1698
Anal. Chem. 1982, 54, 1698-1701
Microporous Membrane Flow Cell with Nonimmobilized Enzyme for Chemiluminescent Determination of Glucose David Piiosof and Timothy A. Meman" School of Chemical Sciences, Unlverslty of Illinois. Urbana, Illinois 6 180 I
A microporous membrane separates a reagent reservoir, containing glucose oxidase buffered at pH 5, from the anaiyte flow stream contalnlng glucose. The enzyme solutlon flows, under pressure, through the membrane at 3.5 pL/mln. Lumlnol, KOH, and Cu(l,lO-phen)c+ (1,lO-phen = 1,lOphenanthrollne) are added to the analyte 80 that the hydrogen peroxide produced by the enzymatic oxidation of glucose can be determined by chemllumlnescence. The membrane allows creation of a pH gradient In the flow cell; the solution Is around pH 5 near the membrane where the enzymatlc reactlon occurs and Is strongly bask In the bulk of the analyte solution where the chemllumlnescent reaction occurs. The membrane llmlts enzyme consumption to amounts that are mlnlscule In comparison to other glucose methods. The detectlon llmlt Is 5 X IO-' M. Preclslon Is 2-3% relatlve standard deviation (RSD). Serum samples were assayed (following deprotelnatlon); resutls correlate well with values obtained In a cllnlcal laboratory using a Beckman Glucose Analyzer 2.
The combination of high specificity and the ability to catalyze reactions of substrates at low concentrationshas made enzymes valuable tools in clinical analysis. Many biologically important substances are routinely determined by using enzymatic reactions in which a product is quantitized electrochemically or spectrometrically. Recently, several enzymatic glucose assay methods have been reported (1-4). As shown in eq 1,glucose is oxidized in the presence of molecular oxygen with glucose oxidase as a catalyst; hydrogen peroxide is produced. The enzymatically generated peroxide can be determined by several methods. Analytical chemiluminescence, the measurement of light produced by a chemical reaction, is particularly useful for the determination of peroxide because of its high sensitivity, its extended dynamic range, and the simple and inexpensive instrumentation required. An extensive review of the chemiluminescent determination of enzymatically generated hydrogen peroxide has recently been published by Seitz (5). The most widely used chemiluminescent reagent is luminol (3aminophthalhydrazide), which reacts with Hz02in the presence of a metal catalyst to produce light (eq 2). p-D-glucose
+ 02
glucose oxidase
PH5
D-gluconic acid
+ H202 (1)
luminol
+ H202
metal
3-aminophthalic acid
+ hv
(2)
As seen in eq 1and eq 2, one of the most severe problems encountered in coupling the enzymatic oxidation of glucose and the chemiluminescent reaction of luminol with peroxide is the very different pH optima required by the two processes. Work done at a compromised intermediate pH (e.g., around 8) does not yield successful results (5), consequently it has been held that the two reactions had to be developed under 0003-2700/82/0354-1698$01.25/0
separate conditions, each one appropriate for the specific reaction. Maloy and co-workers (6) suggested a method based on a "pH jump" from a value at which the enzymatic reaction proceeds momentarily to a more basic pH required by the luminol reaction. Quantitation of glucose was obtained after integration of the chemiluminescent signal over a period of several minutes. Bostick and Hercules (7,8)proposed a flow system that used a column containing immobilized glucose oxidase. The glucose sample was passed through the column and the generated peroxide was mixed downstream with a reagent solution containing luminol, base, and a metal catalyst, producing chemiluminescence measured wiith a photomultiplier tube placed in front of the flow cell. Seitz and coworkers (9) later used similar instrumentation for determination of glucose in urine. Other methods for chemiluminescent determination of glucose were proposed by Seitz and co-workers (10) using peroxyoxalate chemiluminescence in nonaqueous medium and by Veazey and Nieman (II), who described the determination of glucose and other reducing sugars with lucigenin chemiluminescence. The use of microporous membrane flow cells in chemiluminescent analysis was described earlier (12, 13). The instrumentation is schematically shown in Figure 1. In this method a reagent solution is forced by a pressure gradient to flow through a microporous membrane into a compartment in which the analyte is flowing. The reaction between analyte and reagent occurs on the analyte side of membrane close to the surface of the membrane (13). Since the reagent flow rate through the membrane is only a few microliters per minute and the analyte flow rate is on the order of 10 mL/min, there is effectively 3 orders of magnitude dilution of the reagent into the analyte flow stream. This approach provides several analytical advantages for applications in continuous flow: (a) the sample integrity is maintained and contamination of the sample by the reagent is minimal; (b) the volume of reagent used is only a few microliters per assay; this fact is of extreme importance when using enzyme solutions; (c) the light emission is localized close to the microporous membrane (13); (d) it is possible to obtain a pH gradient perpendicular to the membrane so that reactions requiring different pH conditions, as in this case, can be done simultaneously in the same flow cell; (e) the mixing of reagent and analyte occurs rapidly under controlled conditions without the need to stir the solution; (f) the instrumentation is simple and inexpensive. This paper describes the determination of glucose with microporous membrane chemiluminescence flow cells. The method provides a simple and economical way to utilize enzymes in flow streams without resorting to lengthy and complicated immobilization techniques (14,15).A pH gradient is formed in the flow cell and the solution is around pH 5 close to the membrane where the enzymatic reaction occurs arid is strongly basic in the bulk of the analyte solution where the chemiluminescent reaction occurs. This appproach circumvents the necessity to carry out both reactions under different sets of conditions, as suggested earlier. Additionally, the detection limit obtained for glucose and the good correlation 0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982 AN ALY T E rnL/rnin
RINSE+
GLUCOSE OXIDASE
L-J
REAGENT-$*- pL/rnin
'
I -T
0
GLUCOSE J+ 0,5 M KOH 2 I O - ~ MLUMINOL O,I M KHP pn=5,0 2 x IO+M CU(PHEN), (2mg/m~)
6-10
1699
i
2
-+
-I+ I
0
I MEMBRANE CHEMII-UMINESCENCE FLOW SYSTEM
Flgure 1. Microporous membrane chemiluminescence flow system.
with human blood serum assays done by a clinically accepted procedure, make the method attractive for routine analysis of glucose in blood. The work described in this paper dealt with the use of glucose oxidase because of its low price, its stability in solution, and its ease of manipulation. The method can be, and is being, extended to other more complicated or more expensive oxidase enzyme systems.
Flgure 2. Reaction conditions. The left sue represents the pressurized reagent chamber and the right side is the analyte flow stream.
KOH, and C ~ ( p h e n )to ~ ~the + fiial concentrationsshown in Figure 2. Altogether the serum samples were diluted 1000-fold.
RESULTS AND DISCUSSION Reagent Composition. The solution composition shown in Figure 2 was devised by taking into consideration important chemical limitations. As explained earlier, our goal was to create a pH gradient so that the enzymatic reaction would occur a t a low pH close to the membrane, and the peroxide EXPERIMENTAL SECTION would subsequently diffuse into the bulk of the flow stream Apparatus. The flow system shown in Figure 1 consists of containing luminol and a metal catalyst, where the strong a 150-pL flow cell separated from a 1-mL reagent reservoir by medium permits the production of chemiluminescence. a piece of Celgard 5511 (CelaneseCorp.) microporous membrane. The biphthalate buffer, at a relatively high concentration, The membrane is made of polypropylene coated with a nonionic maintains the desired pH for the glucose oxidase reaction close surfactant to achieve wettability. This membrane has a pore size to the membrane. The buffering effect is so strong that the of 0.04 pm and an effective molecular weight cutoff of 200 000. base concentration had to be raised to 0.5 M in order to obtain The reagent reservoir was pressurized to approximately6 psi using significant chemiluminescent signals, normally produced a regulated tank of compressed air. The flow rate of the enzyme around pH 11. solution through the membrane was typically 3.5 pL/min and Generally a free metal cation is used as a catalyst in the depends on the applied pressure. The membrane area was 0.92 cm2. luminol reaction. However, at such a high base concentration A Rheodyne 5020 sample injection valve was used to inject it was obvious that only a complex with a high formation 1.0-mL aliquots of the analyte solution. Between injections, a constant would remain in solution. Other studies have pro0.9% NaCl solution was allowed to flow to rinse the flow cell. posed the use of K3Fe(CN)G(7,8),but this catalyst is inconAnalyte and rinse solutions were delivered at 10 mL/min using venient since it produces a chemiluminescent background even a peristaltic pump (Rainin Instrument Co.). without peroxide (5);ferricyanide probably reacts with disThe light produced in the flow cell was measured with a 1P28A solved oxygen in solution. Among several 1,lO-phenanthroline photomultiplier operated at -875 V and placed directly in front complexes tested in our laboratory, the copper complex gave of the cell. The PMT anlode current was amplified by a Pacific the largest signal and the fastest response and produced little Precision Model 126 photometer and output to a recorder. Reagents. Glucose oxidase (E.C. 1.1.3.4) was obtained from light in the absence of peroxide. All these properties suggested Sigma Chemical Co. as type VI1 with a specific activity of 134000 that C ~ ( p h e n ) was ~ ~ +the best choice as a metal catalyst. units/g. An enzyme reagent solution containing 2 mg/mL of Preliminary Studies. The initial investigations were foglucose oxidase was prepared by dissolving the enzyme in a 0.1 cused on testing the flow of glucose oxidase through the pores M potassium hydrogen ]phthalate buffer at pH 5. The solid of the microporous membrane. Solutions containing glucose enzyme was added within minutes before use to avoid decomconcentrations in the range 10-5-10-3 M and 0.2 M KI were position, even though no substantial loss in enzyme activity was injected on the analyte side, while the reagent reservoir, observed many hours after dissolution. containing 2 mg/mL glucose oxidase in 0.1 M biphthalate Luminol (Aldrich),glucose (Mallinckrodt),1,lO-phenanthroline buffer at pH 5, was pressurized at 6 psi with compressed air. (Baker), trichloroacetic acid (Mallinckrodt) and other chemicals were reagent grade and were used without further purification. The hydrogen peroxide, generated by the enzymatic oxidation All solutions were prepared with distilled deionized water. of glucose, oxidized the iodide in solution, forming a yellow The complex of copper with 1,lO-phenanthroline(C~(phen)~~+) species, 13-, indicating that the enzyme could indeed flow was formed by mixing sufficient copper nitrate and 1 , l O through the membrane pores into the analyte flow stream. phenanthroline to make the total concentrations 2 X M and The yellow color developed only after a few minutes due to 2 X 10" M, respectively. Because the overall formation constant the slow kinetics of the H202/I-reaction. From the chemical for Cu(phen)32+is 2 X laz1(16) and because there is an excess point of view this test was less demanding than using chemof 1,lO-phenanthroline, it is reasonable to assume that all the iluminescence for the detection of peroxide, since the latter copper is complexed. requires high base levels. Additionally, with the iodide test, Serum samples were olbtained from the clinical laboratory of Mercy Hospital, TJrbana, IL. one had the flexibility of being able to wait long times to Glucose standards were prepared by dilution of a 100 mg/L observe 13- production in the waste rather than being limited stock solution. The diluted standards also contained 2 X lo4 M to short time observation in the flow cell. luminol, 0.5 M KOH, and 2 X 10" M C ~ ( p h e n ) ~The ~ + .serum The coupling of the glucose enzymatic oxidation and the samples were diluted 1OOO-fold into a solution containing luminol, chemiluminescent detection of peroxide was then investigted KOH, and Cu(~hen),~+ exactly as the glucose standards. by using 5 X lo4 M glucose, M KOH, and all other Procedures. Serum samples were preserved by adding 5 concentrations as indicated in Figure 2. At this point, no mg/mL of NaF to prevent.glycolysis. To deproteinate the serum, chemiluminescent emission was detected, and the pH of 500-pL portions of the serum samples were added to 5 mL of 4% samples collected downstream of the flow cells was measured trichloroacetic acid and the mixtures were diluted to 10 mL and to be 6.5. This fact indicated that the buffering effect of the centrifuged for several minutes (17). A 2-mL aliquot of the supernatant centrifugatewas then diluted to 100 mL with luminol, biphthalate from the reagent solution was still significant in
1700
ANALYTICAL CHEMISTRY, VOL. 54, NO.
11, SEPTEMBER 1982 GLUCOSE 5 x 1 6M ~ RSD 3,O %
Table I. Comparison of Results Obtained by the Chemiluminescent Procedure and Those Obtained with a Beckman Glucose Analyzer 2 sample
chemiluminescent, mg/dL
Beckman Glucose Analyzer, mg/dL
76
78 180 110
1 2 3 4 5 6 7 8
176 122 108 99
89
65
61
197 188
203 198
spite of the large volume of M KOH present on the analyte side. Obviously this low pH value did not permit the formation of chemiluminescence. Light signals were observed thereafter by increasing the base concentration on the analyte side up to 0.5 M, which was kept as the optimal value for the rest of the studies. Analytical Performance. The chemiluminescent signals were measured as the height of the peaks obtained after glucose injection. The rise time of the peaks was about 1 s and the peak width at half-height was 8-10 s. The signal decayed back to the base line in 20-30 s, and successive injections could be made at that time. The time duration of the peaks is determined by the time for the sample to fill and be flushed from the cell. The half-life for the enzymatic reaction is about 0.5 ms at pH 5 (18). Because the enzymatic reaction is much faster than the solution residence time in the cell, it is probable that essentially all of the injected glucose is converted to hydrogen peroxide. A working curve was obtained with the reagent concentrations indicated in Figure 2 and with aqueous standards containing glucose concentrations in the range of 1 X M to 2 X M. A log-log plot of chemiluminescent intensity vs. glucose concentration shows good linearity (correlation M to 2 X coefficient = 0.9993) over the range 2 X M. Below 2 X M the line curves to asymptotically approach the base line. The least-squares estimates (fstandard deviation) of the parameters for the linear section of the working curve are slope = 0.94 f 0.02 and intercept = 5.9 f 0.1. The value for the slope is in agreement with previous work using copper(I1) and luminol to determine hydrogen peroxide in which slopes from 0.94 to 1.33 were measured and depended on the ratio of the concentrations of Cu(I1) and luminol (19). The detection limit (signal to noise ratio = 2) for our method is 5 X M glucose. This experimental detection limit is 5 orders of magnitude lower than the normal glucose levels in blood serum of 60-110 mg/dL or approximately 5 X M (20). The measurement precision was determined by replicate M glucose, shown in Figure 3. The injections of 5 X relative standard deviation, 3.0%, was slightly higher than the value obtained at higher glucose concentrations. It should be noted that 5 X lo-' M is fairly close to the detection limit of the method. This evaluation was repeated on several different days; the relative standard deviation always fell in the range of 2.1-3.0%. Table 11. Comparison of Enzymatic Methods for Glucose glucose analyzer detection limit, M 6X precision, % 2 enzyme per assay, IU 140 time per assay, s 60 a
Immobilized.
,2 nA
106
Flgure 3. Precision for replicate injections of 5 X
lo-'
M
glucose.
Assay of Clinical Samples. The applicability of the present method in real sample determinations was tested by assaying blood serum samples previously determined for glucose in a clinical laboratory. It was initially believed that in view of the high sensitivity of our method it could be possible to dilute the serum sample 1000-fold and virtually eliminate any matrix interference that might affect the chemiluminescent reaction. When chemiluminescence was measured for the diluted serum samples without previous deproteination,the peaks obtained were lower and took longer to return to base line and were more irreproducible than those of the glucose standards. The addition of known amounts of glucose to the serum samples did not result in sharper and more measurable peaks as expected, indicating severe interference from the serum matrix, even after being diluted by 3 orders of magnitude. A second set of serum samples was deproteinated prior to the assay. The serum samples were still diluted 1000-fold and were compared against glucose standards that contained the same amount of trichloroacetic acid. The glucose standard working curve was constructed over the range 50-250 pg/dL, to facilitate straight calculations compared to the clinical values expressed in mg/hL. After deproteination the peak shapes obtained were identical with these of the glucose standards, suggesting that the main contributor to the matrix interference is the serum protein. This observation is not unusual in clinical methods. Signals obtained for glucose solutions with and without trichloroacetic acid were essentially the same. The serum samples were assayed in triplicate by the chemiluminescent procedure and were also assayed with a Beckman Glucose Analyzer 2. This procedure is based on the glucose oxidase reaction as well but measures oxygen depletion rather than peroxide formation. Table I gives the results for eight serum samples. On the average, the two methods agree within 2%, and in no case do the two methods yield values differing by more than 11%. A t value of 0.28 can be calculated by using the difference between each of the paired
PH jump CL method 6 X low6 2 60 300
enzyme column CL method 2x 0.5 103Q 150
membrane CL method 5 x 10-8 3 0.85 30
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
measurements. The table value of t for 7 degrees of freedom and 95% confidence is 2.365. Therefore, at the 95% confidence level there is no difference between the values obtained by the two methods compared (21). Comparison to Otheir Methods. Table I1 summarizes the performance of the miciroporous membrane chemiluminescence method for glucose as compared to the “pH jump” method reported by Maloy (6),the immobilized enzyme column method reported by Bostick and Hercules (7,8),and the method used by the Beckman Glucose Analyzer 2 (22). Our membrane chemiluminescence technique is advantageous in terms of sensitivity, time required per assay, and enzyme consumption. As a result of the low detection limits, the method would be advantgeous for samples containing glucose at concentrations much below that found in blood serum or for applications where only limited amounts of sample are available and dilution is desirable. Because the measurement time is short and the quantitation is accomplished in a flowing stream, the method is amenable to1 automation and routine analysis. Although deproteination was necessary for blood serum samples, this is not a serious limitation since the trichloroacetic acid method is as simple as the centrifugation required to separate the serum from the whole blood. Furthermore, other assays done in blood serum require protein separation as well; protein removal columns can be routinely incorporated into automated instrumentation (23). The most significant advantage of the microporous membrane chemiluminescence method is the minimal consumption of enzyme. We have reduced the enzyme concentration by a factor of 25 below that reported in Table I1 and used for the bulk of this work (with all other reagent concentrations constant) and found no change in the chemiluminescent signal for 100 mg/dL samples of glucose. This result indicates that it is possible to reduce the enzyme consumption to amounts that are miniscule in coinparisonto previous methods without affecting the performance of the assay. Although it is normally true that immobilization reduces the long term reagent cost and improves the stabiliity of the enzymes, it is less recognized that the immobilization techniques are rather elaborate and time-consuming and often cause a decrease of enzyme activity. Commercially available immobilized enzymes are much more
1701
expensive than the equivalent amount of nonimmobilized enzyme units. The system proposed overcomes the need to immobilize the enzymes and suggests a simple and economical reagent addition method that could also be applied to flow injection analysis and continuous flow techniques where the use of expensive reagent solutions is required.
ACKNOWLEDGMENT We thank Gary Jones of the Mercy Hospital laboratory for kindly supplying the serum samples and Celanese Corp. for providing the membranes used in this study. LITERATURE CITED Kelly, T. A.; Christian, G. D. Anal. Chem 1981, 53, 2110-2114. Gullbault, G. G.; Lubrano, G. T. Anal. Chlm. Acta 1973, 6 4 , 439-455. Updlke, S.J.; Hicks, G. P. Nature (London) 1967, 2 7 4 , 986-988. Thevenot, D. R.; Coulet, P. R.; Sternberg, R.; Laurnet, J.; Gautheron, C. Anal. Chem. 1979, 57, 96-100. Seitz, W. R. I n “Methods in Enzymology”; Deluca, M. A,, Ed.; Academic Press: New York, 1978;Vol. LVII, Chapter 38. Auses, J. P.; Cook, S. L.; Maloy, J. T. Anal. Chem. 1975, 4 7 ,
244-249. Bostlck, D. T.; Hercules, D. M. Anal. Left. 1974, 7 , 347-353. Bostlck, D. T.; Hercules, D. M. Anal. Chem. 1975, 478 447-452. Wllllams, D. C.; Huff, G. F.; Seitz, W. R. Clln. Chem. (Winston-Salem, N . C . ) 1976, 2 2 , 372-374. Williams, D. C.; Huff, G. F.; Seitz, W. R. Anal. Chem. 1976, 48,
1003-1006. Veazey, R. L.; Nleman, T. A. Anal. Chem. 1979, 57, 2092-2096. Nau, V. J.; Nleman, T. A. Anal. Chem. 1979, 57, 424-428, Pilosof, D.; Niemam, T. A. Anal. Chem. 1980, 5 2 , 662-666. Weetall, H. H. Anal. Chem. 1974, 46, 602A-615A. Bowers, L. D.; Carr, P. W. Anal. Chem. 1976, 48, 545A-559A. Inczedy, J. “Analytical Applictions of Complex Equilibria”; Halsted Press: New York, 1976;pp 356-357. Malmstadt, H. V.; Hadjiioannou, T. P. Clln. Chem. (Wlnston-Salem, N.C.) 1959, 5 , 50. Qlbson, Q. H.; Swoboda, B. E. P.; Massey, V. J. Biol. Chem. 1964, 2 3 9 , 3927-3932. Armstrong, W. A., Humphreys, W. G. Can. J. Chem. 1965, 4 3 ,
2576-2584. Diem, K., Lentner, C., Eds. “Sclentific Tables”; Ciba-Giegy: Ardsley, N.Y., 1970;pp 604-605. Christian, G. D. “Analytical Chemistry”, 3rd ed.; Wlley: New York, lg80;pp 70-77. Data sheets for Beckman Glucose Analyzer 2, Irvine, CA, 1981. Data sheets for DuPont aca, Wllmlngton, DE, 1981.
RECEIVED for review February 12, 1982. Accepted May 13, 1982. This work was supported in part by the National Science Foundation (Grant No. CHE-81-08816).
