Amperometric Determination of Glucose at Parts Per Million Levels with Immobilized Glucose Oxidase An Undergraduate Experiment G. Sittampalam and G. S. Wilson University of Arizona, Tucson, AZ 85721 Techniques utilizing immobilized enzymes are becoming popular for routine chemical analysis. Such methods involve interesting new chemistries which are highly selective due to the inherent specificity of an enzyme toward its substrate. In addition, these analytical methods are sensitive, fast, convenient, and revroducible over long.. periods. Hence, we can de. sign instruments incurpuratin:: imniddi7ccl en7ymci t o measure a \.aricty of a~~bsrrutcs ond inhillit, ri. \{?ln\. au,.h instruments are now available on the market and wire reviewed recently in Analytical Chem~stry(1). Clinical laboratories have been using these instruments extensively in the last decade, although analytical chemistry curricula in colleges do not regularly expose students to these techniques. Consequently, we designed an experiment on the operation and utility of an amperometric immobilized enzyme electrode !or probe) for our undergraduate instrumental analvsis course. This experiment demonstrates several advantages of immobilized enzvmes and the am~erometric nrobe in chemical analysis: 1. The reusability of the immobilized enzvme.
temperature, 4. A sensitive amperometric detection system which
measures Ampere),and nanoArnpere level currents (1 nA = 5. Chemical and electronic am~lificationOF the enzymatic reaction which enhances sensitivity (2) Although similar probes have been described in the literature (3),they have not been conveniently adapted for lahoratory teaching to demonstrate their analytical utility. Also, other studies describing enzymatic analysis (4-13) have concerned soluble enzymes, except for that of DeJong and Kumler (12), who utilized immobilized trypsin on glass beads packed in a column. There, the enzyme coupling to the beads was preceded by a 16-hr silanization by refluxing. In our procedure, the enzyme is immobilized in a few minutes, so that the probe may he used within 1-2 hours. Thus, the experiment can be completed within 4 hours.
Principles of Operation This experiment is designed to use the enzyme glucose oxidase. B-D-Glucose, which is a svecific substrate for this enzyme, undergoes the following enzymatic reaction: 0-D-glucose + Oz
glucose oridare
PH 7.3 po4-3b u m r
gluconic acid
+ H202
(1)
T o follow the above reaction, we must detect specifically the formation of one of two products, or the depletion of one of the reactants. In monitoring the products, it is convenient to measure the concentration of Hz02 generated, which will be directly proportional to the glucose concentration in the sample solution. Hz02 concentration can be monitored either hv a votentiometric measurement of iodine vroduced (hv the reaction between Hz02 and excess I- in the sample [ l j ] ) or 70
Journal of Chemical Education
(ni Figure 1. Enzyme immobilization procedure
by a direct anodic oxidation of H202 at a fixed applied potential. The latter approach was chosen in this experiment as described below. The enzyme is "immohilized" or trapped between the inner cellulose acetate and an outer colla~enor a polvcarbonate and a silver cathode (reference electrode) between which a constant potential of +0.700 V is applied. This potential is applied when the probe is plugged into the oxidase meter, which also measures the current due to the oxidation of Hz02 at the platinum anode. Both the amperometric probe with the membrane kit and the oxidase meter can he purchased from
Yellow Springs Instrument Co., Box 279, Yellow Springs, O H
.""". .
AFAR7
When the probe is immersed in a solution of glucose, the latter diffuses through the outer membrane into the enzyme layer and converts t o gluconic acid with the concomitant production of Hz02 as shown in eqn. 1. The Hz02 diffuses selectivelv throueh the cellulose acetate membrane toward the platinum anode, where i t undergoes a n oxidation, while t h e 01 dissolved in solution reduces a t the silver cathode a s shown below: HzOz
-
02
+ 2Ht + 2e
11202 + 2H+ t 2e
-
Hz0
~
(3)
~.~~ ~~
Experimental A detailed account of the enzvme immobilization ~ r o c e d u r e and some precautions for the smooth running of the experiment are described here. This information is by no means comprehensive, and the students and teachers are encouraged t o adapt these procedures to suit their own requirements. The equipment and reagents listed below should be provided for t h e students during the assigned laboratory periods. Equipment
Model 2510 Oxidase probe, Model 25 Oxidase meter, strip chart recorder (with adjustable full scale), 5 vials of 7 or 4-dram capacity, magnetic stirrer, 6 magnetic st~rringbars ('h in. X 'id in.),2-ml and 10-ml pipets calibrated in %o ml, 50-ml beaker and 100-ml wash bottle. Reagents
One mglml stock glucose solution in pH 7.3 POd-Quffer (50 ml), pH 7.3 POdPbuffer (250 ml), d~stilleddeionized HzO and glucose oxidase (Type 11, Sigma Chemical Co., St. Louis, MO). Note: Concentrations of unknown glucose solutions provided by the instructor can be between 2-10 mgldl (dl = deciliter = 100ml).When serum or plasma samples containing 70-80 mgldl glucose are used as unknowns, appropriate dilutions should be made betore measurement (see procedure below). Preparation of P04-3 Buffer
The following ingredients were dissolved in 500 ml distilled deionized water (14): Disodlum phosphate, anhydrous 3.75 g Monosodium phosphate, hydrate 0.95 g Sodium chloride 1.50 g Sodium benzoate 0.50 g Diputsssium EDTA, dihydrate 0.25 g Enzyme lmmobilization
The construction details fur model 2510 enzvme orobe are shown their own procedures whenever appropriate.
~
~
(2)
T h e steadv-state current from the above reactions will be directly proportional t o the Hz02 generated hy the enzymatic reaction, which in turn will he proportional to the glucose concentration in the sample. Thus, the steady-state current is a direct measure of alucose concentration in the sample solution. This enzvmatic reaction can be followed also by monitoring t h e depletion of the 0 2 dissolved in solution using a polar01 graphic oxygen electrode, which is available commercially. In this approach the electrodes measure the pOz in solution. Solubilitv of O7 in aqueous solutions is very sensitive to small variations in temperature and, therefore, requires careful temperature control. (.'oncvntr>iticm of H.O2 I; i ~ l warir,~ttdhy tcmper:nure hut IWI >IStttuch :IS the sdub~ltty m'0:. 'l'hua, the per, ctt! error ~ I I W t~. o :---~ u ~ b i r 1rmneratur( m tlucruatim IS minini:~l.\Ye iound lI1.1l even in t h e absence of thermostating, careful students obtained reproducible results within f5%.Temperature control within fO.Z°C is recommended for more accurate work. ~
(1)Tho cellulose acetate membrane is cast by spreading a thin layer of a cellulose acetate solution made with cyclohexanone and acetone on a clean, dry surface of the probe (see also reference 14). The solvents dry quickly leaving a thin film of cellulose acetate on the electrode surface. This solution was made by mixing cyclohexanone, acetone, and cellulose acetate (39.8% acetyl content) in the ratio of 24241 parts by weight, respectively,and stirring overnight in a closed flask (14). A 10-15 ml supply of thissolution should last a semester. (2) Using an applicator, rubber O-ring, and 1 sq. cm collagen or oolvcarbonate membrane (from the kit orovided bv Yellow Sorinen
The rubber O-ring is positioned on the applicator as shown. A drop of distilled water is in the depression of the applicator (Fig. lb). The collagen or polycarbonate membrane is placed on the applicator just above the O-ring. The drop of water moistens the membrane and makes it pliable. A few crystals olglucase oxidase are now placed on the membrane where they become a tiny drop of liquid in 1-2 min. (Fie , - ~ -l--,. .r i ~ The probe is now held in one hand and the applicator with the membrane, enzyme drop, and O-ring in the other hand. The applicator is inverted and held over the probe, so that the enzyme drop is directly over the platinum anode (Fig. Id). The O-ring is quickly slipped over the probe with thumb and index finger to hold the membrane onto the surface, thus trapping the enzyme as shown (Fig, le). (3) The excess membrane is trimmed, and the probe is carefully washed and then immersed in P04c3 buffer solution. The immabilizatiun procedure described above can be completed in 10-15 min, and the probe can then be plugged into the oxidase meter, which applies a constant f0.700 V between the Pt and Ag electrodes. With this applied potential, the probe is allawed to equilibrate and stabilize for approximately 1-2 hr (at zero offset [14]). During this period, the background current will decrease from values >I00 nanoamperes to about 3-10 nanaamperes. This residual background current can be subtracted using the offset knob on the instrument. The set-up is now ready for use by students.
.
I n our lahoratory the instructors are responsible for the immobilization described above. However, students working on special projects have demonstrated t h a t this rather simple immobilization procedure can be learned very easily and quickly. They also showed t h a t the probe gave useful responses on continuous use a t room temperature for well over 3 weeks. NOTE: The probe can he made in well-equipped machine s h o ~ sb. u t the cost of labor and oarts will be a. ~.~ r o x i m a t e l v $256 (US.) per probe. In many cases, therefore, the outright purchase of the probe from the suggested manufacturer might be convenient. As of July 1980, the retail prices quoted by Yellow Springs Instrument Co., Yellow Springs, Ohio, a r e shown below:
Laboratory Procedure
Ten milliliters of standard glucose solutions (2, 4, 6, 8, and 10 me1100 ml) aremade in I or 4-dram vials withmametic stirrine bars stirred solution,and the steady-state currents are recorded on a chart recorder for 30-60 sec. The prvhe is washed thoroughly between subsequent measurements. An appropriate amount of unknown solution is now pipetted into another vial with a magnetic stirring bar and diluted to 10.0 ml with pH 7.3 phosphate buffer. Current produced bv the unknown solution is then measured as described above. between the electrodes. The calibration curve obtained with the standard solutions is then used to determine the original concentration of glucose in the unknown solution (Fig. 3). Precautions (1) Even a momentary removal of the applied voltage, by unplugging the probe, turning off the oxidase meter, or alVolume 59
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lowing t h e probe t o d r y up, will cat&>arge background eurrents. It m a y t a k e another 1-2 h r before these background currents decrease to negligible levels. This will create problems when more t h a n one s t u d e n t is scheduled t o work o n t h e experiment either in t h e s a m e or suete8sive laboratory periods. Therefore, it should b e emphasized t o each s t u d e n t t h a t t h e experimental set-up should be E f t with (a) t h e oxidase meter ON, (h) t h e enzyme probe immersed in fresh buffer solution a n d plugged into t h e oxidase meter, a n d (c) t h e stirrer motor t u r n e d OFF when t h e assignment i s completed. (2) Whenever a new sample of enzyme is immobilized o n t h e probe, t h e electrode surfaces should b e cleansed thoroughly with acetone and a new film of cellulose acetate should b e cast. (3) A second probe, ready for use, is advisable t o avoid delays if the probe in t h e laboratory becomes inoperational. Results and Discussfon Immobilization by Entrapment
T I N E I N HINUTFS
Figure 2. Typical current-time response of the enzyme probe: A, electrode immersed in glucose solution: b, electrode immersed in buffer solution. (Time interval between A and B = 2 min.)
The immobilization nrocedure described above vields a thin en-
both the electrode and the enzyme. The collagen (or nucleopare) membrane serves to contain glucose oxidase in the enzyme layer. I t is permeable to small molecules, such as " elucose. H9O. and inoreanle ions. Laree molecules . H101. . ., and 0% such as enzvmes cannot oafis throueh the membrane. which forms an
non-recoverable soluble enzymes in the hulk solution. The cellulose acetate membrane, an the other hand, excludes all molecular species, except small ones such as Hz02,02, HzO, and other inorganic ions, from the electrode surface. Experiments have shown that it is not permeable toglucose or e v e n b gluconic acid (15,16,17). Hence the current we observe is due essentially to the HzOz which diffuses in from the enzyme layer to the vicinityofthe working electrode. This selective permeability of the cellulose acetate membrane eliminates interferences from electroactive suhstanees that may originate from the hulk solutio? or even from the enzyme layer. The mechanism of this selective permeabilityaf the cellulose acetate membrane is not fully understood. For example, it is positively impermeable to glucose, ascorbic acid, and uric acid hut allows Hz02 and O2 to diffuse freely. On the other hand, xanthine, which is structurally similar to uric arid, penetrates the membrane in limited amounts hut not as freely as HzOz or 0 2 (15,16,17). Therefore, the membrane's permeability to substances in solution does not seem to depend on molecular size; it appears to be afunction of the net eleceiven nH trical charee on the s~eciesof interest at a . andionic streneth. It should benoted. however. that ascorbic and uric acid are twomiior
0
1
2
3
4
5
the time of the experiment, the analysis of the unknowns was not adversely affected. Enzyme Stability In our experiments, the probe retained useful enzyme activity for annroximatelv 21-30 davs at room temoerature. This hehawor is
strong adsorption of the enzyme onto the membranes is responsible for the extended stability. Probe Response The response of the enzyme probe shows that the initial rapid increase in current is fallowed by a region of relatively constant current
72
Journal of Chemical Education
7
R
9
1
0
1
~
Glucose C o n c e n t r a t i o n m g l d l
Fieure 3. Calibration graph for glucose determination.
Unknown Giucose Concentrations Reported by Students Glucose Concentration in mgldl after Dilution Group B Group A (Unknown = 5.0 mgldi) (Unknown = 3.0 mgldl) Reported Values:
4.6 6.4
2.7 4.4 4.3 4.7
for glucose. Finally, some comments should be made about the amount of e w zyme immobilized on the probe. Readers might have noticed that a "few crystals'' of the enzyme were trapped between themembranes. This means that each new enzvme would have different " nrube . amonnts of enzvmes "immohilized" and thus some differences would
1
Reported Values:
2.9 3.0 2.8 4.0 2.0 77
5.0 Average Standard Deviation = l O 56 moidl
(steady-state) (Fig. 2). A closer inspection reveals a slight slop? in the latter region. This is primarily due to the build-up of HzOzin the bulk solution by diffu~ionas the products are generated in the enzyme layer: Trace amounts of catalase added to the bulk solution will eliminate this effect. For our purposes, however, the "steady-state" current monitored after a fixed time lapse is acceptable for a ealihration curve (Fig. 3). Also, the probe response shows gradual decrease during the second week as demonstrated by the decreasing slopes of the calibration curves. This, however, should nut affect the analysis since each student obtains his or her own calibration curve. The table helow shows the results obtained by two groups of students in our laboratorv. The silver cathode on the probe functions as a reference electrode on which the dissolved 0 2 is reduced (egn. 3). I t is puzzling to think
of a reactive metal like silver as a reference electrode. However, potentiometrie experiments indicate that during the 2-3 hr equilibration period of the prahe in the buffer solution, a layer of AgnO andlor AgCl may be formed on the surface of the cathode. This probably leadsta a Ag/Ag,O and/or AgIAgC1combination which em function as a stable reference electrode (15, 16, 17). Such oxide andlor chloride layer formation will probably account for the large hackground currents during the equilibration process. The uniaue desien of the oxidase meter and the robe allows cur-
behavior of immobilized enzymes. Chemical and Electronic Amplification This experiment illustrates a classic example of the coupling of biochemical and electrochemical reaction systems. The net result should he high selectivity, sensitivity, and convenience, which is typical of hioelectrochemical techniques applied to the measurement of biochemical substances. The most important feature, however, is the signal enhancement derived by the immobilization of the enzyme. Glucose oxidase, localized in a very small volume hetween the two membranes on the prahe, contributes to the chemical amplification of the enzyme reaction. The Hz02 is produced very close to the electrode surface, which "sees" a higher concentration in spite of the slow diffusion of the products into the hulk solution md toward the electrode. This enhancement of the observed current from the oxidation of HzOz again undergoes electronic amplification by the oxidase meter. Thus, a significant chemical and electronic amplification of the signal is achieved without the necessity for catalytic cycling reactions (2).
Further studies A varietv"of suecial nroiects based on this exneriment can . be envisioned for interistkd students. We encokrage readers to devise their own uroiects. Outlines of a few possible . proiects . are mentioned below:. (1) Inhibitors to rlucose oxidase can be determined using this set up. For a s;ccessful determination of the enzyme/ inhibitor interaction, the reaction itself should be reversible. The procedure would, of course, involve a decrease in the enzvme probe resnonse with increasing inhibitor concentra. . tion in excess glucose concentration. Metal ions such as Ag+2,Hg+Z, and C U +reversibly ~ inhibit glucose oxidase and were studied in detail by Nakamura et al. (19);Rogers et al. (20) reported that D-glucal is a competitive inhihitor ~ - ~ -- - -- ~ of the enzvme. Thus. an exueriment based on the procedure described here, to measure these inhibitors (especiallv Hgf2 which is a notorious environmental toxin). . . would u he of special interest to imaginative students. (2) The initial reaction rates can be monitored by measuring currents a t a fixed time interval on the rapidly rising portion of the curve. This can he used to calculate apparent Michaelis Constants (K',) using Lineweaver-Burk plots (15). ~
~
~~
A
~
~
~
A comparison with the K , value of the soluble enzyme described in the literature would give some perspective on the extent of immobilization. I n addition, variation of apparent K, with p H would he useful in determining the optimum working pH of the enzyme probe. (3) The probe can he assembled easily into a flow-through cell to continuously monitor substrate or inhibitor concentration. (4) Other oxidase enzvmes can he "immobilized on the probe for the analysis of their substrates or inhibitors. For examnle. L-amino acid oxidase immobilized on the nrohe can he used to analyze a number of L-amino acids. (5) Finallv. interested students can build an oxidase meter
readers should contact t h e authoryto obtain a copy of the modified desirn. Acknowledgment Data made available by D. Root and L. Cunningham from the undergraduate instrumental analysis laboratory are gratefully appreciated. The authors also express their gratitude to W. T. Lippincott and Dorothy Fuller for valuable suggestions on the manuscript. This work was supported by a course development grant from the Department of Chemistry, University of Arizona, Tucson, AZ 85721. Literature Clted (1) Gray, D.N.,Keyea,M. H., and Watson,B.,Anai.Chem.,49(12), 1067A (1977). (21 Blaedel, W.J., and Baguslaski, R. J., A n d Chem., 50(81,1026(L978). G.G.. in '"Enzyme Electrodes & Solid Surface Fluorescence Methods: Methods in En2yrnoiogy. Vol. XVIV, Academic Pmss, N.Y. 1976, p. 578-633.
(3) Guilbadt,
Ault,A.,J.CHEM.EDUC.,51(6), 381 (1974). (5) Devine, J. E., and Toom, P. M.,J. CHEM. EDUC.. 52(12), 816 (1976). (6) Sp1ittgerber.A. G.,Mitchell, K.,Dahie,G.,Pufter. M.. andBlomquisl,K..J. CHEM. EDUC., S2(10), 680 (1975). (7) Dalne8,T.L..andMor?,K. W., J. CHEM EDUC., S3(2), 126 (19761. (8) Friedman, M. E., and Damn, H. H., J. CHEM. EDUC., 54(4),256 (1977). (9) Hurlbut, J. A., Kavianian, G. R., L. S. Y., Nuttal, K. L..Gentry. S. R., and Hsrsman. T. L., J. CHEM. EDUC.. 54(71,442 119771. (101 Boyer.R.F.,J. CHEM.EDUC.. 54(9).585 (1977). (11) Tay1or.R. P., Broccoli,A. V.,andGrishsm, C. M.,J. CHEM.EDUC., SS(11.63 11978). (4)
(121 D8Jong.P. J.,andKumler,P. L., J.C~~M.E~~c,Sl(3),200(1874). (131 Malm8tadt.H. V.,andPardue, H. L.,Anal. Chrm., 33(8).1040(1961). (14) "Instruction Manual fur Model 25 Oxidasp Meter and Model 2510 Oxidasp Probe.). Yellow Springs lnstrummt Co.. Yellow Springs, OH 45387. (15) Sittampalam, G., "Use of Coordinated Ligands and immobilized Enzymes as Elec~ trochemid Sensors: M. S. Thesls, 1977, Bowling Green State University. Bowling Green. OH 43404. (16) Sriniuasan, V. S., Department of Chemistry, Bowling Green StateUniversity. Bowling Green, OH 43403. Personal Communication. (17) Huntington, J., Yellow Springs Instrument Co.,Yellow Springa, OH 45387, Personal
Communicstjon.
(18) Kissinger, P. T., "Electrochemical Controllex for LCEC,"a bulletin from Bioan&ical Systems Inc.,P O . Box 2206. Weat Lafwatta.IN 47906. (19) Nsksmura, 8.. Ogura. Y., J. Biochrm. (Tokya),64,43911968). (20) Rwers, M. J., and Brandt. K. G.,BiochamiLiy, 10,4624 (1971). (211 Kamin, R. A , Department of Chemistry, University of ~ r i r o n a~, u m nAZ , 85721, Personal Communication.
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