CA1072634A - Polarographic glucose analysis - Google Patents
Polarographic glucose analysisInfo
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- CA1072634A CA1072634A CA238,656A CA238656A CA1072634A CA 1072634 A CA1072634 A CA 1072634A CA 238656 A CA238656 A CA 238656A CA 1072634 A CA1072634 A CA 1072634A
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- glucose
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- polarographic
- electrodes
- glucose oxidase
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Abstract
GLUCOSE ANALYSIS
Abstract of the Disclosure Disclosed is an apparatus and method for analysis of glucose by oxidation thereof in a bed of immobilized glucose oxidase to form hydrogen peroxide and gluconic acid and then directly, polarographically determining the resulting hydrogen peroxide in a polarographic cell and then converting the polarographic measurement to the glucose equivalent of the original specimen.
Abstract of the Disclosure Disclosed is an apparatus and method for analysis of glucose by oxidation thereof in a bed of immobilized glucose oxidase to form hydrogen peroxide and gluconic acid and then directly, polarographically determining the resulting hydrogen peroxide in a polarographic cell and then converting the polarographic measurement to the glucose equivalent of the original specimen.
Description
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This invention relates to a method and apparatus for the determination and analysis of glucose. More particularly, the present invention relates to the analysis of glucose in physiological fluids such as blood and urine, and other aqueous specimens of medical and industrial interest.
There is a great need in the medical field today for a rapid and accurate analytical technique for the determination of glucose in blood serum. As a result of recent advances in enzyme chemistry, there has been a great deal of attention directed towards the development of analytical methods for glucose based on the enzymatic oxidation of glucose in the presence of glucose oxidase. This reaction proceeds according to the equation:
O Glucose oxidas~ GlucOnic Acid~Hydrogen Peroxide.
This equation is a simplification of the complex equilibrium that exists between the alpha and beta anomers of glucose;
hydrogen peroxide; glucolactone, and gluconic acid in the presence of glucose oxidase and suggest that the glucose can be -measured as a function of the consumption of oxygen or the increase -ll iO'7Z~i3i in hydrogen peroxide or gluconic acid.
Many methods based on this equation have been described in the past. As will be apparent from the discussion that follows, these methods have one or more serious limitations which detract from their use in the clinical laboratory.
Many oE these techniques are inherently limite~ by mass transport of either oxygen or glucose through a semipermeable membrane and across a membrane/liquid interface.
One such method is described in the article entitledJ
~0 "An Enzyme Electrode ~or the Amperometric Detexmination of Glucose" by G. G. Guilbault and G. J. Lubrano appearing in Analytica Chimica Acta, 64, (1973) 439-455. This article describes an enzyme electrode method for the determination of glucose by the direct amperometric measurement of hydrogen peroxide. The enzyme electrode is constructed by immobilizing glucose oxidase, in or under a glucose permeable membrane, on a polarographic electrode. When this electrode is placed into a glucose solution, glucose diffuses through the membrane where it is oxidized by the glucose oxidase to yield hydrogen peroxide.
While this device is suitable for many applications~ it is inherently limited by the permeation of glucose throuyh the membrane. Furthe~rmore, the im~obilization of the glucose oxidase directly on the electrode necessitates the replacement or repair of the complete electrode structure in the event I the gluco xidase becomes denacured. This can be time . ' . .
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.., ~ 4 consuming and expensive in a modern analytical laboratory where several hundred blood samples are processed daily.
U.S. Patent 3,539,455 to Leland C. Clark, Jr. issued November 10, 1970 discloses another polarographic membrane electrode method for analyzing glucose. According to ~his technique r glucose from the specimen permeates a membrane to react with a confined solution of soluble glucose oxidase to generate hydrogen peroxide which is polarographically measured.
This technique is membrane dependent and limited, and does not employ immobilized enzymes.
Many~ other membrane polarographic techniques have been proposed wherein the enzymatic oxidation of glucose to gluconic acid and hydrogen peroxide is measured as a function of the amount of oxygen consumed. Such techniques usually employ a membrane type oxygen electrode of the type disclosed in U.S.
Patent 2,913,386 to Leland C. Clark, Jr., issued November 17, 1959. Such techniques are illustrated in U.S. Patents 3,~12,517 ;~i to Kadish et al issued May 19, 1970; 3,542,662 to Hicks et al issued November 24, 1970; and in the articles "A New Principle ~-of Enzymatic Analysis" by H.U. Bergmeyer and A. Hagen appearing in the Z. Anal. Chen. 261, 333-336, (1972); "The Enzyme Elect-rode" by S.J. Updike and G.P. Hicks appearing in Nature, Vol.
214, 986-988, (1967); "Studies on Conditions ~or the Polaro~
graphic Determination of Glucose with Glucose Oxidase" by M. Jemmali and R. Rodriquez-Kabana appearing in the Clinica Chimica Acta, 42 (1972) 153-159; and "I~mobilized Enzymes.
A Prototype Apparatus for Oxidase Enzymes in Chemical Analysis Utilizing Covalently Bound Glucose Oxidase", by M.K. Weibel, W. Dritschilo, ~.J. Bright, and ..... . .. .
l~Y~ 3 A. E. Humphrey appearing in the Analytical siochemistry, 52l 402-414 (1973). These measurement techniques are inherently limited by the diffusion of oxygen through a permselective membrane.
In U.S. Patent 3,707,455 to Derr et al issued December 26, 1972, a potentiometric, rather than a polarographic, method is shown wherein the glucose permeates a membrane to a reaction chamber containing soluble glucose oxidase and the resulting increase in gluconic acid is potentiometrically detected.
O~her techniques of more general interest which measure glucose as a function of various secondary chemical reactions are disclosed in U.S. Patents 3,591,480 to Neff et al issued July 6, 1971; 3,623,960 to David L. Williams issued November 30, 1971; 3,770,607 to David L. Williams issued November 6, 1973;
and in the articles "Electrochemical-Enzymatic Analysis of Blood Glucose and Lactate" by D~L. Williams, A~R. Doig, Jr.
and A. Korosi appearing in Analytical Chemistry, Vol. 42, No. 1, (1970); "Ion-Electrode Based Automatic Glucose Analysis System"
by R.A. Lienado and G.A. Rechnitz appearing in Analytical 20 Chemistryt Vol. 45, No. 13, (1973); and "Enzyme Electrode for Glucose Based on an Iodide Membrane Sensor" by G. Nagy, L.H.
Von Storp, and G.G. Guilbault appearing in Analytica Chimica Acta, 66, (1973), 443-455.
In the above described polarographic systems of the ~ -prior art for measuring glucose where hydrogen peroxide is directly determinedt a membrane is always required because of the possible interference and the poisoning of the electrode with extraneous materials in the specimen. The present invention provides an analytical technique for glucose by the direct polarographic determination of hydrogen peroxide that does not require a membrane.
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.- ' ~ -.-, In the present invention, a small glucose specimen size compared with the volume of a constant flowing aqueous buffered diluent stream passes through a reaction chamber containing a bed of immobilized glucose oxidase where it is oxidized to hydrogen peroxide as it permeates or per-colates through the bed~ The reaction chamber has a specimen inlet and reaction product outlet. The reaction product stream passes from the chamber into a polarographic cell. This results in a short contact tirne between the electrodes and the glucose specimen and electrode inter-ference and poisoning is not a factor and a membrane is not required. The electrode response is much faster and the sensitivity and reproducibility is increased in the present invention as compared with processes that are ;
limited by a membrane diffusion step. ~
Accordingly, the present invention overcomes these ~ -disadvantages of the prior art by providing an apparatus -~
and method for analysis of glucose by oxidation thereof in a bed of immobilized glucose oxidase to form hydrogen peroxide and gluconic acid and then directly, polarographical-ly determining the resulting hydrogen peroxide in a polarographic cell and then converting the polarographic measurement to the glucose equivalent of the original specimen.
Thus, by the present teachings, there is provided a method for determining glucose in an aqueous specimen containing glucose which comprises the sequential steps of diluting the specimen in an aqueous buffered diluent buffered to a pH in the range of about 4 to about 8, passing the diluted solution into a bed of glucose oxidase immobilized on a solid support, retaining the diluted specimen in :
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contact with the glucose oxidase for a time suf~icient to oxidize the glucose to form hydrogen peroxide, removing the resulting reaction mixture from the bed, passing the resulting reaction mi~ture into direc-t contact with a set of polarographic electrodes, polarographically deter-mining hydrogen peroxide in the reaction mixture while in direct contact with the polarographic electrodes to produce an electrical current proportional to the hydrogen peroxide concentration of the specimen, dete~nining the electrical current and translating the current determination to the glucose conaentration of the specimen.
In accordance with a urther embodiment of the present teachings, an apparatus is pruvided for determining glucose in an aqueous specimen which contains glucose,and comprises in combination, a reaction chamber .
having an inlet for introducing the specimen and an outlet for removal of a reaction product~ the reaction chamber containing a bed of glucose oxidase. A polarographic cell is interconnected with the reaction product outlet, the cell comprises a tubular sample chamber having a sample inlet and outlet and the sample chamber has a set of :
polarographic electrodes which are positioned therein for -direct èlectrical communication with a sample in the sample chamber, the electrodes are electrically connect-ed ~or amperometric response -to electrical current gener-ated in the cell.
One of the primary eatures of the present in~ention is that the bed of immobilized glucose oxidase is physically separated ~rom the polarographic cell. This represents a marked improvement over the enzyme electrode types discussed above, where the hydrogen peroxide must 5a -" ~
' ' ' permeate through a membrane which covers the sensing portion of the polarographic electrode.
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The present invention will be further described with referenee to the drawings wherein Figure 1 is a schematic proeess flow diagram for praetieing the present invention and Figs. 2 and 3 are schematic 1~w diagrams illustrating the details of polarographiecells whieh ean be used in eonjunction with Figure 1.
Referring now to Fig. 1~ an aqueous specimen eontaining glueose flows into a reaetion chamber eontaining a bed o immobilized glueos~ oxidase where it is maintained for a time and at a temperaturè sufficient to oxidize the glueose to glueonie aeid and hydrogen peroxide. The reaetion chamber is equipped with a specimen inlet and reaetion produet outiet.
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lU'7~2fi34 Typically, this oxidation is completed within a few seconds to 30 minutes or longer at temperatures ranging from 0C to about 50Co A time period of less than 1 minute at room t~mperature is usua]ly sufficient for the oxidation of glucose.
S Because the glucose oxidase is most efficient in the oxidation of glucose in the 4 to 8 pH range, the glucose specimen, prior to contact with the glucose oxidase, is diluted with an aqueous diluent which is buffered to prevent the pH thereof from driftlng out of the pH range where glucose oxidase is most efficient for oxidizing glucose. Preferably for efficiency in oxidation of glucose by glucose oxidase the pH of the buffered diluent is in the range of about 5 to 7~
Particularly suitable aqueous bufered diluents are solutions of 50dium acetate and acetic acid. Other acceptable bufers include sodium citrate, and other water soluble phosphate, and phthlate salts in the concentration of 0.001 to 0.5M.
rrhe oxygen required for this oxidation reaction is provided by that oxygen concentration which is normally dissolved in the glucose specimen and buffered diluent at room temperature. This concentration usually provides enough oxygen to oxidize sufficient glucose for reliable and reproducible polarographic detection. It is not necessary for all of the glucose to be oxidized to form hydrogen peroxide. It is only necessary that ~the percentage conversion is reliable and reproducible from a standard to unknown specimens within a given set of laboratory conditions. Thus a reprcducible value of 60~ conversion of . .
. , .
. /,-10'7Z~;34 G~13336 glucose provides precise polarographic response for the purpose of calibration and analysis~ In the preferred embodiment essentially all of the gluoose is oxidized to proctuce hydrogen peroxide so that any possible error of variation in conversion from sample to sample is eliminated. To achiev,e such complete conversion of glucose9 supplemental oxygen from an external source (e.g. a sparge of oxyyen or an oxygen-containing gas) can be introduced into the specimen if desireda although this is seldom,required.
Slight fluctuations can occur in the dissolved oxygen con~
centration in the glucose specimen and buffered diluent as a function of changes in pressure or temperature. Such fluctuations are minor and the presence of su~ficient oxygen is assured when using the detection method, sample size and 15 , concentrations herein specified. This is an important advàntage over methods measuring glucose as a function of oxygen depletion.
The ratio of dilution of the glucose specimen in the buffered diluent varies with tha concentration of glucose in the specimen. For physiological fluids such as blood and urine having unknown concentration within the expected concentration range9 dilution of 2.5 microliters of specimen ~, into a stream of bu~fered diluent flowing at the rate of 0.1 to 5 and preferably 0.1 to 2 ml per minute is suitable for a significant polarographic response~ Usually, for efficiency and economy, a small glucose specimen (e~g. about 2 to 10 microliters) is ln3ected into a stream of buffered diluent . .
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lQ'~Z634 flowing at the rate of 0.1 to 10 ml per minute for introduction ¦ into the bed of immobilized glucose oxidaseO The buffered dilute is caused to flow by a small metering pump of conventional ¦ design. ~ .
In addition to buffer it is desirable to add salts such ¦ as potassium chloride or sodium chloride which serve to ¦ esta~lish the reference potential when silver-silver chloride reference electrodes which use the sample as a f illing solution are employed. A bacterial inhibitor can be incorporated in the buffered diluent to retard bacterial interferenceO
Any of the known methods or immobiliæing glucose oxidase on an insoluble support to form a bed of immobilized glucose oxidase can be used in practicing the present invention. For . instance; glucose oxidase can be covalently coupled to a porous : 15 glass support with an amino-functional silane coupling agent as disclosed in t~e article entitled, ~Immobilized Enzymes: A Prototype Apparatus for Oxidase Enzyme in ¦ Chemical A lys is Util iz ing Cova lently Bound GluFos e ¦
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Oxidase," by M. K. Weibel et al appear`ing in Analytical ._ Biochemistry, 52, 402~414 (1973); glucose oxidase can be . immobilized on column packing as in the article entitled, "A New Principle of Enzymatic Analysis" by H~ V. Bergmeyer and A. Hagh appearing in Z~ Anal. Chem. 261, 333-336 (1972);
glucose oxidase can be immobilized in polyacrylic polymers as in the article entitled, "Enzyme Electrodes for Glucose Based on an Iodide Membrane Sensor" by G. ~agy et al appearing in Analytica Chemica Acta, 66, (1973~, 443-455; glucose oxidase can be immobilized in a polyacrylamide gel as in the article entitled, "An Enzyme Electrode for the Amperometric Determination of Glucose" by G. Guilbault et al appearing in ~ Analytica Chemica Acta 64~ tl973), 439-455 or U. S. Patent : 3,542,662 to Hicks et al issued November 24, 1970; glucose ~:~
oxidase immobilized on nickel-silica alumina as in the article entitled, "Immobilization of Glucose Oxidase on Nickel-Silica ~ :
. Alumina" by W. M. Herring et al appearing in Biotechnology and ~
Bioengineering, Vol. XIV, pages 975~984 (1972); and glucose I ~ :
oxidase can be immobilized with cyanogen bromide according to the method of U. S. Patent 3,645,852 entitled, "Method of -Binding Water-soluble Proteins and Water-soluble Peptides to Water-insoluble Polymers Using Cyanogen Halide," by R. Axen, : ~:
. J. Porath, and E. Ernbach issued February 29, 1972. Thus, in formin~ the bed of immobilized slucose oxidase the selection of the support from materials such as . ~ -10- ;"~
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. ., ~ - . . ,:' - . . .
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iO~63 porousglass; particulate and preferably porous refractory oxides such as alumina, titania, zirconia~ silica, magnesia, talc, and thoria; glass frit; particulate procelain; compacted and sintered reractory oxides; clays; water insolu~le polymers, and immobilizing the glucose oxidase thereon by chemical or p~ysical means is well known in the art.
The only requirement is that the bed must be permeable to the sample specimen while p~oviding a high surface area to volume ratio to assure adequate contact between the glucose oxidase and the glucose specimen. A packed bed of glucose oxidase immobilized on an inert particulate, porous, fibrous or gelled support is preferred for this puxpose~ When using particulate refractory oxides,particle size and percent porosity are not particularly cxitical as long as adequate contact between glucose and glucose oxidase is provided. Volume porosity in the range cf about 10~ to about 60~ with an average pore-size diameter in the range of about 0.01 to lo micrcns have been found to be effective and readily available. Particulate refractory oxides having a particle size diameter in the range of about 1 to about 1000 microns are readily available with a particle size diameter in the rangeof about 100 microns to 400 microns being quite practical for the present purposes.
Aftex oxidation of the glucose in the bed of immobilized ~lucose oxidase the resulting reaction mixture flows to the ~25 polarographic cel~l.
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According to the present invention theaqueous buffered diluent is continuously pumped through the reaction chamber containing the bed of immobilized glucose oxidase and polarographic cell. The glucose specimen is injected from a hypodermic syringe into the bed of immobilized glucose oxidase through an injection port which can be in the Eorm of a mixing "tee" covered with a rubber diaphragm.
While flowing through and permeating the bed vf immobilized glucose oxidase the glucose is oxidized to hydrogen peroxide and gluconic acid which remain in the specimen as reaction products.
This reaction mixture then flows into the polarographic cell when the hydrogen peroxide is polarographically detected by direct contact with the polarographic electrodes. The polarographic response from the measurement of the hydrogen peroxide is measured by a current measuring device such as a current follower. This value is then coverted to the glucose equivalent of the original specimen. The glucose equivalent of the original specimen is usually reported in mg glucose/100 ml ~i.e. mg percent) of specimen. These units are conventional in clinical applications.
Two embodiments of polarographic cell illustrated in Fig. 1 are shown schematically in Figs. 2 and 3~ These cells are called "flow-through" cells because the sample under analysis continuously flows through the cell.
In Fig, 2 the cell body 10 is constructed of a rigid, inert7 electrically insulating material such as glass or plastic. Pol~methyl methacrylate plastics have been found to be quite satisfactory for this purpose. The cell body 10 is provided with a narrow passageway 11 through which . ,,--. ~.''.
lO~Z63 ¦ G-13336 ¦ the specimen of reaction mixture containing the hydrogen ¦ peroxide flows into cell cavity 15. The cavity 15 is of I such dimensions to assure effective contact between the sample ¦ to the polarographic electrodes while minimizing dead volume.
¦ The cell used in the Examples is plastic and has a c~vity I about 0 1 inch in width, 0.5 inch in length, and about 0.02 ¦ inch in depth. ~he electrodes enter ~rom the top o~ the cell ¦ and are positioned along the "length" dimensionO
¦ In Fig. 2 a set of two polarographic electrodes 12 and 13 ¦ are mounted in cell body 10 so the sensing tips thereo are ¦ in direct contact with the specimen in cavity 15. ~lectrode 12 ¦ functi.ons as both the counter electrode and reference electrode.
Electrode 12 is of conventional design and can be a silver wire ¦ coated with silvar chloride; or calomel. The silver-silver ¦ chloride reference electrode is preferred because the sample being measured can function as the reference electrode filling ¦ solution by incorporation of chloride ions therein. The other ¦ types of reference electrodes using their own contained filling ¦ solution can be used if desired. Electrode 12 is connected ¦ to a source of constant DC potential (usually about 0.6 volts) as in conventional polarography. Electrode 13 is the working electrode and is in the form of a platinum, palladium, gold, yraphite, or carbon or other inert conductive material.
The hydrogen peroxide in the specimen readily depolarizes the polarographic electrodes and current flow, which is measured ¦¦ by the cu ent measuring device, at the given applied vol=age, . .' . ''. .
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is proportional to the hydrogen peroxide concentration~ This cuxrent flow is converted to the glucose equivalent o the original specimen. In the polarographic cell shown in Fig. 2 the reference electrode must also function as a counter S electrode and participate in the oxidation reduction reaction taking place in the cell. The IR drop ic; not compensated and th potential difference between the reference elec~rode and the working electrode may change.
The cell of Fig. 3 is preferred over the cell of Fig. ~ .
In the cell of Fig. 2 the xeference electrode is the only other ~lectrode in the circuit, so that current i~ forced to flow through it. This can cause a vaxiation in the potential difference between the electrodes due to the ~R drop through the sample. Furthermore, when a silver-si~ver chloride reference electrode is used, the silver chloride coating is eventua~ly depleted by thP oxidation-reduction reaction~
Accordingly, the use of such a cell, even when the electrode is positioned closely to the working electrode, is not as precise as the cell of Fig. 3.
When using the cell of Fig. 3 in a routine laboratory environment, a peak detector and a sampling and hold circuit can be used to measure the maximum current above a base line current and this diffexence will be proportional to the hydro-gen peroxide concentration in the specimen and is~ therefore, proportional to the glucose concentration.
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In a preferred polarographic cell shown in Figure 3, the polarographic cell is potentiostatic. ~his is the so-called "three electrode polarographic" as disclosed in the article, "The Renaissance in Polarographic and Vol-tammetric Analysis"
by Jud B. Flato, appearing in Analytical Chemistry, Vol. 44, September 1972, the disclosures of which are incorporated by reference.
Figure 3 has a cell body 10 and cavity 15 like the cell of Fig. 2. The cell of Frig. 3 is equipped with a reference electrode 23 in the form of a silver wire coated with silver chloride positioned as closely as possible to the working electrode 21 which is a platinum wire. The applied potential ;
(+0.6 volts DC) is applied to the input of a control amplifier 30 to which the reference electrode 23 is also connected through voltage follower 31. The output of the control ampli-fier 30 is connected to counter electrode 20 which is a platinum -wire. By this design essentially no current flows through ~
reference electrode 23 and sufficient compensating potential is `
applied to counter electrode 20 to maintain the potential dif-erence between the reference electrode 23 and the working electrode 21. Working electrode 21 is connected to a small conventional current measuring device which provides a current measurement which is converted to the glucose equivalent of the original specimen. Other conventional electrodes such as described in conjunction with Fig. 2 can be used in the embodiment of Fig. 3 if desired.
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In the Examples that follow all parts are parts by weight, all percentages are weight percentages, and all temperatures are in C unless stated otherwlse.
Example I
Part A
Immobilized Glucose Oxidase on Alumina Powder _ . _ _ _ _ _ One gram of particulate alumina is washed thoroughly with distilled water. The particulate alumina has a particle size in the range of -40 to ~70 mesh (U.S. sieve screen) and an average pore size diameter of 0.1 to 0.2 microns.
Fifty mg o~ glucose oxidase (obtained from Worthington Biochemical Corporation having a reported activity of 140 International Units per milligram) is added to the wet particulat~
alumina in 40 ml of an aqueous solution which has been bufexed to pH 5.5 with standard buffer comprising a mixture of potassium dihydrogen phosphate and disodium hydrogen phos,~hate.
The resulting mixture is stirred gently for one-half hour at 6-8C to sorb enzyme.
To this mixture is added a crosslinking reagen-t formed by mixing 20 ml methanol; 10 ml distilled water; O.lS ml concentrated hydrochloric acid; 0008 ml diaminopropane; and O.02 ml dibromoethane~ The combined mixture of alumina, glucose oxidase and crosslinking reagent is stirred gently with a magnetic stLrrer~at 6 to 8C overnight to immobilize the glucose oxidase on the alumina, The resulting i~mobilized glucose oxidase~alumina composite is washed with about 2 liters of distilled water and stored in distilled water, . . . ' ~
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ioqz~,34 Part B
Assay of Immobilized Glucose Oxi~ase~Alumina Compo~i-te The catalytic activity o the immobilized glucose oxidase/alumina composite of Part A is calculated from the measured rate of oxidation of ~ -D-ylucose to ~luconic acid by para~benzoquinone in the presence of the glucose oxidase/
alumina composite. I~he reaction is represented hy:
~D-glucose~p-benzoquinone~o GlUc05e Oxidase) 1 i hydroquinone The reaction is followed by measuring potentiometrically the change in concentration of hydroquinone with time. A
platinum detector ~lectrode (Beckman model 39273) is used with a double junction calomel-silver/silver chloride reference electrode (Orion model 90-20 00). Standard solutions or calibrating the elec~rode system are prepared -fxom hydroquinone with about a 100 molar excess of p-benzoquinone also present in the aqueous solutions, A calibration graph is drawn by plokting hydroquinone concentration against millivolt readings from the potentiometer.
The reaction medium in which the oxidation of glucose takes place~is an aqueous solution which is 0.1 molar in : dextrose and 0.01 molar in phosphate buffer at pH 5.5.
It is stirred overnight to assure that equilibrium has been ~ reached between khe~ and ~ -D-glucose forms. Suficient 1~ p-ben-oqu ne and hydroquinone is added to make the final . ' . ' ' '.
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¦ solution 1.0 x 10-2M and 1.0 x 10 4M in these latter two ¦ components. A known quantity of the glucose oxidase/alumina ¦ composite is added to a given volume of the reaction medium ¦ and the change in the potential of the electrode immersed ¦ in ~he solutionisfo~owedwith time using the electrode system :
described above. From the millivolt readings the corresponding ¦ concentration of hydroquinone is determined from the ¦ calibration graph and a plot is made of test solution con-¦ centration versus time. The initial slope of this curve ¦ represents the rate of oxidation of~ -D-glucose catalyzed by ¦ the immobilized enzym0. The activity is calculated from the ¦ ralationship:
Rate of oxidation ~ctivit~
I ~' Y Volume of alumina ¦ Using this assay procedure the glucose oxidase/alumina ¦ composite of Part A is found to have an activity of 995 ~ International Units of glucose oxidase per ml of glucose ¦ oxidase/alumina composite. Ten days later, after being stored ¦ in distilled water at 4C , the activity is 825 International ¦ Units of glucose oxidase per ml of glucose oxidase/alumina ¦ composite.
An International Unit of biological activity has been ? defined as the amount of active enzyme which converts sub-strate to product at the rate of one micromole per minute.
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Part C
A glass column is prepared from a 50 mm borosilicate, glass tu~e with an inside diameter of 2.8 mm and an outside .
. 5 diameter of 6 mm. A 4no mesh ny].on disc is attached to one .
end of the column. The immobilized glucose oxidase/alumina composite of Part A is charged thereto to fill the tube. The glucose oxidase/alumina packs into the column by gravity and .
the othex end of the column is also fitted with a 400 mesh nylon disc after the column is filled. The volume of the glucose oxidase/alumina composite is approximately 0.3 cc. , The column ends are then fitted with plastic tubing fittings one of which is in the form of a "tee" for sample injection. The injection tee is provided with a rubber membrane for sampl.e injection with a hypodermic needle.
. Part D
Anal,ysis of ~lucose A buffered diluent for the glucose specimen is prepared . from: 50 ml of saturated aqueous solution of 2-chloro-4-phenylphenol; 10 ml o l.ON potassium chloride; 8~2g (0.10 molej sodium acetate; sufficient l.OM acetic acid to adjust ., the pH to 5.6; and additional water to make one liter of solution. The buffer is thus approximately 0.1 m~lar in acetate, 0.01 molar in potassium chloride and contains a trace ¦¦ of the ba ricide, 2-chloro-4-pheny1phenol.
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:IU''~Z63'1 The polarographic cell is as shown in Fig~ 3 and has been described above. The electrodes are connected to a PAR Model 174 polarographic analyzer.
The standard solution used to calibrate the polarographic S cell is a 0.01 molar aqueous glucose solution which is 0.1 molar in sodium chloride as a reference electrode ~illing solution. ~oth chemicals are reagent grade. The standard solution is prepared well in advance of its use to assure chemical equilibrium. Expressed in terms commonly used in the clinical laboratory this standard represents a g~ucose concentration of 180 mg of glucose per 100 ml of specimen (i.e. 180 mg percent glucose).
¦ The buffered diluent is pumped through the apparatus ¦ described i~ Fig. 1 at a rate of 1.0 ml/min. This produces ¦ a base li~e current of approximately one nanoampere. A 3.0 ¦ microliter sample of the O.lOM glucose standard solution is ¦ injected with a hypodermic needle through the injection "tee"
¦ into the immobilized enzyme column as shown in Fig. 1. The ¦ current rapidly increases to a maximum current of 250 nanoamps I over a 10 second period and then rapidly decreases over a 30 second period to a value approaching the base line value.
The current to concentration calibration factor is thus l.39 nanoamps/mg percent glucose for a three microliter sample.
~en separate 3,0 microliter specimens of a~standard ~lood serum sample (reported to contain 83.5 mg percent glucose) are sequ'entially injected into the stream of buffered diluent flowing into the bed of immobilized glucose oxidase and the resulting .. , . ' ,~
1i~7Z63 ¦ G-13336 ¦ peroxide is polarographically analyzed as described above ¦ and the corresponding current follower reading are recorded.
¦ Using the calibration factor to convert current to concentration, ¦ the samples are found to contain an av~rage value of 80 mg ¦ glucose/100 ml specimen (80 mg percent) with a coefficient of ¦ variance of 0.7~o.
l A second, abnormal, standard blood serum sample (reported ¦ to contain 197 mg percent glucose) is analyzed by the above ¦ procedure~ From a total of 20 analyses an average value of ¦ 200.5 mg glucose/100 ml is obtained with a standard coeffici2nt ¦ of variance of 1.3%.
I Example II
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¦ Part A
l Preparation of Immob _ zed Glucose Oxidase Five grams of particulate alumina having a particle size in the range of -70 to +80 mesh (U.S. sieve screen) and an average pore diameter of 0.1 to 0~2 microns is dried by heating at 1060C for two hours. After cooling, thealumina is soaked in lD0N HCl for 2-4 hours and washed with-distilled water to remove adsorbed air. The washed al~l~a is placed in a beaker with about 10 ml of 0.01M phosphate buffer (pH 6.0).
Glucose oxidase, as in Example I (250 mg) is added to tne - particulate alumina with enough distilled water to bring the volume of the mixture to about 40 ml. The glucose oxidase/alumin 25 ~ I mixture sti/Fed ~ently w~th a magnetic stirr~r at roOA
Il I
. '" ' , , , :10'7Z634 temperature for about one-half hour.
A crosslinking reagent is prepared from 20 ml methanol, 10 ml water, 0.15 ml concentrated hydrochloric acid, 0.05 ml diaminopropane and 3 ml of a 10~ by weight solution of gluteraldehyde. Th~s reagent is added dropwise to the glucose oxidase/alumina mixture described above. The addition is made at room temperature and the final reaction mixture left standing overnight a~ 0. It is then washed in O.OOlM phosphate bufer (pH 5.5) and stored in distilled water until assayed.
~he assay procedure is the same as that described in Example I, Part B. The activity is found to be about 150 International Units per ml of glucose oxidase/alumina composite.
Part B
.
. Glucose AnalYsis ;
A bed of immobilized glucose oxidase i5 prepared as descri~ed in Example I using the glucose oxidase/alumina composite described in Part A aboveu The standardization procedure for the analytical system is essentially the same as that in Part D o Example I.
The flow rate for the buffered diluent is 1.0 ml/min., and it is the same composition as in Example I. A 3.0 microliter standard glucose~solution (containing 180 mg percent glucose) gives a reading of 590 nanoamps. The calibration factor i5 thus 3.28 nanoamps/mg percent ylucose for the 3 microliter sample. -~ -22- ;
. . :
10'7Z~
Ten separate 3.0 microliter specimens of a standard blood serum sample (reported to contain 83.5 mg glucose/100 ml) are sequentially injected into the bed of immobilized glucose oxidase as above, and the corresponding current readings record-ed. Using the calibration factor to convert current to glucose concentration, the samples are analyzed to contain an average value of 82 mg glucose/100 ml with a coefficien~ of variance of 1~5%.
A second, abnormal, standard blood serum sample (reported to contain 197 m~ percent glucose) is analyzed as above. From a total of 10 tests an average value of 197.2 mg glucose/100 ml is obtained with a standard coefficient o variance of 1.0C~.
Example III
Part A
Preparation of Immobilized Glucose Oxidase Five grams of particulate alumina having a particle size in the range of -60 to ~70 mesh (U.SO sieve screen) and an average pore diameter of 0.1 to 0.2 microns is heated at 1150C
for two hours. After cooling, the particulate alumina is soaked in 1.0~ Hcl overnight. It is then washed with distilled water and placed i~ a beaker with 50 ml o~ 0.001 phos~hate buffer (pH
t. . 6.0) for one-half hour before adding 25 ml of glucose oxidase solution. The glucose oxidase (Asperilligus niger) is obtained from Pierce Chemical Company in a buffered solution (pH 4.0) and has a reported activity of 1000 International Units/ml.
The resulting gluc~se oxidase/alumina mixture is stirred . :
~1 ' , I
O~Zi,3 gently for one-half hour at 3-8C.
A crosslin~ing reagent is prepared by mixing 20 ml methanol~ 0.01 ml diaminopropane, 0.15 ml concentrated HCl, 0.03 ml dibromoethane and 10 ml water. This reagent is adcled at a rate of 0.15 to 0.20 ml/min to t.he glucose oxidase/alumina mixture while stirring gently with a magnetic stirrer and main-taining the temperature at 3 to 8C forl~ ~ hours, The result-ing immobilized glucose oxidase/alumina composite is washed with 2 liters of distilled water and stored in distilled water until assayed, The~assay procedure i5 the same as that described in Example I. Theactivity of the sample is 2,300 International Units/ml immobilize~ glucose/alumina composite.
Part ~
Glucose Anal~sis ~ bed of immobilized glucose oxidase i5 prepared as described in Example I using the glucose oxidase/alumina composite described in Part A above. '~he volume of the column in this case however is only 0.2 cc.
The standardization procedure for the analytical system is essentially the same as that in Part D of Example I. The flow rate for the buffered diluent of the compasition of Example I is 0.7 mI/min. A 2.5 microliter sample of the glucose standard containing 180 mg percent of glucose gives a current of 150 nanoamps. The calibration factor is thus
This invention relates to a method and apparatus for the determination and analysis of glucose. More particularly, the present invention relates to the analysis of glucose in physiological fluids such as blood and urine, and other aqueous specimens of medical and industrial interest.
There is a great need in the medical field today for a rapid and accurate analytical technique for the determination of glucose in blood serum. As a result of recent advances in enzyme chemistry, there has been a great deal of attention directed towards the development of analytical methods for glucose based on the enzymatic oxidation of glucose in the presence of glucose oxidase. This reaction proceeds according to the equation:
O Glucose oxidas~ GlucOnic Acid~Hydrogen Peroxide.
This equation is a simplification of the complex equilibrium that exists between the alpha and beta anomers of glucose;
hydrogen peroxide; glucolactone, and gluconic acid in the presence of glucose oxidase and suggest that the glucose can be -measured as a function of the consumption of oxygen or the increase -ll iO'7Z~i3i in hydrogen peroxide or gluconic acid.
Many methods based on this equation have been described in the past. As will be apparent from the discussion that follows, these methods have one or more serious limitations which detract from their use in the clinical laboratory.
Many oE these techniques are inherently limite~ by mass transport of either oxygen or glucose through a semipermeable membrane and across a membrane/liquid interface.
One such method is described in the article entitledJ
~0 "An Enzyme Electrode ~or the Amperometric Detexmination of Glucose" by G. G. Guilbault and G. J. Lubrano appearing in Analytica Chimica Acta, 64, (1973) 439-455. This article describes an enzyme electrode method for the determination of glucose by the direct amperometric measurement of hydrogen peroxide. The enzyme electrode is constructed by immobilizing glucose oxidase, in or under a glucose permeable membrane, on a polarographic electrode. When this electrode is placed into a glucose solution, glucose diffuses through the membrane where it is oxidized by the glucose oxidase to yield hydrogen peroxide.
While this device is suitable for many applications~ it is inherently limited by the permeation of glucose throuyh the membrane. Furthe~rmore, the im~obilization of the glucose oxidase directly on the electrode necessitates the replacement or repair of the complete electrode structure in the event I the gluco xidase becomes denacured. This can be time . ' . .
..
. ~
.., ~ 4 consuming and expensive in a modern analytical laboratory where several hundred blood samples are processed daily.
U.S. Patent 3,539,455 to Leland C. Clark, Jr. issued November 10, 1970 discloses another polarographic membrane electrode method for analyzing glucose. According to ~his technique r glucose from the specimen permeates a membrane to react with a confined solution of soluble glucose oxidase to generate hydrogen peroxide which is polarographically measured.
This technique is membrane dependent and limited, and does not employ immobilized enzymes.
Many~ other membrane polarographic techniques have been proposed wherein the enzymatic oxidation of glucose to gluconic acid and hydrogen peroxide is measured as a function of the amount of oxygen consumed. Such techniques usually employ a membrane type oxygen electrode of the type disclosed in U.S.
Patent 2,913,386 to Leland C. Clark, Jr., issued November 17, 1959. Such techniques are illustrated in U.S. Patents 3,~12,517 ;~i to Kadish et al issued May 19, 1970; 3,542,662 to Hicks et al issued November 24, 1970; and in the articles "A New Principle ~-of Enzymatic Analysis" by H.U. Bergmeyer and A. Hagen appearing in the Z. Anal. Chen. 261, 333-336, (1972); "The Enzyme Elect-rode" by S.J. Updike and G.P. Hicks appearing in Nature, Vol.
214, 986-988, (1967); "Studies on Conditions ~or the Polaro~
graphic Determination of Glucose with Glucose Oxidase" by M. Jemmali and R. Rodriquez-Kabana appearing in the Clinica Chimica Acta, 42 (1972) 153-159; and "I~mobilized Enzymes.
A Prototype Apparatus for Oxidase Enzymes in Chemical Analysis Utilizing Covalently Bound Glucose Oxidase", by M.K. Weibel, W. Dritschilo, ~.J. Bright, and ..... . .. .
l~Y~ 3 A. E. Humphrey appearing in the Analytical siochemistry, 52l 402-414 (1973). These measurement techniques are inherently limited by the diffusion of oxygen through a permselective membrane.
In U.S. Patent 3,707,455 to Derr et al issued December 26, 1972, a potentiometric, rather than a polarographic, method is shown wherein the glucose permeates a membrane to a reaction chamber containing soluble glucose oxidase and the resulting increase in gluconic acid is potentiometrically detected.
O~her techniques of more general interest which measure glucose as a function of various secondary chemical reactions are disclosed in U.S. Patents 3,591,480 to Neff et al issued July 6, 1971; 3,623,960 to David L. Williams issued November 30, 1971; 3,770,607 to David L. Williams issued November 6, 1973;
and in the articles "Electrochemical-Enzymatic Analysis of Blood Glucose and Lactate" by D~L. Williams, A~R. Doig, Jr.
and A. Korosi appearing in Analytical Chemistry, Vol. 42, No. 1, (1970); "Ion-Electrode Based Automatic Glucose Analysis System"
by R.A. Lienado and G.A. Rechnitz appearing in Analytical 20 Chemistryt Vol. 45, No. 13, (1973); and "Enzyme Electrode for Glucose Based on an Iodide Membrane Sensor" by G. Nagy, L.H.
Von Storp, and G.G. Guilbault appearing in Analytica Chimica Acta, 66, (1973), 443-455.
In the above described polarographic systems of the ~ -prior art for measuring glucose where hydrogen peroxide is directly determinedt a membrane is always required because of the possible interference and the poisoning of the electrode with extraneous materials in the specimen. The present invention provides an analytical technique for glucose by the direct polarographic determination of hydrogen peroxide that does not require a membrane.
..
.- ' ~ -.-, In the present invention, a small glucose specimen size compared with the volume of a constant flowing aqueous buffered diluent stream passes through a reaction chamber containing a bed of immobilized glucose oxidase where it is oxidized to hydrogen peroxide as it permeates or per-colates through the bed~ The reaction chamber has a specimen inlet and reaction product outlet. The reaction product stream passes from the chamber into a polarographic cell. This results in a short contact tirne between the electrodes and the glucose specimen and electrode inter-ference and poisoning is not a factor and a membrane is not required. The electrode response is much faster and the sensitivity and reproducibility is increased in the present invention as compared with processes that are ;
limited by a membrane diffusion step. ~
Accordingly, the present invention overcomes these ~ -disadvantages of the prior art by providing an apparatus -~
and method for analysis of glucose by oxidation thereof in a bed of immobilized glucose oxidase to form hydrogen peroxide and gluconic acid and then directly, polarographical-ly determining the resulting hydrogen peroxide in a polarographic cell and then converting the polarographic measurement to the glucose equivalent of the original specimen.
Thus, by the present teachings, there is provided a method for determining glucose in an aqueous specimen containing glucose which comprises the sequential steps of diluting the specimen in an aqueous buffered diluent buffered to a pH in the range of about 4 to about 8, passing the diluted solution into a bed of glucose oxidase immobilized on a solid support, retaining the diluted specimen in :
~ .
~ ~ - 5 - ~
contact with the glucose oxidase for a time suf~icient to oxidize the glucose to form hydrogen peroxide, removing the resulting reaction mixture from the bed, passing the resulting reaction mi~ture into direc-t contact with a set of polarographic electrodes, polarographically deter-mining hydrogen peroxide in the reaction mixture while in direct contact with the polarographic electrodes to produce an electrical current proportional to the hydrogen peroxide concentration of the specimen, dete~nining the electrical current and translating the current determination to the glucose conaentration of the specimen.
In accordance with a urther embodiment of the present teachings, an apparatus is pruvided for determining glucose in an aqueous specimen which contains glucose,and comprises in combination, a reaction chamber .
having an inlet for introducing the specimen and an outlet for removal of a reaction product~ the reaction chamber containing a bed of glucose oxidase. A polarographic cell is interconnected with the reaction product outlet, the cell comprises a tubular sample chamber having a sample inlet and outlet and the sample chamber has a set of :
polarographic electrodes which are positioned therein for -direct èlectrical communication with a sample in the sample chamber, the electrodes are electrically connect-ed ~or amperometric response -to electrical current gener-ated in the cell.
One of the primary eatures of the present in~ention is that the bed of immobilized glucose oxidase is physically separated ~rom the polarographic cell. This represents a marked improvement over the enzyme electrode types discussed above, where the hydrogen peroxide must 5a -" ~
' ' ' permeate through a membrane which covers the sensing portion of the polarographic electrode.
. .
~ :
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.... ",... ...
:
-. '. ' ' - 5b -' ' .
. ' . . .
3'~
The present invention will be further described with referenee to the drawings wherein Figure 1 is a schematic proeess flow diagram for praetieing the present invention and Figs. 2 and 3 are schematic 1~w diagrams illustrating the details of polarographiecells whieh ean be used in eonjunction with Figure 1.
Referring now to Fig. 1~ an aqueous specimen eontaining glueose flows into a reaetion chamber eontaining a bed o immobilized glueos~ oxidase where it is maintained for a time and at a temperaturè sufficient to oxidize the glueose to glueonie aeid and hydrogen peroxide. The reaetion chamber is equipped with a specimen inlet and reaetion produet outiet.
' ; ' ' ';.:"
lU'7~2fi34 Typically, this oxidation is completed within a few seconds to 30 minutes or longer at temperatures ranging from 0C to about 50Co A time period of less than 1 minute at room t~mperature is usua]ly sufficient for the oxidation of glucose.
S Because the glucose oxidase is most efficient in the oxidation of glucose in the 4 to 8 pH range, the glucose specimen, prior to contact with the glucose oxidase, is diluted with an aqueous diluent which is buffered to prevent the pH thereof from driftlng out of the pH range where glucose oxidase is most efficient for oxidizing glucose. Preferably for efficiency in oxidation of glucose by glucose oxidase the pH of the buffered diluent is in the range of about 5 to 7~
Particularly suitable aqueous bufered diluents are solutions of 50dium acetate and acetic acid. Other acceptable bufers include sodium citrate, and other water soluble phosphate, and phthlate salts in the concentration of 0.001 to 0.5M.
rrhe oxygen required for this oxidation reaction is provided by that oxygen concentration which is normally dissolved in the glucose specimen and buffered diluent at room temperature. This concentration usually provides enough oxygen to oxidize sufficient glucose for reliable and reproducible polarographic detection. It is not necessary for all of the glucose to be oxidized to form hydrogen peroxide. It is only necessary that ~the percentage conversion is reliable and reproducible from a standard to unknown specimens within a given set of laboratory conditions. Thus a reprcducible value of 60~ conversion of . .
. , .
. /,-10'7Z~;34 G~13336 glucose provides precise polarographic response for the purpose of calibration and analysis~ In the preferred embodiment essentially all of the gluoose is oxidized to proctuce hydrogen peroxide so that any possible error of variation in conversion from sample to sample is eliminated. To achiev,e such complete conversion of glucose9 supplemental oxygen from an external source (e.g. a sparge of oxyyen or an oxygen-containing gas) can be introduced into the specimen if desireda although this is seldom,required.
Slight fluctuations can occur in the dissolved oxygen con~
centration in the glucose specimen and buffered diluent as a function of changes in pressure or temperature. Such fluctuations are minor and the presence of su~ficient oxygen is assured when using the detection method, sample size and 15 , concentrations herein specified. This is an important advàntage over methods measuring glucose as a function of oxygen depletion.
The ratio of dilution of the glucose specimen in the buffered diluent varies with tha concentration of glucose in the specimen. For physiological fluids such as blood and urine having unknown concentration within the expected concentration range9 dilution of 2.5 microliters of specimen ~, into a stream of bu~fered diluent flowing at the rate of 0.1 to 5 and preferably 0.1 to 2 ml per minute is suitable for a significant polarographic response~ Usually, for efficiency and economy, a small glucose specimen (e~g. about 2 to 10 microliters) is ln3ected into a stream of buffered diluent . .
.' .
. "
lQ'~Z634 flowing at the rate of 0.1 to 10 ml per minute for introduction ¦ into the bed of immobilized glucose oxidaseO The buffered dilute is caused to flow by a small metering pump of conventional ¦ design. ~ .
In addition to buffer it is desirable to add salts such ¦ as potassium chloride or sodium chloride which serve to ¦ esta~lish the reference potential when silver-silver chloride reference electrodes which use the sample as a f illing solution are employed. A bacterial inhibitor can be incorporated in the buffered diluent to retard bacterial interferenceO
Any of the known methods or immobiliæing glucose oxidase on an insoluble support to form a bed of immobilized glucose oxidase can be used in practicing the present invention. For . instance; glucose oxidase can be covalently coupled to a porous : 15 glass support with an amino-functional silane coupling agent as disclosed in t~e article entitled, ~Immobilized Enzymes: A Prototype Apparatus for Oxidase Enzyme in ¦ Chemical A lys is Util iz ing Cova lently Bound GluFos e ¦
~ : ' .
' . ., ~; ~ .
: I ~ _g_ . '' Z6'~
Oxidase," by M. K. Weibel et al appear`ing in Analytical ._ Biochemistry, 52, 402~414 (1973); glucose oxidase can be . immobilized on column packing as in the article entitled, "A New Principle of Enzymatic Analysis" by H~ V. Bergmeyer and A. Hagh appearing in Z~ Anal. Chem. 261, 333-336 (1972);
glucose oxidase can be immobilized in polyacrylic polymers as in the article entitled, "Enzyme Electrodes for Glucose Based on an Iodide Membrane Sensor" by G. ~agy et al appearing in Analytica Chemica Acta, 66, (1973~, 443-455; glucose oxidase can be immobilized in a polyacrylamide gel as in the article entitled, "An Enzyme Electrode for the Amperometric Determination of Glucose" by G. Guilbault et al appearing in ~ Analytica Chemica Acta 64~ tl973), 439-455 or U. S. Patent : 3,542,662 to Hicks et al issued November 24, 1970; glucose ~:~
oxidase immobilized on nickel-silica alumina as in the article entitled, "Immobilization of Glucose Oxidase on Nickel-Silica ~ :
. Alumina" by W. M. Herring et al appearing in Biotechnology and ~
Bioengineering, Vol. XIV, pages 975~984 (1972); and glucose I ~ :
oxidase can be immobilized with cyanogen bromide according to the method of U. S. Patent 3,645,852 entitled, "Method of -Binding Water-soluble Proteins and Water-soluble Peptides to Water-insoluble Polymers Using Cyanogen Halide," by R. Axen, : ~:
. J. Porath, and E. Ernbach issued February 29, 1972. Thus, in formin~ the bed of immobilized slucose oxidase the selection of the support from materials such as . ~ -10- ;"~
. , . .
. ., ~ - . . ,:' - . . .
- ~ .. . , : .
iO~63 porousglass; particulate and preferably porous refractory oxides such as alumina, titania, zirconia~ silica, magnesia, talc, and thoria; glass frit; particulate procelain; compacted and sintered reractory oxides; clays; water insolu~le polymers, and immobilizing the glucose oxidase thereon by chemical or p~ysical means is well known in the art.
The only requirement is that the bed must be permeable to the sample specimen while p~oviding a high surface area to volume ratio to assure adequate contact between the glucose oxidase and the glucose specimen. A packed bed of glucose oxidase immobilized on an inert particulate, porous, fibrous or gelled support is preferred for this puxpose~ When using particulate refractory oxides,particle size and percent porosity are not particularly cxitical as long as adequate contact between glucose and glucose oxidase is provided. Volume porosity in the range cf about 10~ to about 60~ with an average pore-size diameter in the range of about 0.01 to lo micrcns have been found to be effective and readily available. Particulate refractory oxides having a particle size diameter in the range of about 1 to about 1000 microns are readily available with a particle size diameter in the rangeof about 100 microns to 400 microns being quite practical for the present purposes.
Aftex oxidation of the glucose in the bed of immobilized ~lucose oxidase the resulting reaction mixture flows to the ~25 polarographic cel~l.
. ,~ ' .
. . ' ' '' 63~
According to the present invention theaqueous buffered diluent is continuously pumped through the reaction chamber containing the bed of immobilized glucose oxidase and polarographic cell. The glucose specimen is injected from a hypodermic syringe into the bed of immobilized glucose oxidase through an injection port which can be in the Eorm of a mixing "tee" covered with a rubber diaphragm.
While flowing through and permeating the bed vf immobilized glucose oxidase the glucose is oxidized to hydrogen peroxide and gluconic acid which remain in the specimen as reaction products.
This reaction mixture then flows into the polarographic cell when the hydrogen peroxide is polarographically detected by direct contact with the polarographic electrodes. The polarographic response from the measurement of the hydrogen peroxide is measured by a current measuring device such as a current follower. This value is then coverted to the glucose equivalent of the original specimen. The glucose equivalent of the original specimen is usually reported in mg glucose/100 ml ~i.e. mg percent) of specimen. These units are conventional in clinical applications.
Two embodiments of polarographic cell illustrated in Fig. 1 are shown schematically in Figs. 2 and 3~ These cells are called "flow-through" cells because the sample under analysis continuously flows through the cell.
In Fig, 2 the cell body 10 is constructed of a rigid, inert7 electrically insulating material such as glass or plastic. Pol~methyl methacrylate plastics have been found to be quite satisfactory for this purpose. The cell body 10 is provided with a narrow passageway 11 through which . ,,--. ~.''.
lO~Z63 ¦ G-13336 ¦ the specimen of reaction mixture containing the hydrogen ¦ peroxide flows into cell cavity 15. The cavity 15 is of I such dimensions to assure effective contact between the sample ¦ to the polarographic electrodes while minimizing dead volume.
¦ The cell used in the Examples is plastic and has a c~vity I about 0 1 inch in width, 0.5 inch in length, and about 0.02 ¦ inch in depth. ~he electrodes enter ~rom the top o~ the cell ¦ and are positioned along the "length" dimensionO
¦ In Fig. 2 a set of two polarographic electrodes 12 and 13 ¦ are mounted in cell body 10 so the sensing tips thereo are ¦ in direct contact with the specimen in cavity 15. ~lectrode 12 ¦ functi.ons as both the counter electrode and reference electrode.
Electrode 12 is of conventional design and can be a silver wire ¦ coated with silvar chloride; or calomel. The silver-silver ¦ chloride reference electrode is preferred because the sample being measured can function as the reference electrode filling ¦ solution by incorporation of chloride ions therein. The other ¦ types of reference electrodes using their own contained filling ¦ solution can be used if desired. Electrode 12 is connected ¦ to a source of constant DC potential (usually about 0.6 volts) as in conventional polarography. Electrode 13 is the working electrode and is in the form of a platinum, palladium, gold, yraphite, or carbon or other inert conductive material.
The hydrogen peroxide in the specimen readily depolarizes the polarographic electrodes and current flow, which is measured ¦¦ by the cu ent measuring device, at the given applied vol=age, . .' . ''. .
. ' ,--~
is proportional to the hydrogen peroxide concentration~ This cuxrent flow is converted to the glucose equivalent o the original specimen. In the polarographic cell shown in Fig. 2 the reference electrode must also function as a counter S electrode and participate in the oxidation reduction reaction taking place in the cell. The IR drop ic; not compensated and th potential difference between the reference elec~rode and the working electrode may change.
The cell of Fig. 3 is preferred over the cell of Fig. ~ .
In the cell of Fig. 2 the xeference electrode is the only other ~lectrode in the circuit, so that current i~ forced to flow through it. This can cause a vaxiation in the potential difference between the electrodes due to the ~R drop through the sample. Furthermore, when a silver-si~ver chloride reference electrode is used, the silver chloride coating is eventua~ly depleted by thP oxidation-reduction reaction~
Accordingly, the use of such a cell, even when the electrode is positioned closely to the working electrode, is not as precise as the cell of Fig. 3.
When using the cell of Fig. 3 in a routine laboratory environment, a peak detector and a sampling and hold circuit can be used to measure the maximum current above a base line current and this diffexence will be proportional to the hydro-gen peroxide concentration in the specimen and is~ therefore, proportional to the glucose concentration.
, ~:
. ' .
.
~rJ'~3~
In a preferred polarographic cell shown in Figure 3, the polarographic cell is potentiostatic. ~his is the so-called "three electrode polarographic" as disclosed in the article, "The Renaissance in Polarographic and Vol-tammetric Analysis"
by Jud B. Flato, appearing in Analytical Chemistry, Vol. 44, September 1972, the disclosures of which are incorporated by reference.
Figure 3 has a cell body 10 and cavity 15 like the cell of Fig. 2. The cell of Frig. 3 is equipped with a reference electrode 23 in the form of a silver wire coated with silver chloride positioned as closely as possible to the working electrode 21 which is a platinum wire. The applied potential ;
(+0.6 volts DC) is applied to the input of a control amplifier 30 to which the reference electrode 23 is also connected through voltage follower 31. The output of the control ampli-fier 30 is connected to counter electrode 20 which is a platinum -wire. By this design essentially no current flows through ~
reference electrode 23 and sufficient compensating potential is `
applied to counter electrode 20 to maintain the potential dif-erence between the reference electrode 23 and the working electrode 21. Working electrode 21 is connected to a small conventional current measuring device which provides a current measurement which is converted to the glucose equivalent of the original specimen. Other conventional electrodes such as described in conjunction with Fig. 2 can be used in the embodiment of Fig. 3 if desired.
' '~
.: .
' .
1~ :IO~Z63~1 ~
. . -:
In the Examples that follow all parts are parts by weight, all percentages are weight percentages, and all temperatures are in C unless stated otherwlse.
Example I
Part A
Immobilized Glucose Oxidase on Alumina Powder _ . _ _ _ _ _ One gram of particulate alumina is washed thoroughly with distilled water. The particulate alumina has a particle size in the range of -40 to ~70 mesh (U.S. sieve screen) and an average pore size diameter of 0.1 to 0.2 microns.
Fifty mg o~ glucose oxidase (obtained from Worthington Biochemical Corporation having a reported activity of 140 International Units per milligram) is added to the wet particulat~
alumina in 40 ml of an aqueous solution which has been bufexed to pH 5.5 with standard buffer comprising a mixture of potassium dihydrogen phosphate and disodium hydrogen phos,~hate.
The resulting mixture is stirred gently for one-half hour at 6-8C to sorb enzyme.
To this mixture is added a crosslinking reagen-t formed by mixing 20 ml methanol; 10 ml distilled water; O.lS ml concentrated hydrochloric acid; 0008 ml diaminopropane; and O.02 ml dibromoethane~ The combined mixture of alumina, glucose oxidase and crosslinking reagent is stirred gently with a magnetic stLrrer~at 6 to 8C overnight to immobilize the glucose oxidase on the alumina, The resulting i~mobilized glucose oxidase~alumina composite is washed with about 2 liters of distilled water and stored in distilled water, . . . ' ~
. .
. .
ioqz~,34 Part B
Assay of Immobilized Glucose Oxi~ase~Alumina Compo~i-te The catalytic activity o the immobilized glucose oxidase/alumina composite of Part A is calculated from the measured rate of oxidation of ~ -D-ylucose to ~luconic acid by para~benzoquinone in the presence of the glucose oxidase/
alumina composite. I~he reaction is represented hy:
~D-glucose~p-benzoquinone~o GlUc05e Oxidase) 1 i hydroquinone The reaction is followed by measuring potentiometrically the change in concentration of hydroquinone with time. A
platinum detector ~lectrode (Beckman model 39273) is used with a double junction calomel-silver/silver chloride reference electrode (Orion model 90-20 00). Standard solutions or calibrating the elec~rode system are prepared -fxom hydroquinone with about a 100 molar excess of p-benzoquinone also present in the aqueous solutions, A calibration graph is drawn by plokting hydroquinone concentration against millivolt readings from the potentiometer.
The reaction medium in which the oxidation of glucose takes place~is an aqueous solution which is 0.1 molar in : dextrose and 0.01 molar in phosphate buffer at pH 5.5.
It is stirred overnight to assure that equilibrium has been ~ reached between khe~ and ~ -D-glucose forms. Suficient 1~ p-ben-oqu ne and hydroquinone is added to make the final . ' . ' ' '.
. ,-.
.
lO';~Z63~
¦ solution 1.0 x 10-2M and 1.0 x 10 4M in these latter two ¦ components. A known quantity of the glucose oxidase/alumina ¦ composite is added to a given volume of the reaction medium ¦ and the change in the potential of the electrode immersed ¦ in ~he solutionisfo~owedwith time using the electrode system :
described above. From the millivolt readings the corresponding ¦ concentration of hydroquinone is determined from the ¦ calibration graph and a plot is made of test solution con-¦ centration versus time. The initial slope of this curve ¦ represents the rate of oxidation of~ -D-glucose catalyzed by ¦ the immobilized enzym0. The activity is calculated from the ¦ ralationship:
Rate of oxidation ~ctivit~
I ~' Y Volume of alumina ¦ Using this assay procedure the glucose oxidase/alumina ¦ composite of Part A is found to have an activity of 995 ~ International Units of glucose oxidase per ml of glucose ¦ oxidase/alumina composite. Ten days later, after being stored ¦ in distilled water at 4C , the activity is 825 International ¦ Units of glucose oxidase per ml of glucose oxidase/alumina ¦ composite.
An International Unit of biological activity has been ? defined as the amount of active enzyme which converts sub-strate to product at the rate of one micromole per minute.
: ' .
Part C
A glass column is prepared from a 50 mm borosilicate, glass tu~e with an inside diameter of 2.8 mm and an outside .
. 5 diameter of 6 mm. A 4no mesh ny].on disc is attached to one .
end of the column. The immobilized glucose oxidase/alumina composite of Part A is charged thereto to fill the tube. The glucose oxidase/alumina packs into the column by gravity and .
the othex end of the column is also fitted with a 400 mesh nylon disc after the column is filled. The volume of the glucose oxidase/alumina composite is approximately 0.3 cc. , The column ends are then fitted with plastic tubing fittings one of which is in the form of a "tee" for sample injection. The injection tee is provided with a rubber membrane for sampl.e injection with a hypodermic needle.
. Part D
Anal,ysis of ~lucose A buffered diluent for the glucose specimen is prepared . from: 50 ml of saturated aqueous solution of 2-chloro-4-phenylphenol; 10 ml o l.ON potassium chloride; 8~2g (0.10 molej sodium acetate; sufficient l.OM acetic acid to adjust ., the pH to 5.6; and additional water to make one liter of solution. The buffer is thus approximately 0.1 m~lar in acetate, 0.01 molar in potassium chloride and contains a trace ¦¦ of the ba ricide, 2-chloro-4-pheny1phenol.
. .
. .
.
:IU''~Z63'1 The polarographic cell is as shown in Fig~ 3 and has been described above. The electrodes are connected to a PAR Model 174 polarographic analyzer.
The standard solution used to calibrate the polarographic S cell is a 0.01 molar aqueous glucose solution which is 0.1 molar in sodium chloride as a reference electrode ~illing solution. ~oth chemicals are reagent grade. The standard solution is prepared well in advance of its use to assure chemical equilibrium. Expressed in terms commonly used in the clinical laboratory this standard represents a g~ucose concentration of 180 mg of glucose per 100 ml of specimen (i.e. 180 mg percent glucose).
¦ The buffered diluent is pumped through the apparatus ¦ described i~ Fig. 1 at a rate of 1.0 ml/min. This produces ¦ a base li~e current of approximately one nanoampere. A 3.0 ¦ microliter sample of the O.lOM glucose standard solution is ¦ injected with a hypodermic needle through the injection "tee"
¦ into the immobilized enzyme column as shown in Fig. 1. The ¦ current rapidly increases to a maximum current of 250 nanoamps I over a 10 second period and then rapidly decreases over a 30 second period to a value approaching the base line value.
The current to concentration calibration factor is thus l.39 nanoamps/mg percent glucose for a three microliter sample.
~en separate 3,0 microliter specimens of a~standard ~lood serum sample (reported to contain 83.5 mg percent glucose) are sequ'entially injected into the stream of buffered diluent flowing into the bed of immobilized glucose oxidase and the resulting .. , . ' ,~
1i~7Z63 ¦ G-13336 ¦ peroxide is polarographically analyzed as described above ¦ and the corresponding current follower reading are recorded.
¦ Using the calibration factor to convert current to concentration, ¦ the samples are found to contain an av~rage value of 80 mg ¦ glucose/100 ml specimen (80 mg percent) with a coefficient of ¦ variance of 0.7~o.
l A second, abnormal, standard blood serum sample (reported ¦ to contain 197 mg percent glucose) is analyzed by the above ¦ procedure~ From a total of 20 analyses an average value of ¦ 200.5 mg glucose/100 ml is obtained with a standard coeffici2nt ¦ of variance of 1.3%.
I Example II
I , .
¦ Part A
l Preparation of Immob _ zed Glucose Oxidase Five grams of particulate alumina having a particle size in the range of -70 to +80 mesh (U.S. sieve screen) and an average pore diameter of 0.1 to 0~2 microns is dried by heating at 1060C for two hours. After cooling, thealumina is soaked in lD0N HCl for 2-4 hours and washed with-distilled water to remove adsorbed air. The washed al~l~a is placed in a beaker with about 10 ml of 0.01M phosphate buffer (pH 6.0).
Glucose oxidase, as in Example I (250 mg) is added to tne - particulate alumina with enough distilled water to bring the volume of the mixture to about 40 ml. The glucose oxidase/alumin 25 ~ I mixture sti/Fed ~ently w~th a magnetic stirr~r at roOA
Il I
. '" ' , , , :10'7Z634 temperature for about one-half hour.
A crosslinking reagent is prepared from 20 ml methanol, 10 ml water, 0.15 ml concentrated hydrochloric acid, 0.05 ml diaminopropane and 3 ml of a 10~ by weight solution of gluteraldehyde. Th~s reagent is added dropwise to the glucose oxidase/alumina mixture described above. The addition is made at room temperature and the final reaction mixture left standing overnight a~ 0. It is then washed in O.OOlM phosphate bufer (pH 5.5) and stored in distilled water until assayed.
~he assay procedure is the same as that described in Example I, Part B. The activity is found to be about 150 International Units per ml of glucose oxidase/alumina composite.
Part B
.
. Glucose AnalYsis ;
A bed of immobilized glucose oxidase i5 prepared as descri~ed in Example I using the glucose oxidase/alumina composite described in Part A aboveu The standardization procedure for the analytical system is essentially the same as that in Part D o Example I.
The flow rate for the buffered diluent is 1.0 ml/min., and it is the same composition as in Example I. A 3.0 microliter standard glucose~solution (containing 180 mg percent glucose) gives a reading of 590 nanoamps. The calibration factor i5 thus 3.28 nanoamps/mg percent ylucose for the 3 microliter sample. -~ -22- ;
. . :
10'7Z~
Ten separate 3.0 microliter specimens of a standard blood serum sample (reported to contain 83.5 mg glucose/100 ml) are sequentially injected into the bed of immobilized glucose oxidase as above, and the corresponding current readings record-ed. Using the calibration factor to convert current to glucose concentration, the samples are analyzed to contain an average value of 82 mg glucose/100 ml with a coefficien~ of variance of 1~5%.
A second, abnormal, standard blood serum sample (reported to contain 197 m~ percent glucose) is analyzed as above. From a total of 10 tests an average value of 197.2 mg glucose/100 ml is obtained with a standard coefficient o variance of 1.0C~.
Example III
Part A
Preparation of Immobilized Glucose Oxidase Five grams of particulate alumina having a particle size in the range of -60 to ~70 mesh (U.SO sieve screen) and an average pore diameter of 0.1 to 0.2 microns is heated at 1150C
for two hours. After cooling, the particulate alumina is soaked in 1.0~ Hcl overnight. It is then washed with distilled water and placed i~ a beaker with 50 ml o~ 0.001 phos~hate buffer (pH
t. . 6.0) for one-half hour before adding 25 ml of glucose oxidase solution. The glucose oxidase (Asperilligus niger) is obtained from Pierce Chemical Company in a buffered solution (pH 4.0) and has a reported activity of 1000 International Units/ml.
The resulting gluc~se oxidase/alumina mixture is stirred . :
~1 ' , I
O~Zi,3 gently for one-half hour at 3-8C.
A crosslin~ing reagent is prepared by mixing 20 ml methanol~ 0.01 ml diaminopropane, 0.15 ml concentrated HCl, 0.03 ml dibromoethane and 10 ml water. This reagent is adcled at a rate of 0.15 to 0.20 ml/min to t.he glucose oxidase/alumina mixture while stirring gently with a magnetic stirrer and main-taining the temperature at 3 to 8C forl~ ~ hours, The result-ing immobilized glucose oxidase/alumina composite is washed with 2 liters of distilled water and stored in distilled water until assayed, The~assay procedure i5 the same as that described in Example I. Theactivity of the sample is 2,300 International Units/ml immobilize~ glucose/alumina composite.
Part ~
Glucose Anal~sis ~ bed of immobilized glucose oxidase i5 prepared as described in Example I using the glucose oxidase/alumina composite described in Part A above. '~he volume of the column in this case however is only 0.2 cc.
The standardization procedure for the analytical system is essentially the same as that in Part D of Example I. The flow rate for the buffered diluent of the compasition of Example I is 0.7 mI/min. A 2.5 microliter sample of the glucose standard containing 180 mg percent of glucose gives a current of 150 nanoamps. The calibration factor is thus
-2~-.
.' l~Z~3~
0.83 nanoamps/mg percent glucose for the 2.5 microliter sample.
~ine separate 2.5 microliter specimens of a standard ~lood sample are sequentially injected into the bed o~ i~mobilized glucose oxidase as above and the corresponding current readings S recorded~ Using the calibration factor to con~ert current to concentration, the samples are found to produce an average value of 79.7 mg glucose/lO0 ml with a standarc1 coefficient of variance of l.5~. The reported value is 83.5 mg glucose/lO0 ml.
A second~ abnormal, standard blood serum sample containing 197 m~ percent of glucose is analyzed as above. From a total of ll tests an average value of 205.6 mg glucose/lO0 ml is obtained with a standard coefficient of variance of 1.3~.
Similar results are obtained in the above procedure for glucose analysis when the bed of immobilized glucose oxidase is prepared by immobilizing glucose oxidase on a crosslinked polydextran obtained from Pharmacia Fine Chemicals Inc. under the trade name of Sephadex G-200.
A 1.25 g sample of the polydextran is washed with 2 liters of distilled water and a uniform gel is prepared b~ mixing with water. Four grams of cyanogen bromide is crushed in a mortar and transferred quickly to the polydextran gel. The pH of the dispersed gel is quikly adjusted to about lO.5 with a 3~ sodium h~droxide solution and maintained at this pH
by the gradual addition of more 3N sodium hydroxide.
' ,:., ' ~:
. , '' ~
. , .
.
l(~q;~634 Periodically, crushed ice prepared from deionized water is added to maintain the temperature of the reaction mi~ture at about 20C. The introduction of base is stopped when there is no further change in p~l and the cyanogen bromide crystals are consumed. The mixture is stirred con~inuously throughout the reaction. A total of about 13 ml of sodium hydroxide solution is required.
A large amount of ice is added and the mix~ure is trans-ferred to a sintered glass filter funnel. The product is washed under vacuum using first 300 ml of O.OlM tris (hydroxymethyl) aminomethane buffer (adjusted to pH 7 with HCl) and then 300 ml of 0.01 ~ phosphate buffer (pH 5.6). Both wash solutions were previously cooled in an ice bath. The bottom of the filter funnel is then closed with a piece of wax film.
I~theresulting cyanogen bromide activated polydextran still on the filter funnel, is added a cold solution of 50 mg glucose oxidase in 20 ml of 0.01 M phosphate buffer (pH 5.6).
The resulting product is stirred with a glass rod on the filter funnel and then transerred to a beaker. The washing of the cyanogen bromide activated polydextran and the subsequent - . mixing with the glucose oxidase solution on the funnel is done vexy quickly, i.e. in a total time of about 90 seconds.
The glucose oxidase/polydextran mix-ture is stirred gently with a magnetic stirrer for 16-20 hours at 0C. It is then washed with 1-2 liters of distilled water and stored in O~lM
. . .' .~qZ,63f~
. G-13336 phosphate buffer, pH 5.6. After 65 days the activity is 450 International Unit~/ml glucose oxidase/polydextran composite gel. The glucose oxidase/polydextran composite gel is packed in a column and glucose specimens are analyæed as in Example I. -Similar results are also obtainecl in the above procedure for glucose analysis when the immobilized enzyrne bed comprises glucose oxidase immobilized on porous particulate anion exchange resin. The resin used is the chloride form of a styrene-divinylbenzene copolymer with quarternary amine ion exchange groups (available from Bio-Rad Laboratories under their designation AG-MP-l).
A 250 mg sample of the anion resin is stirred vigorously with 30 ml of O.lOM sodium borate buffer (pH 8.5). It is then centrifuged for 20 minutes at 12,000 g's. ~ne supernatant is decanted and this-washing procedure repeated twice. A
final wash is carried out in a similar manner using O.lOM
acetate buffer, pH 5Ø
The washed resin is dispersed in about 50 ml of the acetate buffer and stirred ~ently for 15-20 minutes at 5-10C before adding 100 mg of glucose oxidase in small increments. To this mixture is then added 0.5 ml of dlbromo-propane dissolved in 2 ml of acetone~ This final mixture is stirred gently for one hour at 5-10C. The reaction product is centrifuged at 120 gravity forces and the supernatant .
lO~Z63 discarded. The glucose oxidase/ion exchange resin composi.te is fuxther treated with 40 ml of an acetate buffer solution (pH S.O) which is 0.05 molar in 2~aminoethanol. It is stirred for one hour at 0, filtered and washed with 1-2 liters of distilled deionized water in which it is then subsequently .
stored at 4C. After one week the activity is 15 International Units/ml of immobilized glucose oxidase/resin composite. The composite gel is pac~ed in a column and glucose specimens are .
analyzed as in Example 1. ~
..
.' . .
.' l~Z~3~
0.83 nanoamps/mg percent glucose for the 2.5 microliter sample.
~ine separate 2.5 microliter specimens of a standard ~lood sample are sequentially injected into the bed o~ i~mobilized glucose oxidase as above and the corresponding current readings S recorded~ Using the calibration factor to con~ert current to concentration, the samples are found to produce an average value of 79.7 mg glucose/lO0 ml with a standarc1 coefficient of variance of l.5~. The reported value is 83.5 mg glucose/lO0 ml.
A second~ abnormal, standard blood serum sample containing 197 m~ percent of glucose is analyzed as above. From a total of ll tests an average value of 205.6 mg glucose/lO0 ml is obtained with a standard coefficient of variance of 1.3~.
Similar results are obtained in the above procedure for glucose analysis when the bed of immobilized glucose oxidase is prepared by immobilizing glucose oxidase on a crosslinked polydextran obtained from Pharmacia Fine Chemicals Inc. under the trade name of Sephadex G-200.
A 1.25 g sample of the polydextran is washed with 2 liters of distilled water and a uniform gel is prepared b~ mixing with water. Four grams of cyanogen bromide is crushed in a mortar and transferred quickly to the polydextran gel. The pH of the dispersed gel is quikly adjusted to about lO.5 with a 3~ sodium h~droxide solution and maintained at this pH
by the gradual addition of more 3N sodium hydroxide.
' ,:., ' ~:
. , '' ~
. , .
.
l(~q;~634 Periodically, crushed ice prepared from deionized water is added to maintain the temperature of the reaction mi~ture at about 20C. The introduction of base is stopped when there is no further change in p~l and the cyanogen bromide crystals are consumed. The mixture is stirred con~inuously throughout the reaction. A total of about 13 ml of sodium hydroxide solution is required.
A large amount of ice is added and the mix~ure is trans-ferred to a sintered glass filter funnel. The product is washed under vacuum using first 300 ml of O.OlM tris (hydroxymethyl) aminomethane buffer (adjusted to pH 7 with HCl) and then 300 ml of 0.01 ~ phosphate buffer (pH 5.6). Both wash solutions were previously cooled in an ice bath. The bottom of the filter funnel is then closed with a piece of wax film.
I~theresulting cyanogen bromide activated polydextran still on the filter funnel, is added a cold solution of 50 mg glucose oxidase in 20 ml of 0.01 M phosphate buffer (pH 5.6).
The resulting product is stirred with a glass rod on the filter funnel and then transerred to a beaker. The washing of the cyanogen bromide activated polydextran and the subsequent - . mixing with the glucose oxidase solution on the funnel is done vexy quickly, i.e. in a total time of about 90 seconds.
The glucose oxidase/polydextran mix-ture is stirred gently with a magnetic stirrer for 16-20 hours at 0C. It is then washed with 1-2 liters of distilled water and stored in O~lM
. . .' .~qZ,63f~
. G-13336 phosphate buffer, pH 5.6. After 65 days the activity is 450 International Unit~/ml glucose oxidase/polydextran composite gel. The glucose oxidase/polydextran composite gel is packed in a column and glucose specimens are analyæed as in Example I. -Similar results are also obtainecl in the above procedure for glucose analysis when the immobilized enzyrne bed comprises glucose oxidase immobilized on porous particulate anion exchange resin. The resin used is the chloride form of a styrene-divinylbenzene copolymer with quarternary amine ion exchange groups (available from Bio-Rad Laboratories under their designation AG-MP-l).
A 250 mg sample of the anion resin is stirred vigorously with 30 ml of O.lOM sodium borate buffer (pH 8.5). It is then centrifuged for 20 minutes at 12,000 g's. ~ne supernatant is decanted and this-washing procedure repeated twice. A
final wash is carried out in a similar manner using O.lOM
acetate buffer, pH 5Ø
The washed resin is dispersed in about 50 ml of the acetate buffer and stirred ~ently for 15-20 minutes at 5-10C before adding 100 mg of glucose oxidase in small increments. To this mixture is then added 0.5 ml of dlbromo-propane dissolved in 2 ml of acetone~ This final mixture is stirred gently for one hour at 5-10C. The reaction product is centrifuged at 120 gravity forces and the supernatant .
lO~Z63 discarded. The glucose oxidase/ion exchange resin composi.te is fuxther treated with 40 ml of an acetate buffer solution (pH S.O) which is 0.05 molar in 2~aminoethanol. It is stirred for one hour at 0, filtered and washed with 1-2 liters of distilled deionized water in which it is then subsequently .
stored at 4C. After one week the activity is 15 International Units/ml of immobilized glucose oxidase/resin composite. The composite gel is pac~ed in a column and glucose specimens are .
analyzed as in Example 1. ~
..
.' . .
Claims (10)
1. A method for determining glucose in an aqueous specimen containing glucose, comprising the sequential steps of:
diluting said specimen in an aqueous buffered diluent buffered to a pH in the range of about 4 to about 8, passing the diluted specimen into a bed of glucose oxidase immobilized on a solid support, retaining said diluted specimen in contact with said glucose oxidase for a time sufficient to oxidize said glucose to form hydrogen peroxide, removing the resulting reaction mixture from said bed, passing the resulting reaction mixture into direct contact with a set of polarographic electrodes, polarographically determining hydrogen peroxide in said reaction mixture while in direct contact with said polarographic electrodes to produce an electrical current proportional to the hydrogen peroxide concentration of said specimen, determining said electrical current, and translating said current determination to the glucose concentration of said specimen.
diluting said specimen in an aqueous buffered diluent buffered to a pH in the range of about 4 to about 8, passing the diluted specimen into a bed of glucose oxidase immobilized on a solid support, retaining said diluted specimen in contact with said glucose oxidase for a time sufficient to oxidize said glucose to form hydrogen peroxide, removing the resulting reaction mixture from said bed, passing the resulting reaction mixture into direct contact with a set of polarographic electrodes, polarographically determining hydrogen peroxide in said reaction mixture while in direct contact with said polarographic electrodes to produce an electrical current proportional to the hydrogen peroxide concentration of said specimen, determining said electrical current, and translating said current determination to the glucose concentration of said specimen.
2. The method of claim 1 wherein essentially all of said glucose is oxidized to form hydrogen peroxide.
3. The method of claim 1 wherein said aqueous buffered diluent is buffered to a pH in the range of about 5 to 7.
4. The method of claim 1 wherein said aqueous specimen containing glucose is blood serum.
5. The method of claim 1 wherein said solid support is a particulate refractory oxide.
6. Apparatus for determining glucose in an aqueous specimen containing glucose, comprising in a combination, a reaction chamber having an inlet for introducing said specimen and an outlet for removal of a reaction product, said reaction chamber containing a bed of glucose oxidase, a polarographic cell interconnected with said reaction product outlet, said cell comprising a tubular sample chamber having a sample inlet and outlet, said sample chamber having a set of polarographic electrodes positioned therein for direct electrical communication with a sample in said sample chamber, said electrodes being electrically connected for amperometric response to electrical current generated in said cell.
7. The apparatus of claim 6 wherein said set of electrodes includes three electrodes comprising a working electrode, a counter electrode, and a reference electrode.
8. The apparatus of claim 6 wherein said set of electrodes comprises a pair of electrodes including a reference electrode and a working electrode.
9. The apparatus of claim 7 wherein said working electrode is platinum, said counter electrode is platinum, and said reference electrode is silver coated with silver chloride.
10. The apparatus of claim 6 wherein said solid support comprises a particulate refractory oxide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA238,656A CA1072634A (en) | 1975-10-30 | 1975-10-30 | Polarographic glucose analysis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA238,656A CA1072634A (en) | 1975-10-30 | 1975-10-30 | Polarographic glucose analysis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1072634A true CA1072634A (en) | 1980-02-26 |
Family
ID=4104400
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA238,656A Expired CA1072634A (en) | 1975-10-30 | 1975-10-30 | Polarographic glucose analysis |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1072634A (en) |
-
1975
- 1975-10-30 CA CA238,656A patent/CA1072634A/en not_active Expired
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