detector embodying an immobilized enzyme electrode which can be operated
in a rate detection mode. The high performance of the detector is based,
in part, on the development of an electrode covered with a hydrophobic
membrane, such as Teflon, having glucose oxidase bonded onto the surface
thereof in an immobilized state.
The invention will be described with reference to an instrument,
diagrammatically illustrated in FIG. 1, for analysis of blood glucose at
frequent intervals for substantially continuous monitoring of glucose
levels in patients' blood. While description will be made for operation
over 2 minute intervals, it will be understood that the frequency of
determinations for blood glucose levels can be made on more frequent or
less frequent intervals, but on an intermittent basis, as will hereinafter
be described.
The instrument diagrammed in FIG. 1 comprises a small bore plastic tube 10
fitted with an intravenous single lumen luer lock 11 connected catheter
12. Connected to this tube via line 14, fitted with a pinch valve 16, is
an intravenous infusion bag 18 adapted to be filled with a sterile
pharmacological saline solution containing sufficient heparin, about
400.mu. per liter, for infusion to prevent any thrombus formation in the
catheter between cycles for drawing patient's blood. Also connected to the
tube 10 is a second line 20 leading to a container 22 adapted to be filled
with a solution containing a calibrated amount of glucose for calibrating
the instrument, the flow of which into the tubing system is controlled by
a pinch valve 24.
A blood sample loop 26 of a measured volume is interposed in the line
between a blood sample pump 28 and the catheter, preferably between the
inlet from the source of calibrating solution and the pump, with pinch
valves 30 and 32 on opposite sides of the loop for closing off the loop.
One end of the blood sample loop 26 is connected by line 34 to the
analyzer 36 with a pinch valve 38 to isolate the loop from the analyzer,
while the other end of the loop is connected by line 40 to the outlet of a
syringe 42. The inlet of the syringe communicates through line 44 to a
source 46 of a buffer solution 48 such as a phosphate buffer, with pinch
valve 50 in line 40 to isolate the blood sample loops from the syringe.
Line 52 connects the outlet from the pump 28 to waste which may be in the
form of a waste container 54.
In the illustrated modification, the analyzer 36 is formed with an upper
compartment 56 for first bringing the blood sample to oxygen and
temperature equilibrium before the sample is displaced from the upper
compartment to a lower electrode sensing cuvette 58 containing the enzyme
electrode glucose sensor 60, hereinafter to be described in greater
detail, and a magnetic stirring bar 62 for rapid mixing of the sample
during analysis.
The cuvette is provided with an outlet in the bottom portion which is
connected by line 64 to the inlet of syringe 66 while the outlet from the
syringe 66 is connected by line 68 to waste, such as container 54. The
cuvette is also provided with an inlet connected by line 70 to the outlet
from syringe 72 while the inlet to the syringe 72 is connected by line 74
to a source 48 of a buffer solution, such as a phosphate buffer. The
cuvette is provided with an orifice 90 to maintain the cuvette at
atmospheric pressure.
The upper compartment 56 has an outlet 76 in the upper portion connected by
line 78 to a source of negative air pressure, indicated by the numeral 80,
and to a source of positive air pressure, indicated by the numeral 82. The
line 78 is provided with a valve 84 for controlling communication of the
negative air pressure line and a valve 86 for controlling communication
with the positive air pressure line.
The syringes 42, 66 and 72 are preferably joined in a syringe table 88 for
conjoint actuation of the piston type actuator in each of the three
syringes.
In operation, with valves 16, 24, 38 and 50 closed, and valves 30 and 32
open, the catheter 12 is connected into a blood vessel of the patient and
the pump 28 is operated over a 20 second period for withdrawal of
patient's blood into and through the sample loop 26 to fill the loop. In
the illustrated modification, when use is made of a canula 10 having a
diameter of 0.03 inch and a length of 3 feet, 200 microliters of blood is
drawn over a 20 second interval to fill the sample loop 26 with the
patient's blood. At the same time, the syringe table is raised to draw
buffer into the syringes 42 and 72 and to empty the electrode cuvette by
withdrawal of the contents thereof into the syringe 66.
During the remainder of the blood analysis cycle, and until the next cycle
occurs, with valves 30 and 32 closed and valve 16 open, heparinized saline
drains back through line 10 at a rate of 1/2 to 1 ml/minute to flush the
catheter to prevent any thrombus formation in the catheter.
Meanwhile, with valves 38 and 50 open and valves 30, 32, and 86 closed and
valve 84 switched to negative air pressure, the syringe table is operated
to displace fluids from the syringes to (1) deliver waste from syringe 66
to the waste container 54, (2) deliver buffer from the syringe 72 through
line 70 to the electrode cuvette 58, and (3) to flush the sample from the
sample loop 26 through line 34 into the equilibration chamber 56. The
blood sample trapped in the sample loop, in a measured amount of 40
microliters, is thus washed into the chamber 56 with 200 microliters of
buffer, such as a 0.2 M phosphate buffer at pH 6.0.
After the blood sample has been delivered to the equilibration chamber 56,
equilibration of the whole blood sample to 37.degree. C. and the oxygen
tension to that of ambient air is achieved by opening valve 84 to activate
the negative air pressure system which draws room air which has been
equilibrated to 36.degree. C. upwardly through orifice 90 through a
distributor at the base of chamber 56 whereby the air rises through the
whole blood sample as bubbles. Equilibration can be achieved by bubbling
air through the sample and buffer solution in the chamber for 40 seconds
to equilibrate the sample to room air pO.sub.2.
About 2 seconds after equilibration has been achieved, valve 84 is closed
and valve 86 is opened to activate the positive pressure system (under
about 5 psi) which causes rapid transfer (in about 0.5 second) of the
equilibrated whole blood sample from the equilibration chamber 56 to the
electrode sensing cuvette 58 which contains buffer solution, such as 2 ml.
of pH 6.0 phosphate buffer delivered from the syringe 72. The enzyme
electrode sensor then makes a rate determination of the glucose
concentration.
In the presence of glucose, oxygen tension rapidly falls as oxygen is
consumed at the electrode tip. The maximal rate of fall in oxygen tension
is recorded and detected in less than 3 seconds. This rate is proportional
to glucose concentration because of the stoichiometric relationship
between oxygen and glucose that is apparent from the following equation:
##EQU1##
Following each analysis the system is adapted automatically to wash the
sample loop 26, the equilibration chamber 56 and the reaction cuvette 58
before another cycle.
In the illustrated modification, a cycle is carried out over a period of
150 seconds, making it possible to monitor the blood by separate analysis
every 21/2 minutes.
In operation, patient's blood is drawn in an amount to flush the heparnized
solution from the tubing and to fill the loop, with the interim fluids
being pumped to waste.
The apparatus is periodically standardized by opening valve 24 before or
after the catheter 12 is inserted so that, upon operation of the pump 28,
standard solution with a known amount of glucose can be drawn into the
loop 26, after which the described normal sequence of operations are
carried out to flush the calibrating solution from the loop 26 into the
equilibration chamber 56 for equalization of temperature and oxygen and
then from the chamber 56 into the enzyme electrode glucose sensor 60 for
analysis.
The described sequence of operations of the valve, pump and syringe table
can obviously be connected with suitable electrical controls for automatic
sequencing of the operations on a controlled time basis for testing for
blood glucose levels on repeated cycles of uniform duration whereby
reliable comparisons can be made for following the course of medical
procedure and/or the patient's well being.
The reliability of the test results depends somewhat on the equilibration
of the blood samples and calibrating solutions for temperature and oxygen
levels before the rate determination is made. Use can be made of other
means for temperature and oxygen equilibration of the fluids subjected to
the test. For example, instead of making use of an equilibration chamber
56 of the type illustrated in FIG. 1, use can be made of an oxygen
equilibration coil of the type illustrated in the portable glucose monitor
illustrated in FIG. 2 of the drawing.
Briefly described, in the illustrated portable unit, blood is drawn by pump
100 from the patient 102 through the canula 104 in an amount to fill the
blood sample loop 106. The outlet from the pump 100 is connected by line
108 to a waste bag 110. A standard solution for calibrating the unit is
provided in container 112 connected to a portion of the canula 104 in
advance of the loop, as in the apparatus illustrated in FIG. 1, and a bag
114 of IV solution is also provided as in the apparatus illustrated in
FIG. 1, for flow of IV solution to the patient while the removal of whole
blood is stopped. Suitable valve members 116 and 118 are provided for
controlling the flow from the calibration container and the IV bottle
respectively.
A compact motorized syringe table 120 is provided for operating syringes
122, 124 and 126. The inlet to syringe 122 is connected to one end of the
blood sample loop for drawing the measured amount (40 microliters) of
blood from the loop plus buffer wash solution from buffer bag 132. The
outlet is connected to an oxygen equilibration coil 128 of the type well
known to the skilled in the art and made of a gas pervious-fluid
imprevious fabric which allows oxygen from the environment to penetrate
into the coil for equilibrating the oxygen concentration in the blood
sample. The oxygenated blood sample is drawn by syringe 124 from the coil
128 and displaced into the receiving coil which is also formed of a fiber
oxygenating tubing and from which it is flushed with additional buffer
drawn from the buffer bag 132 through line 134 into the syringe 126 for
displacement to the differential oxygen-glucose electrode 136 for
evaluation, as previously described. The material is flushed to the waste
bag 110 before a next cycle of operation is initiated.
Temperature equilibration is effected by housing the buffer bag 132 and the
coils 128 and 130 in a temperature controlled environment, such as at a
temperature of 37.degree. C., as outlined by the broken lines in FIG. 2 of
the drawing.
In the modification illustrated in FIG. 2, the mixing coil 130 may be
dispensed with along with syringe 124 whereby syringe 126 operates to draw
the equilibrated blood sample from the coil 128 along with the additional
buffer for admixture therewith before introduction into the analyzing
unit.
To the present, the determination of blood (whole blood plasma or serum)
has been made with a glucose oxidase enzyme reagent, as described in Clin.
Chem. 14 116(1968). When glucose is added to the enzyme reagent in a
stirred thermostated cell, glucose reacted with the oxygen in the presence
of glucose oxidase in accordance with the reaction
Glucose+O.sub.2.sup.Glucose Oxidase Gluconic Acid+H.sub.2 O.sub.2
The amount of glucose is measured indirectly by measuring the amount of
dissolved oxygen in the reaction solution.
Such technique, which makes use of glucose oxidase enzyme reagent, finds a
number of objections from the standpoint of cost of the reagent and the
time consumed for making a determination.
An important factor in the test procedure and apparatus described resides
in the utilization of an electrode in which a small amount of glucose
oxidase enzyme is immobilized on the membrane of the oxygen electrode
thereby to eliminate the need for an enzyme reagent and markedly to
increase the response and speed for making a determination.
An important inventive concept resides therefore in the fabrication of an
electrode having a membrane of hydrophobic material, such as Teflon, a
portion of which, preferably at the electrode tip, is converted to a
hydrophilic surface on which the glucose oxidase enzyme can be immobilized
as by a stable covalent bond. The result is an oxygen sensor in which the
only element that would penetrate the membrane is oxygen since others of
the elements are either not volatile enough and/or are too polar.
Oxygen at the electrode tip is consumed in the presence of glucose oxidase.
Under such circumstances, use can be made of a rate determination based
upon the current output of the electrode due to the presence of glucose in
the sample being tested. Under such circumstances, the electrode can
exhibit some slow baseline drift while an accurate determination can be
made within a few seconds. All that is required is the blood sample be
diluted in a suitable buffer and equilibrated to room temperature and
ambient oxygen level before the analysis is made.
Thus the blood sample is drawn from the patient and equilibrated from the
standpoint of temperature and oxygen by exposing the sample to oxygen in
air, as by the bubble method of the equilibration chamber 56 or by the
diffusion method of the equilibration coils 128 to give the sample the
oxygen tension that is ambient. Thus when initially pulled by the
electrode, the electrode will read the starting oxygen tension. If glucose
is present, the oxygen at the electrode tip is consumed, the effect of
which is to enable a rate determination to be made from the flow of
electrons based on oxygen penetration to the cathode tip of the electrode.
Briefly described, the hydrophobic electrode membrane, such as a membrane
of Teflon or other hydrophobic plastic material, is etched to convert the
hydrophobic surface to one that is hydrophilic. This can be accomplished,
for example, with a commercial preparation of metallic sodium in
naphthalene/tetrahydrofuran, such as marketed by Loctite Corporation of
Newington, Connecticut. The smaller the area covered by the glucose
oxidase, the more effective the analysis, since surface area is not
critical when use is made of a stirred solution of the material being
tested. In fact, it is sufficient if only the tip, such as a round area
25-200 microns in diameter, is treated.
For this purpose, a drop of the etchant can be placed on the membrane tip
for 1 minute and then rinsed with acetone and water, followed by drying.
Next, the enzyme is mobilized as by covalent bonding the treated surface.
Attachment is made with the aid of a thin layer of protein gel containing
glucose oxidase enzyme. A solution of gelling protein, glucose oxidase and
fixative is prepared immediately before application to the etched surface
of the membrane. The following is an example of a formulation which may be
used:
4 volumes of Glucose Oxidase Solution, Type VI, available from Sigma
Chemical Company, St. Louis, Mo.
1 volume of 20% by weight Bovine Albumin in solution in water
1 volume of a 4% of a Paraformaldehyde solution in water
A drop of the solution is placed on the etching area of the membrane, as
with a plastic micropipet tip. The tip of the pipet is emptied of solution
and used to remove as much solution as possible from the drop on the
membrane. The remaining thin layer of solution is dried at room
temperature for 1 minute and then drying is continued while the treated
membrane is refrigerated at 4.degree. C. for several hours.
The prepared glucose oxidase membrane is used in place of regular Teflon
membrane in a commercial glucose analyzer instrument, such as marketed by
Beckman Instrument Company of Fullerton, Calif., with no substantial
alteration of the analyzer or the sample size of the plasma required for
analysis. The membrane, stored at 4.degree. C., has a shelf life of at
least 8 months and its useful life will depend somewhat on the steps taken
to prevent growth of microorganism.
Covalent bonding of the enzyme-gel preparation occurs by reaction with
groups on the treated hydrophilic surface of the hydrophobic membrane,
such as with carbonyl, hydroxyl and carboxyl groups. Instead of
paraformaldehyde for immobilization and fixing of the enzyme, the enzyme
can be immobilized on the membrane by the use of other bifunctional
compounds having one group that attached to the etched surface and one or
more other groups that provide sites for attachment of the enzyme.
Representative of such other fixatives or immobilizing agent are
butyraldehyde, carboxiimide, formaldehyde and other well known
bifunctional coupling agents. The protein carrier gel may not be essential
to the fixation of the enzyme onto the treated surface, since it can be
entirely eliminated or other carriers can be employed.
EXAMPLE 1
The precision, accuracy and linearity of the automated glucose analyzer is
demonstrated, using standards made up in distilled water and whole blood
by addition of appropriate amounts of gravimetrically determined glucose.
The aqueous standards were prepared by dissolving 10 grams of dextrose in
1 liter of distilled water. 24 hours was allowed for mutarotation. Serial
dilutions were then prepared to produce 50, 100, 200, 300, 400 and 500
mg/dl standard. The whole blood samples were anti-coagulated with 200 mg
ethylene diamine tetramine (EDTA) per liter of whole blood.
An anesthetized dog was used for the analysis. The analyzer was connected
to the right jugular vein of the dog with a 21 gauge pediatric scalp vein
needle. The left jugular vein was canulated to allow discrete blood
sampling for reference glucose analyzing, and for infusion of glucose and
insulin. Reference glucose determinations were made on a Beckman glucose
analyzer.
At the start of the experiments, the automated glucose analyzer was
calibrated with 100 mg/dl glucose standard. A graph representing the data
from a typical experiment is presented in FIG. 3 of the drawings. As
indicated in the graph, 37 minutes after the experiment started, a 16 gram
glucose bolus was given intravenously. 67 minutes after the start of the
experiment, 16 units of regular insulin was given intravenously. At 90
minutes after the start of the experiment, 16 grams of glucose was
infused.
It will be noted from the graph that the amount of glucose detected in the
blood rose precipitously each time upon infusion of glucose and that the
amount of glucose in the blood was determined as having fallen to lower
levels in response to the infusion of insulin.
With daily use of the instrument described, sufficient enzyme activity
remained immobilized on the membrane having the electrode so that it was
necessary to change the membrane only after two to four weeks, providing
the membrane surface contact and antibacterial solution such as a solution
of benzoic acid in 0.2 M phosphate buffer at pH 7.4. If the same enzyme
membrane is used briefly daily and stored at 4.degree. C. when not in use,
loss of enzyme activity over a one month period will be found to be less
than 5%.
The electrode employed in the practice of this invention, with the enzyme
immobilized on the surface of the membrane, is illustrated in FIG. 4
wherein the base of the electrode comprises a glass rod 150 having a
cathode in the form of a platinum electrode 152 which extends to the tip
of the rod 150, and anodes in the form of silver chloride reference
electrodes 154 which extend into the space between the end portion of the
glass rod and a Teflon membrane 156 that is secured onto the end of the
electrode with a space 158 in between that is filled with electrolyte 160.
The membrane of Teflon is releasably mounted onto the end portion of the
glass rod by means of an O-ring 162 which seats in an annular groove 164
of the rod sealably to engage portions of the membrane 156 which span the
groove. The enzyme is immobilized on the gel layer 166 bonded, as
heretofore described, on the outer surface of the membrane layer, at the
tip portion of the electrode. The electrode is similar in construction to
that described in U.S. Pat. No. 3,542,662 except for the membrane with the
enzyme immobilized in a layer bonded to the outer surface of the membrane
at the tip of the electrode.
As previously described, the blood glucose analyzer of this invention is
based on a polargraphic detection system in which the specificity for
oxygen is excellent since oxygen is the only electro-active substance in
blood defusible through a Teflon membrane for reaction at the electrode.
The specificity of glucose oxidase for glucose has also been established.
By operation of the glucose sensor in a rate determination mode, any base
line drift in the oxygen electrode is eliminated.
There are a number of distinct advantages to the invention described and
claimed, namely: the blood sampling can be carried out on frequent
intervals, such as 150 seconds. This minimizes the amount of blood removed
from the patient for analysis and facilitates the maintenance of a
clot-free sampling system by comparison with continuous sampling. The
amount of heparin infused into the patient between sampling cycles is not
sufficient to cause any significant degree of systematic heparnization. By
avoiding infusion of the sample blood back into the patient, the chance of
sepis is greatly reduced. The use of an immobilized enzyme electrode in
the glucose detector permits miniaturization and simplification of
operation with minimal reagent requirement. Within seconds after sampling,
the result can be secured for the glucose concentration in the patient's
blood.
It will be apparent from the foregoing that a significant improvement is
provided in a system for blood glucose analysis and in elements employed
in analysis equipment whereby rapid and accurate determinations of blood
glucose can be made on site for monitoring glucose levels in a patient's
blood.
It will be understood that changes may be made in the details of
construction, arrangement and operation, without departing from the spirit
of the invention, especially as defined in the following claims.
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