(15) C. E. Harvey, Spectrochim. Acta, Part B, 25, 73 (1970). (16) H. Kaiser, Spectrochim. Acta, 2, 1 (1941).
(10) R. J. Decker and D. J. Eve, Spectrochim. Acta, Part 6, 25, 479 (1970). (11) C. E. Harvey, "Spectrochernical Procedures." Applied Research Laboratories,Glendale, Calif., 1950. (12) J. R. Churchill. lnd. Eng. Chem., Anal. Ed., 16, 653 (1944). (13) "Methods For Emission Spectrochemical Analysis," 3rd ed., American Society for Testing Materials, Philadelphia, Pa., 1960. (14) R . Gerbatsch and H. Scholze, Spectrochim. Acta, Part E, 25, 101 (1970).
RECEIVEDfor review August 2, 1974. Accepted November 11, 1974* This work was supported in part by Science Foundation Grant, GP-37026X.
Quantitative Determination of Blood Glucose Using Enzyme induced Chemiluminescence of Luminsl Debra T. Bostick and David M. Hercules Department of Chemistry, University of Georgia, Athens, Ga. 30602
Blood glucose is measured using enzymatic conversion of P-D-glucose to D-gluconic acid and hydrogen peroxide in an immobilized glucose oxidase (EC 1.1.3.4) column. The peroxide subsequently reacts with a mixed luminol-ferricyanide reagent to produce chemiluminescene, proportional to P-D-glucose concentration. The method is linear between lo-' and 10-4M glucose, and correlates well with standard methods for glucose determination. With prior adsorption of uric acid, the chemiluminescent technique may be used for urine glucose analysis. The system may also be applied to the analysis of hydrogen peroxide in the lo-* to lO+M range.
Estimation of true blood glucose has been hampered by the relative nonspecificity of most analytical techniques. Glucose analysis based on the inherent specifity of an enzymatic reaction has provided the most accurate means for obtaining blood glucose concentration. T h e glucose oxidase method, originally described by Keston ( I ) , is the most commoniy employed enzymatic t,echnique for routine blood glucose analysis. T h e method i s based on the following reaction sequence: [3-o-glucose H,02
+-
+
O2
glucose oxidase - _ _ f
o-gluconic acid
chromogenic oxygen acceptor
+
H,O,
peroxidase
chromogen
in which the chromogen most frequently is 0-dianisidine or 0-toluidine. T h e first reaction is highly specific for glucose (2); however, the second reaction is subject to several interferences. These include reducing substances, such as bilirubin, ascorbic acid, uric acid, and drug metabolites, which may depress results by either competing with the chromogen for peroxide or by reducing the chromogen ( 3 ) .Negative error may also be observed if the p H is too acidic for the enzymatic reactions. Under these conditions, peroxidase is inhibited by fluoride and chloride ions which may be present in the reaction media as serum preservatives ( 4 1. Glucose estimation may he p H dependent if the p H of the final solution remains above four, since the absorption maximum of the oxidized chromogen is p H dependent above this value ( 5 ) . To circumvent many of the interferences associated with the peroxidase-coupled glucose oxidase method, the present technique monitors hydrogen peroxide concentration using the chemiluminescence of luminol (5-amino-2,3-dihy-
drophthalazine-1,4-dione).In the presence of certain metals, peroxide reacts with luminol in basic media to form an excited aminophthalate anion, which returns to ground state by the emission of a photon (6, 7). In the glucose oxidase-luminol coupled reaction sequence, the amount of light emitted is proportional to @-D-glucoseconcentration. Generally, metal ions possessing oxidation states requiring a one-electron transfer are capable of promoting the chemiluminescent reaction between peroxide and luminol in water (8). These have included Fe(I1)-containing compounds, such as hemin (9, 1 0 ) and hematogen ( I 1 ). Copper(I1) (12-16), as well as mixed Cu(I1)-persulfate ( 1 7 )and Cu(T1)-hemin (18) solutions, have also been employed in the luminol reaction. Cobalt(I1) (19). Fe(II1) ( 2 0 ) , Fe(CN)e3- (21, 2 2 ) and SbC16- ( 2 3 ) have been cited as reagents capable of producing chemiluminescence in the presence of luminol and hydrogen peroxide. T h e present paper summarizes the chemiluminescent response promoted by several of the above metals, observed during attempts to establish a procedure for peroxide analysis based on the luminol reaction. I t further describes the adaptation and development of this analysis for the determination of blood glucose. A preliminary communication of this work has appeared ( 2 4 ) .Other workers have independently reported a similar method (25 ).
EXPERIMENTAL Apparatus. The chemiluminescence produced by the oxidation of luminol is followed in a continuous flow system using the apparatus shown in Figure 1. The system uses three 50-ml plastic syringes, containing luminol dissolved. in 0.1M H3B03-KOH buffer. KaFeiCN)e, or another metal, and an aqueous background of 0.004M acetate buffer. The syringes are driven by a Harvard Model 600-2-200 infusion pump, .capable of maintaining uniform flow against back pressures greater than 250 psi produced by the enzyme column. Solutions from the ferricyanide and luminol syringes are joined by a glass Y-tube containing a platinum coil t o enhance mixing. A platinum gauze plug is located father down the flow line for the same purpose. Samples are introduced into the acetate flow line by a Chromatronix SV-8031 sample injection valve. Either the acetate background or the same slug flows into the glucose oxidase column. The column itself is a 16-cm Pyrex tube with an i.d. of 4 mm; 2 cm from each end of the column, the i.d. is decreased to 3 mm to accommodate Chromatronix column fittings. As the glucose sample enters the column, hydrogen peroxide generated and carried in the column effluent to the cell where it reacts with the luminol-ferricyanide reagent. Nitrogen gas is bubbled through the cell to ensure uniform mixing. The chemiluminescence produced by the luminol-peroxide-ferricyanide reaction is detected by an RCA 1P21 photomultiplier ANALYTICAL CHEMISTRY, VOL. 47,
NO. 3,
M A R C H 1975
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to the diluted serum and vortexed. The mixture was allowed t o stand for 10 minutes before being centrifuged. Two ml of the centrifugate were diluted to 100 ml wit,h 0.004M acetate buffer. The prepared samples were then introduced into the flow system and the chemiluminescent response recorded.
9ACL(GROUND SAMPLE
RESULTS AND DISCUSSION
LNITROGEN K 3 Fe(CNj6
Figure 1. Diagram of the flow system for the chemiluminescent determination of blood glucose
tube attached to the face of the cell. The signal is amplified by a Princeton Applied Research (PAR) Model 270 D.C. photometerpreamplifier in conjunction with. a PAR Model 280 power supply. The response is recorded on a Hewlett-Packard Model 7101B potentiometric recorder. The effect of column temperature on chemiluminescent response was determined by submerging the enzyme column, sample loop, samples and 2-meter X '&inch background equilibration coil in a ten-gallon water bath. The water temperature was controlled to within 40.1 "C by a Yellow Springs Instrument Co., YSI-B3RC, temperature regulator and a 150-watt light bulb. Chemicals. Luminol (Eastman Kodak) was converted to the sodium salt with reagent grade sodium hydroxide. The salt was twice purified by double recrystallization from basic aqueous solution at 0 O C (26). A 4 X M luminol stock solution was prepared by dissolving 0.8 g luminol salt, 73 g potassium hydroxide, and 61.8 g boric acid in one liter of water. The luminol stock solution was allowed to stand for three days to stabilize before use. Metal solutions were prepared from reagent grade salts. Hydrogen peroxide standards were prepared by diluting a 3% H202 stock solution (Baker) which was previously standardized against standard permanganate; sodium oxalate (Mallinckrodt) was the primary standard (27). The diluted peroxide standards were maintained at pH 4.5 to minimize decomposition (28). The peroxide standards were prepared daily since dilute solutions are stable for only 24 hours at room temperature ( 1 4 ) . Sigma Chemical Company standard glucose solutions, 10 mg/ml and 1 mg/ml, were diluted to prepare glucose standards. Glucose oxidase immobilized on Sepharose beads was purchased from Worthington Biochemical Corporation. The immobilized enzyme contains approximately 50 mg of active enzyme, corresponding to about 103 units. Soluble glucose oxidase has a broad pH profile with a reported optimum at pH 5.6 (29). Therefore, the 0.004M acet,ate background was prepared to maintain the enzyme column at pH 5.6 (30 ). Blind stahlized control sera were obtajned from the Center for Disease Control (CDC),Atlantq, Ga. for the correlation study. The samples are pooled calves sera which have been preserved by millipore filtration to remove bacteria and by addition of NaF to prevent glycolysis. The samples are reported to be stable for years when stored at room temperature. The control sera had previously been characterized by the glucose oxidase, Somogi-Nelson, o- toluidine, and ferricyanide methods for glucose analysis. Sigma Glucose Kits and Worthington Glucostat Kits were purchased from Sigma Chemical Co. and Worthingtoq Biochemicals, respectively. The effect of uric acid (2,6,8-trioxypurine)on the chemiluminescent reaction was observed by adding weighed quantities of potassium urate (Sigma) to glucose standards. All of the above reager:ts were prepared using water from a Continential Water Conditioning Company deionization system. The effect,of reaction pH on chemiluminescent response was studied by adjusting the pH of 2 X 10-4M luminol, dissolved in the H:$O:{-KOH buffer, with either 4M HCI or 1M NaOH before adjusting the working luminol reagent to its final volume. Luminol and metal concentration profiles were' accomplished by loading the respective syringes with the desired concent,ration of the reagents. Peak height was then observed with a fixed H202 concentration. The CUC' serum samples were prepared for the correlation study by measuring 100 pl of either serum or standard by an automatic pipet into 1.9 ml of water. Two ml of the Somogi filtrate, containing 1 ml of 1.8%Ra(OHI2and 1 ml of 2% %So4 (31), were added 448
.
T h e development of the chemiluminescent glucose technique was divided into two stages. T h e goal of the first stage was to establish and optimize the quantitative aspects of the peroxide-luminol reaction. For this study, t h e enzyme column was absent from the flow line and hydrogen peroxide standards were introduced into t h e flow system. Selection of the Chemiluminescent System. T h e reaction of luminol in the presence of Cu(II), Co(II), Ni(II), and SzOs2- was investigated. Initial studies with Cu(I1) were done in the presence of 0.1M ammonia since previous literature reported t h a t copper required NH3 for linearity (8). A t a reaction p H of 10.5 and 4 X 10-4M luminol, the chemiluminescent response (peak height) was observed in the range 10-6-10-2M Cu(I1). T h e response was that of mixed kinetic order with peroxide concentration. Armstrong and H u m p h r e y (14 ) suggested that linearity could be achieved by equating peak area rather than peak height with peroxide concentration. However, our results did not support this conclusion. Because t h e highest sensitivity, without precipitate formation, occurred at 10-3M Cu(II), this concentration was used for the remaining studies involving copper. Attempts were made t o determine the parameters governing the kinetics of the luminol-peroxide-Cu(I1) response. Flow rate and the presence of NH? had no effect on reaction kinetics; however, t h e reaction p H decidedly controlled the first-order kinetics of the chemiluminescent reaction. Between p H 7.5 and 12.3, the reaction became increasingly linear with peroxide concentration as the p H was increased. At p H 12, a linear least squares analysis of the H202 calibration curve gave a sinusoidal change in the sign of the Y-residuals; however, their absolute magnitude was less than the error incurred in measuring peak height. Therefore, peroxide analysis based on a luminol- CutII) reaction may be performed on samples over a small concentration range. However, analysis of peroxide samples with concentrations varying 6ver several orders of magnitude could produce significant error due to the slight curvature in the standard curve. 'In addition, a more suitable luminolmetal reaction was desirable since the high pH, required for linearity, causes the formation of Cu(OH)>precipitates which give spurious fluctuations of chemiluminescent intensity. T h e luminol-peroxide reaction in the presence of Co(1I) was observed t o be twenty times more sensitive to peroxide than with Cu(I1) at p H 10.5. However, the maximum concentration of Co(I1) that could be used was below 10-4M because of precipitate formation. T h e chemiluminescent response was characterized by extraneous side peaks just following t h e major peak. T h e peaks were not reproducible between runs and had significant noise at the tops of the peaks. T h e luminol-Ni(I1) reaction gave a response that resembled a step function. With increasing peroxide concentration, chemiluminescene intensity assumed one value before shifting to a second value with more concentrated peroxide samples. T h e peroxide concentration at which t h e shift in response occurred was not reproducible. Neither flow rate nor p H affected this behavior. Nickel(I1) also could not be used in concentrations greater than 10-4M because of precipitate formation.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, MARCH 1975
P
I
l a
P
d
Figure 4. Figure 2.
chemiluminescence intensity vs. K3Fe(CN),jconcentration
Conditions: 9.6 X lO-'M ml/min/syringe
H 2 0 p ; 2 X 10-4M luminol; pH 10.8; flow rate. 4.41
Chemiluminescence intensity vs. luminol concentration
Conditions: 9.6 X lO-'M ml/rnin/syringe
H202; 10-'M
K3Fe(CN)& pH 10.4; flow rate, 4.41
160
3
*
L
r ^..
i
C
'
- r d
^ j
I_
Figure 3.
Chemiluminescence intensity vs reaction pH oi-_Ip,-L.
Conditions 9 6 X lO-'M H 2 0 2 , 10-*M K ? F ~ S ( C N2) ~ X, 10-4M luminol, flow rate 4 41 ml/min/syringe
1
2
3
.
4
FLOW
T o eliminate problems encountered with metal hydroxide precipitates. the luminol-persulfate reaction was investigated. A t 10-:IM K:&O8, the sensitivity was observed to double between p H 10-11; however, the noise also increased significantly. In addition, persulfate gave irreproducible peaks and had a very unstable background. Ferricyanide gave linear chemiluminescent response, had good sensitivity. and had soluble reaction products. Therefore, the ferricyanide svstem was adopted for the chemiluminescent determination of glucose.
Optimization of the Luminol-Ferricyanide-Peroxide System. T h e sensitivity of the luminol reaction was markedly affected by ferricyanide concentration. Holding pH, flow rate, luminol and peroxide concentrations constant, a profile of chemiluminescent intensity us. ferricyanide concentration was obtained as shown in Figure 2 . Maximum light, emission occurred a t 10-2M ferricyanide; therefore this concentration was adopted for the method. Because the kinetics of chemiluminescent reactions are pH dependent, a p H profile was prepared as shown in Figure 3 . A h e a r least squares analysis of the peroxide calibratinn curve was performed a t each pH. From this analysis, the reaction order was observed to remain first order throughout the p H range studied. T h e chemiluminescent response remained essentially constant between p H 10.410.8; a reaction p H of 10.5 was used in further experiments. Of all factors studied, luminol concentration had the
5
L
6
A
-
7
8
9
RATE
~mls/min/syringeI
Figure 5.
Chemiluminescence intensity vs. flow rate
(0)Data collected with a Harvard Model 600-2-200 infusion pump. (A)Data collected with a Harvard Model 975 infusion pump. Conditions: 9.6 X lO-'M H202, 10-*M K$Fe(CN)e. 2 X 10-4M lurninol, pH 10.6
most significant effect on reproducibility. In the range 5 X 10-"-3 X 10-2M luminol, the relative peak height a p proached a limit only a t high luminol concentrations. This can be seen in Figure 4.At luminol concentrations greater than ca. lOP3M, a significant increase in noise was seen. T h e optimum signal-to-noise ratio occurred a t ca. 2 X 10-4M luminol concentration which was adopted for the method. Therefore, the method could be made more sensitive to peroxide concentration, but only a t the sacrifice of precision. Relative light emission increased with increasing flow rate as seen in Figure 5 . Two different pumps were used to collect the data. The triangular points represent data collected from the pump used for the peroxide studies. T h e circular points were obtained with a more powerful pump which was required to pump against the back pressure produced when the enzyme column was in the flow line. The increasing sensitivity suggests that the chemiluminescent reaction is going to completion within the cell; a t higher flow rates, more light is emitted per unit time, resulting in
ANALYTICAL C H E M I S T R Y , VOL. 47, NO. 3 , M A R C H 1975
449
Table I. Correlation Study between CDC Reference and the Chemiluminescent Values for Six Control Samples Average CL
Sample
value, m q / d l
Std dev in CL
value, m g i d l Q
CDC control value, m d d h
1 42.8 1.45 42.0 2 79.6 1.72 78.0 3 79.8 1.66 81.0 4 106.4 1.26 110.0 5 136.1 3.89 135.8 6 212.4 2.31 210.0 a Based on four replicates. * Based on the glucose oxidase technique. -7
-6
-5
-4
LOG [GLUCOSE]
Figure 6. Calibration curve for glucose determination Conditions: 10-*M K3Fe(CN)6, 2 X 10-4M luminol, cell pH 10.3, column pH 5.6, t = 22 OC;flow rate, 2.76 ml/min/syringe. aqueous glucose standards were used
250
200
,
I
150
IOmin
I
Figure 7. Chemiluminescence response of a glucose standard run Numbers above duplicate peaks are glucose concentration (mg/dl); 10-*M K3Fe(CN)6,2 X 10-4M luminol: cell pH 10.5,column pH 5.6; flow rate, 2.76 ml/min/syringe, aqueous glucose standards were used
greater peak height ( 3 2 ) .A flow rate of 4.41 ml/min/syringe was arbitrarily chosen for the peroxide studies although a flow rate of 2.76 ml/min/syringe was the fastest that could be obtained once the glucose oxidase column was placed in the system. With conditions optimized for the luminol-peroxide-ferricyanide system, the linear range for peroxide was 10-5M. T h e limit of detection was 7 X 10-9M peroxide a t a signal-to-noise ratio of 2. T h e lower limit of detection is imposed by background light emission. T h e reproducibility in peak height was determined by measuring ten replicates of an 8 X lO-7M peroxide sample; the relative standard deviation was f0.7%. The Chemiluminescent Determination of Glucose. Once the parameters for the chemiluminescent determination of peroxide were optimized, the glucose oxidase column was placed in the flow line. Using glucose standards in the 10-7M range, the observed chemiluminescent intensity 450
ANALYTICAL C H E M I S T R Y , VOL. 47, NO. 3, M A R C H
was approximately one-half the value calculated for complete conversion of glucose, using identical flow rates. However, the calibration curve was both linear and reproducible as is seen in Figure 6. T h e linear range for glucose analysis was 10-s-10-4M. T h e limit of detection was 2 x 10-8M glucose. T o define the cause for reduced sensitivity in glucose analysis, the temperature dependence of the efficiency of the glucose oxidase column was evaluated. Since the activity of the soluble enzyme increases significantly with temperature (33), greater sensitivity should be realized a t higher column temperature if glucose is not totally being converted a t room temperature. Using a water bath controlled to hO.1 "C, the peak height of chemiluminescence remained independent of column temperature from 24.138.0 "C. Above 38 "C, peak height slowly began to decrease. This behavior suggests that glucose is totally oxidized in the enzyme column. The decrease in peak height above 38 "C may indicate slow denaturation of the enzyme and/or temperature catalyzed enolization of glucose standards (34). The lower sensitivity probably results because glucose oxidase reacts only with 6-n-glucose. D-Glucose exhibits an equilibrium concentration of 35% a-D-glucose. Therefore, a significant amount of glucose in the sample is not being analyzed. This may be rectified by addition of the enzyme, mutarotase (EC 5.1.3.3) to the glucose oxidase column to catalytically convert the a-anomer to the fl form. If doubling the sensitivity is of significant importance, glucose oxidase may be isolated from Penicillum nonatum, rather than from Aspirgillus niger as used in the present column. Glucose oxidase from P. notatum contains mutarotase as a contaminant (35). A correlation study with blind control serum samples, having a broad therapeutic glucose range, was performed to determine the accuracy of the chemiluminescent method for blood glucose analysis. The samples were deproteinated and diluted by a factor of 2000. The chemiluminescent response for the glucose standard run appears in Figure 7 . A linear least squares analysis was done between the CDC reference values based on the glucose oxidase method and the chemiluminescent values for serum glucose in the six samples. These data are summarized in Table I. The equation for the least squares line was y = 1.01 x - 1.1mg/dl. T h e accuracy of the chemiluminescent method was also checked against two known methods for blood glucose, using a Versatol standard serum. Sigma Glucose Kits were used to check against trichloroacetate deproteination and the 0-toluidine colorimetric method. T h e Worthington Glucostat method uses ZnS04-Ba(OH)z deproteination and the glucose oxidase-peroxide system coupled with 3,3'-dimethoxybenzidine as a color-producing agent. These represent two of the most widely used methods for clinical
1975
Table 11. Comparison of T h r e e Glucose Methods Using a Reconstituted S e r u m Samplea
L1
So. of
Mean
Xlethod
analyses
value, mg/dl
Sigma Glucose Kit (doluidine) Worthington Glucostat (enzymatic) Chemiluminescent
5
75.7
2.8
5
75.4
1.2
5
75.1
0.5
Re1 std dev,
e
