CA1138311A - Apparatus for monitoring blood glucose levels and elements - Google Patents

Abstract

Abstract of the Disclosure A means for monitoring blood glucose levels at frequent intervals, which includes a means for equal-izing the temperature and oxygen level in the blood and sensing the rate of oxygen consumption by the glucose contained in the blood in the presence of glucose oxidase enzyme immobilized on a hydrophobic membrane covering a measurement electrode and elements employed therein including the equalizing means and the electrode and method for preparing same.

Description

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This invention relates to a means and method for monitorlng blood glucose levels and to elements employed in the practice of same.
Referring to diabetes, as illustra-tive of the need for rapid and frequent analysis of blood glucose levels, diabetes is characterized by elevated blood glucose. The severity o~
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 manifesta-tions of the disease.
In di.abe~ic patients dependent on insulin injections, total absence of endogenous insulin, antibody agains~ insulin, or less understood types of "insulin resistance", control o~
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 inter.se study is required.
Furthermore, determining glucose spill into the urine can sometimes lead to an erroneous judgment abou-t the patient's current insulin requirement~ Thi.s is because some diabetics spill glucose into the urine from ~he effects of too much, rather than too li~tle, insulin, The cause of this seeming paradox is recurrent hypoglycenic insulin reactions. Each of these reactions is ~ransient and may or may not cause symptoms, but is ~ollowed by a massive rebound hyperglycemia, ~ediated through adrenalin and glucagon reIease and less well understoo~ mechanism, i~ .

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To evaluate and successfully -treat ~hese patients, -the physici.an must obtain frequent blood ylucose determinations.
Frequent blood glucose de-termination is also desirable when a diabetic patient is acutely ill, undergoing surgery, childbirth, or su~fering from severe ketoa~idosis~ occasionally, non~diabetic patien-ts such as the acutely.il:L patient treated with a pharmacoloyic dose of corticosteroid, or the patient with recurrent fainting spe~ls who is suspect of having func-tional hypoglycemia needs to have frequent serial blood glucose determinations madeO

In summary~ there is a need for an instrument, pxefer-ably 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 clinica]. use. Problems encountered have included poor precision of the glucose detector, clotting and drift in khe blood sampling system, and non-linearity of the signal output. Such instruments tend to be complicated and have required ~requent and sometimes complex calibration.

It is an object of this invention to provide an instrument and method for fxequent and rapid analysis of blood glucose, which is relatively free of the problems heretof~re encountered, which is portable to enable use for a bedside 2S instrument for continuous monitoring of blood glucose levels in a patient, which can be automated for maintaining a pre-determined analysis program, and which is relatively free of clotting and/or dri~t in the system for blood sampling and linear in the signal output, and it is a related ob~ect to produce and to provide elements for use in the successful operation of the blood glucose analyzer for monitoring blood glucose levels.

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Thus, ln accordance with the present invention, -there is provided a method for monitoring blood glucose levels comprising the steps of equalizing the oxygen concentration in a bloocl sample by con-tacting the sample with a:ir for a sufficient length of time to effect oxygen equalization~ transferring the sample after air-oxygen equalization to a sensor having an elect-rode covered with a hydrophobic membrane hav:ing a surface on which glucose oxidase is immohilized whereby oxygen is consumed by reaction with glucose in the blood in the presence of the glucose oxidase, and measuring oxygen consumption resulting from the reaction of glucose in the blood in determining the level of glucose in the blood.
In another aspect, the invention provides an apparatus for monitoring blood glucose levels comprising a sensor having an electrode, at least a portion of which is covered with a hydrophobic membrane having glucose oxidase enzyme immobilized to a surface portion thereof, a canula adapted to mount a catheter for withdrawing blood from a patient, a source of IV
solution and means connecting the source of IV solution with -the canula for draining the IV solution back through the canula to the patient when blood is not being withdrawn, a pump having an inlet connected to the canula and an outlet connected to waste, a source of buffer solutionl an equalizing member for equalizing the temperature and oxygen level of an increment of blood to be sensed, a syringe having an inlet connected to the source of -the buffer and an outlet connected to the canula for flushing said increment of withdrawn blood to the equalizing member, another syringe having an inlet connected to the sensor for withdrawing liquid that has been sensed and an outlet connected to waste~

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and a passage communicating the equalization member with the sensor for the -transmission of an equalized increment of blood to the sensor for determination of the leve:L of glucose in the blood in the presence of the sensor.
Particular embodiments of the present invention will now be described for purposes of illustration, but not of limitation, wi-th reference to the accompany:ing drawings, in which:
Figure 1 is a flow diagram of a blood glucose analyzer embodying the features of this invention;
Figure 2 is a flow diagram similar to that of Figure 1 with modifications in the analyzer;
Figure 3 is a graph of the determination for blood glucose levels derived from the example in the application; and Figure ~ is a schema-tic 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 Figure 1, for analysis of blood glucose at frequent intervals for substantially con-tinuous 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.

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The instrument diagrammed in Figure 1 comprises asmall bore plastic tube 10 fitted with an inLravenous single lumen luer lock ll connected catheter 12. Connected to this tube via line l~, fitted wl-th a pinch val.ve 16, is an intravenous infusion bag 1.8 adapted to be filled with a s~erile pharma-col~gical saline solution containing sufficient heparin, about 400 ~ 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 inter-posed in the line between a blood sample pump 28 and the catheter, preferably between the inlet from the source of calibrating solu-tion 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 connec~ed 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 communi.cates 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 o-f a waste container 54.
In the illustrated modification, the analyzer 36 is ~ormed with an upper compartment 56 for first bringing the blood sample to oxygen and temperature equilibrium before the ~3~31~
sample is displaced from the upper compartment to a lower electrode sensing c~vette 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~
~ he cllvette is provided with an out:let 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 _ 5 _ ~ ~ 3 ~ 3~

is raised to draw buffer into the syringes 4~ 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 1~ 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 Erom the syringes to (1) deliver waste from syringe 66 to the waste container 54,

(2) deliver buffer from ~he 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 wi~h 200 microliters of buffer, such as a 0,2 M phosphate buffer at pH 6Ø
After the blood sample has been delivered to the equllibration chamber 56, equilibration of the whole blood sample to 37C and ~he oxygen tension to that of ambient air is achieved by opening valve 84 to activate the negative air pres-sure system which draws room air which has been equilibrated to 36C upwardly through orifice 90 through a distributor a~ the base of chamber 56 whereby the air rises through the whole blood sample as bubbles. Equilibration can be achieved by ~ubbling air through the sample and buffer solu~ion in the chamber for 40 seconds to equi~ibrate the sample ~o room air P02,

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Ahout 2 seconds a:E-ter equilibration has been achieved, valve 84 isclosed 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 eq~lilibration cham~er 56 to the electrode sensing cuvette 58 which contains bufer 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 -ti.p. The maximal rate of fall in oxyten tension is recorded and detected in less than 3 seconds. This rate is proportional to glucose concentra-tion because of the stoichiometric relationship between oxygen and glucose that is apparent from the following equation:

Glucose + 2 glucose gluconic acid + ~22 oxidase Following each analysis the system is adapted auto-matically to wash the sample loop 26, the equilibration chamber 56 and the reaction cuvette 58 be~ore another cycle.
In the illustrated modification, a cycle is carried out over a period of 150 seconds, makiny it~possible to monitor the blood by separate analysis every 2-1/2 minutes.
In operation, patient's blood is drawn in an amount to ~lush the heparnized solution from the tu~ing and to fill the loop, with the interim fluids being pumped to waste ~he apparatus is periodicall~ standardized by opening valve 24 before r 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 ~hich the described normal sequence of operations are csrried out ~3~3~
to flush the calibrating sol.ution from the loop 26 into the equilibration chamber 56 ~or equal.ization of temperature and oxyyen and then from the chamber 56 into the enzyme electrode glucose sensor 60 ~or analysis.
The.described sequence o-E operations of the valve, pump and svringe table can obviously be connected with suitable electrical controls for automatic seguencing of the operations on a controlled time basis for testing for blood glucose levels on repeated cycles of uniform duration whereby reliable compari-sons can be made ~or following the course of medical procedure and/or the patient's well being.
The reliability o~ the test results depends somewhat on the eguil.ibration of the blood samples and calibrating solu-tions for temperature and oxygen levels before the rate determina-tion is madeO Use can be ~ade of other means for temperature and oxygen equilibration of the fluids subiected to the test.
For example, instead of making use of an equilibration chamber 56 of the type illustrated in Figure l, use can be made o~ a.n oxygen equilibration coil of'the type illustrated in the port-able glucose monitor illustrated in Fiyure 2 of the drawingO
Brie~ly described, in t'he illustrated porta'ble unit, blood is drawn by pump 100 from the patient 102 throuyh the canula 10 inan amount to fill the blood sample loop 106.
The outlet from the pump lO0 i5 connected by line 108 to a waste bag llO. 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 illus-tra-ted in Figure 1, and a bag 114 of IV solution is also provided as in the apparatus illustrated in Figure 1, for Elow of IV solution to the patient while the removal of whole blood 3~
is stopped Suitable valve members 116 and llS are provided for controlling ~he flow from the calibration container and the IV bottle respectively.
A compact motorized syringe table L20 :is provided ~or 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 buf~er 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 penetrat~ into the coil for equilibrating the oxyyen concen-trat~on 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 evaluation7 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 con-trolled environment~such as at a temperature of 37C, as outlined by the broken lines in Figure 2 of the drawingO
In the modification illustrated in Figure 2, themixing 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.

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To -the presen-~ th~ deter~in~tion of blood (~hole blood plasma or serum) has been made wi-tn a ~lucose o~idase enzyme reac3ent, as described in Clin~ Chem. 1~ 116(1968). ~;hen glucose is added to the enzyme reagent in a stirred thermostated cell, ylucose reacted with -the oxygen in the pxesence of glucose o~idase in accordance with the reaction O Glucose oxidase > Glucc?nic Acid ~ H2O2 The amount of glucose is measured indirectly by measuring the amount of dissolved oxygen in the reaction solution.
Such techni~ue, which makes use of glucose oxidase enzyme reagent, finds a number of objections from the stand--point 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 irnrnobilized 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 there-fore in the -fabrication o-f 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 imrnobilized 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 elernents are either no_ volatile enouyh-andJor are too polar.
Oxygen a-t the electrode tip is consumed in the pres-* Trade Maxk ~- 10 --~=3 .~. ,, 3~

ence of glucose oxidase. Under such circumstances, use can be made of a rate de~ermination based upon the c:urrent output of the electrode due to the presence of glucose in the sample being tested. Under such circumstances, the electrode can exhi.bit some slow baseline drift while an accurate determination can be made within a few seconds. All that is required is the blood sampl be diluted in a suitable buffer and equilibrated to room tempera-ture 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 oE the equilibration chamber 56 or by the diffusion method of the equilibration coils 12~ 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 ~lucose is present, the o~ygen 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 penetra-tion to the cathode tip of the electrode.
Briefly described, the hydrophobic electrode membrane~
such as a membrane of Teflon* or otherhydrophobic plastic mater=
ial, 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 sur-face 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.

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For this purpose, a drop of the etchant can be placed on the men~rane tip for l minute and then rinsed with acetone and ~ater,~ollowed by drying.
Next, the enzyme is mobiliæed as by covalent bonding the treated surEace. Attachment is made with f_he aid of a thin layer of protein gel containiny glucose ox dase enzyme.
A solution o~ yelling protein, glucose oxidase and fixative is prepared immediately before application to the etched surface of the membrane. The following is an example of a ~ 10 formulation which may be used:

4 volumes o-f Glucose Oxidase solution, Type VI, available from Sigma chemffcal Company, St. I,ouis, Missouri 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 etcF~lng area of the membrane, as with a plastic micropipet tip. The tip o~ 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 aried at room tem-peratu~e for l minute and then drying is continued while the treated membrane is refrigerated at 4~C ~or several hours.
The prepared glucose oxidase membrane is used in place of regular Te~lon*membrane in a commercial glucose analyzer instrument, such as marke.ed by seckman Instrument Company o, Fullerton, California, wlth no substantial alteration of the analyzer or the sample size of the plasma required for analysis The membrane, stored at 4C, has a shelf life of at least 8 n~onths and its useful life will depend some~hat on the steps taken to prevent growth oE microorganism * Trade Mark ~....

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Covalent bonding of the enz~ne-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 o-E paraformaldehyde for immobilization and fi~ing of the enzyme, the enzyme can be immobilized on the membrane by the use of o-ther 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 o~ appro~riate amounts of gravimetrically determined glucose. The aqueous standards were prepared by dissolving lO grams of dextrose in l liter of distilled water, 24 hours was allowed for muta-rotation. Serial dilutions were then prepared to produce 50 ?
100, 200, 300, 400 and 500 mg/dl standard. The whole blood samples were anti-coagula-ted 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 ~he right jugular vein of the dog with a 21 gauge pediatric scalp vein needle, The left jugular ~ein was canulated to allow discrete blood sampling for refer-ence glucose analyzing~ and for infusion of glucose and insulin.

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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 s~andard. A graph representing the data from a typical experirnent is presented in Figure 3 of the drawings. As indicated in the graph, 37 minu-tes 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 oE
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.
~ ith daily use of the instrument described, sufficient enzyme activity remained îmmobilized on the membrane having the electrode so that it was necessary to change the membrane only after two to :Eour wee~s, 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 4C when not in use, loss of enzyme activi~y over a one month period will be found to be less than 5%.
The electrode employed in the practice of this inven-tion, with the enzyme immobilized on the surface of the membrane, is illustrated in Fig. 4 wherein the base of the electrode com-prises a glass rod 150 having a cathode in the form of a platinum 3 ~ ~

electrode 152 whicl~ extends to the tip of the rod 150, and anodes in ~he form of silver chl.orlde reference electrodes 154 ~hich extend into the space between the encl port:ion of the glass rod a-nd a Teflon membrane ]56 that is secured onto the end of the electrode ~ith a space 158 in between that is filled with elect~olyte 160. Tl~e 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 con-struction to that described in U.S, Pa-tent No. 3,5~l2,662 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 e~cellen-t since oxygen is the only electro-active substance in blood defusible through a Teflon*membrane for reaction at the elec-trode. The specifi-city of glucose oxidase for glucose has also been established.
By operation of the glucose sensor in a rate determination mode, any base line drif-t in the oxygen electrode is eliminated, -There are a number of distinct advantages to the invention described and claimed? namely: the blood samplingcan be carried out on frequent intervals, such as 150 seconds~
This minimi.zes the amount of blood removed from the patient for analysis and facili~ates the maintenance of a clot-free sampling system by comparison with continuous sampling, The * Trade Mark 3~

amount of heparin infused into the patient between sampling cycles is not sufficient to cause any significant deyree 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 enæyme clectrode 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 e~uip-ment 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.

Claims (17)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for monitoring blood glucose levels comprising the steps of equalizing the oxygen concentration in a blood sample by contacting the sample with air for a sufficient length of time to effect oxygen equalization, transferring the sample after air-oxygen equalization to a sensor having an electrode covered with a hydrophobic membrane having a surface on which glucose oxidase is immobilized whereby oxygen is consumed by reaction with glucose in the blood in the presence of the glucose oxidase, and measuring oxygen consumption resulting from the reaction of glucose in the blood in determining the level of glucose in the blood.

2. The method as claimed in claim 1 which includes the step of equalizing the blood sample as to temperature as well as to oxygen levels before measurement of oxygen consumption.

3. The method as claimed in claim 1 in which the blood sample is equalized for oxygen level by bubbling the air through the sample for a period of time.

4. The method as claimed in claim 1 in which the blood sample is equalized for oxygen level by holding the blood sample in an oxygen equilibration coil exposed to atmospheric air for diffusion of oxygen therethrough from the atmosphere of the blood.

5. The method as claimed in claim 2 in which the blood sample is equalized to a temperature of about 37°C.

6. The method as claimed in claim 1 in which the sensor operates in a rate determination mode based upon the rate of oxygen consumption by the glucose present in the blood at the electrode.

7. The method as claimed in claim 1 in which the blood sample is flushed to the sensor with the buffer.

8. An apparatus for monitoring blood glucose levels comprising a sensor having an electrode, at least a portion of which is covered with a hydrophobic membrane having glucose oxidase enzyme immobilized to a surface portion thereof, a canula adapted to mount a catheter for withdrawing blood from a patient, a source of IV solution and means connecting the source of IV solution with the canula for draining the IV
solution back through the canula to the patient when blood is not being withdrawn, a pump having an inlet connected to the canula and an outlet connected to waste, a source of buffer solution, an equalizing member for equalizing the temperature and oxygen level of an increment of blood to be sensed, a syringe having an inlet connected to the source of the buffer and an outlet connected to the canula for flushing said increment of withdrawn blood to the equalizing member, another syringe having an inlet connected to the sensor for withdrawing liquid that has been sensed and an outlet connected to waste, and a passage communicating the equalization member with the sensor for the transmission of an equalized increment of blood to the sensor for de-termination of the level of glucose in the blood in the presence of the sensor.

9. An apparatus as claimed in claim 8 which includes a source of calibrating solution containing a known amount of glucose and means communicating the source of calibrating solution with the canula for substituting calibrating solution for the increment of blood to calibrate the apparatus.

10. An apparatus as claimed in claim 8 in which the glucose oxidase is immobilized on the surface of the membrane at the tip of the electrode.

11. An apparatus as claimed in claim 8 in which the equalizing member is in the form of an equalizing chamber which overlies the sensor and has an inlet for receiving the increment of blood and an outlet in communication with the sensor, and means for bubbling air through the chamber.

12. An apparatus as claimed in claim 11 which includes a source of positive pressure and a source of negative pressure, an outlet at the upper portion of the chamber in communication with said sources of positive and negative pressure and valve means in between, and having an inlet in the lower portion of the chamber in communication with atmospheric air whereby when the outlet communicates with the source of negative pressure air is drawn through the inlet for bubbling through the increment of blood in the chamber, and when in connection with the source of positive pressure the increment of blood is displaced from the equalizing chamber to the sensor.

13. An apparatus as claimed in claim 8 in which the equalizing chamber comprises an equilibration coil through which oxygen penetrates from the air to equalize oxygen levels in the blood contained in the coil.

14. An apparatus as claimed in claim 13 in which the equilibration coil comprises a tubular member formed of a fabric through which oxygen can diffuse.

15. An apparatus as claimed in claim 8 which includes a blood sample loop in the canula between the pump and the catheter.

16. An apparatus as claimed in claim 8 which includes a source of calibrating solution and means communicating the source with the canula, a syringe having an outlet communicating with the sensor and an inlet communicating with the source of buffer solution or flushing the sensor after a sensor operation has been completed, a blood sample loop in the canula between the catheter and pump, a syringe having an inlet communicating with the buffer source and an outlet communicating with the blood sample loop for flushing the increment of blood in the blood sample loop to the equalization member.

17. An apparatus as claimed in claim 16 which includes valve means between the end of the canula and the blood sample loop, valve means between the blood sample loop and the pump, valve means in the line communicating the IV solution with the canula, valve means in the line communicating the calibrating solution with the canula, a valve means in the line connecting the syringe with the blood sample loop, valve means in the line communicating the blood sample loop with the equalizing chamber, and automatic means for operating valves in sequence for automotive analysis of the blood glucose level.