100
Anal. Chem. 1981, 53, 190-193
Immunoelectrochemical Assay for Creatine Kinase Isoenzyme MB Chang-Li Yuan,‘ Shia S. Kuan, and George G. Gullbault” Department of Chemistry, Universky of New Orleans, New Orleans, Louisiana 70122
A novel creatine kinase Isoenzyme MB (CK-MB) electrode has been developed, based on the principle that after immunolnhibltion with goat-anti-human CK-M antibodies the residual activity of CK-B in serum Is detected with a platinum electrode by coupling the NADH (generated from the HK-GPD reactlons) to the ferricyanide-diaphorase indicator reaction; the electrochemical oxidation of ferricyanide ion is monitored at t0.36 V vs. SCE. The whole assay takes only 10 mln and the linearity of a calibration plot of serum CK-MB enzyme activity extends up to 875 U/L. The CV and recovery value of this method are 3.0% and 97.8%, respectively. The results obtained from the proposed method correlate very well with those obtalned by the Helena eiectrophoresis-fiuorodensitometric method.
Scheme I
,
C K j Y n t i b o d y CK-Y CK-B CrPvADPvG-6-P
I
HK
G-6-PD
Diaphorase Fe( CN)6-4
, : : BA( ,
I
Y /
+
H’
6-P-Gl u c o n a t e
introduction of antibody CK-M. The residual CK-B activity is measured by monitoring the rate of current increase at 0.36 V by use of the HK-GDPH system and ferricyanide as redox agent in the presence of diaphorase. The coupled reaction scheme is shown in Scheme I.
EXPERIMENTAL SECTION The activity of creatine kinase (E.C. 2.7.3.2) isoenzyme MB (CK-MB), a cardiac specific isoenzyme in serum, is now widely used as an index of acute myocardial infarction (1-3). Among the methods currently used, the immunoinhibition test appears to be the best technique for fast determination of CKMB (4). Conventionally the CK-MB activity after inhibition is monitored spectrometrically by observing the UV change originating from the reduced form of nicotinamide adenine dinucleotide (NADH) produced from the hexokinase glucose 6-phosphate (HK-GPD) coupled sections. Because of many inherent advantages such as simplicity, rapidity, adaptibility to interfacing with other instruments, and freedom of interferences from turbidity, electrochemical enzymatic methods have recently become useful analytical tools for the assay of biologically active materials and metabolites in the living organism. Electrochemical methods for the determination of NADH or NADPH (reduced form nicotinamide adenine dinucleotide phosphate) have been well developed. Methods include direct amperometric measurement of NADH at solid electrodes (5-8)and coupling NADH (or NADPH) to different redox agents (such as 2,6-dichlorophenolindophenol (9),Bindschedler’s Green (IO),and ferricyanide (11)) for indirect amperometric monitoring. The measurement of oxygen consumption by NADH in the presence of horse-radish peroxidase (12,13) has been used as well. Other electrochemical methods based on potentiometric electrodes and miscellaneous enzyme sensors have been discussed (14,15).The electrochemistry of the Fe(CN)63-complex ion in biological media has been established in this system. Both NADPH and ferricyanide are stable in the presence of oxygen (16,17), and the voltammetric half-wave potential of the Fe(CN)t-/Fe(CN)64-system is moderately positive, whereas amperometric residual currents in the presence of serum are low (18). On the basis of the information described above, a novel immunoelectrochemical system was then designed for fast and simple assay of CK-MB. In this system, the CK-M activity is inactivated after the Current address: E. I. du Pont de Nemours, La Place, LA. 0003-2700/81/0353-0190$01.00/0
Apparatus. The rate of current change at a fixed potential was monitored with a polarographic analyzer (PAR 174A, Princeton Applied Research Corp., Princeton, NJ) using a platinum electrodepoised at +0.36 V vs. SCE (Saturated Calomel Electrode) and an auxiliary platinum foil electrode. A linear chart recorder (Cole-ParmerInstrument Co., Chicago, IL) was used to record the rates of reaction. An EC Motomatic motor control stirrer (Model E550M, Electro Chart Corp., Hopkins, MN) and a ministirrer (2 X 8 mm) were used to control the stirring speed during assay. A circulating water bath with a laboratory immersion heater (Blue M Electric Co., Blue Island, IL) and a circulating pump (BrinkmanIC-2, Brinkman,Westbury,NY)were used to maintain a constant temperature within fO.l OC. pH adjustments were performed with a Beckman Research pH meter (Beckman Instrument, Inc., Fullerton, CA). Autopipeta (FinnpipetteKy., Helsinki, Finland) of 5-50,50-200, and 200-1000 ~ Lcapacities L were used to deliver sera, substrates, and reagents. Reagents and Solutions. A conventional Oliver-RosalkiCK reagent solution modified by Dade was prepared as described below by use of chemicals obtained from Sigma Chemical Co. (St. Louis, MO). Cardiozymekit is as follows: triethanolamine buffer (pH 7.01, 100 m M creatine phosphate, 35 mM; glucose, 20 mM, magnesium acetate, 10 mM; adenosine diphosphate (ADP), 1mM; nicotinamide adenine dinucleotide phosphate, 0.6 mM; adenosine monophosphate, 10 mM, glucose 6-phosphatedehydrogenase,2.4 U/assay; hexokinase, 2.4 U/assay; reduced glutathione, 0.3 mM. CK-M inhibiting antibodies (from goat) were obtained from E. Merck (Darmstadt, Germany). Potassium ferricyanide crystals [K4Fe(CN)B.3H20] were obtained from Matheson Coleman and Bell (Norwood,OH). Potassium chloride was purchased from Fisher Scientific co. (Fair Lawn, NJ). Procedure, Immerse the three electrodes into 2 mL of Cardiozyme kit solution in a 10-mL minibeaker maintained at 37 OC using a thermal cell circulated with water from a constant-temperature water bath, Pipet 0.1 mL of 0.1 M potassium chloride solution, 0.1 mL of 5.4 U/assay of diaphorase, and 0.1 mL of creatine kinase MB isoenzyme control of serum and preincubate 2 min at 37 O C . Stir the assay solution with a ministirrer at a constant rate of 800 rpm. Fix the potential of the polarographic analyzer at +0.36 V vs. SCE, push the control button, and initiate 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
l@l
w
POTENTIAL, V
O
L
W
2 5
t-.4
40
-
20
-
w
I I
1 -.a I
Figure 1. The cyclic voltammogram of ferricyanide-ferrocyanide species In TEA buffer ((3.1 M, pH 7). Scan rate = 5 mV/s.
0
0.4
0.2
0.6
0.8
1.0
KC1.M
Figure 3. The effect of potassium chloride on the relative activity for the electrochemical assay of CK-MB with lmmunoinhibitionunder optimal conditions.
0
2.7
DIAPHORASE.
6 TIME, M I N
the electrochemical reaction by introducing 0.1 mL of freshly prepared 3.4 X low3M €erricyanidesolution into the assay solution. Record the rate of current change with time and calculate the activity of creatine kinase MB after immunoinhibition from a calibration curve of A i l A t vs. activity. Rinse the electrodes with dilute nitric acid between each assay to ensure that the electrode surfaces are clean and the results are reproducible. The calibration plot of A i / A t vs. CK-MB activity is linear up to 875 U/L.
RESULTS AND DISCUSSION Preliminary Study. The current-voltage curve of a 3.4 X loT3M solution of ferricyanide in 0.1 M, pH 7, triethanolamine buffer, with 0.1 M potassium chloride as a supporting electrolyte, was recorded by use of the threeelectrode cell with a platinum working electrode (Figure 1). The potential was scanned from 0.6 to -0.15 V vs. SCE in an unstirred solution at a scan rate of 5 mV/s. The curve illustrates feasibility of using ferricyanide in TEA buffer system (19-21). In order to ensure that the applied potential is high enough to effect the anodic oxidation
* Fe(CN)63-+ e-
IJ/ASSAY
Figure 4. The effect of diaphorase on the relative activity for the electrochemical assay of CK-MB with lmmunoinhlbiton under optimal conditions.
12
Figure 2. The effect of NADPH on the reduction current of ferricyanide. The current was observed after adding 0.1 mL of NADPH (10 mg/mL) at each point (asterisk)while the voltage was fixed at 4-0.36 V. The other ingredients, Fe(CW),3-and diaphorase, were kept at suitable excess amounts.
Fe(CN),"
10.8
8.1
5.4
(1)
we applied a potential of +0.36 V in the assay. The electron transfer reactions Fe(CN):-
+ NADPH
diaphorase
' Fe(CN)a-
+ NADP' (2)
and Fe(CN)64-+ Fe(CN)$-
+ e-
(1)
were studied at a fixed potential of +0.36 V and at a constant rate of stirring. The current level was proportionally changed (Figure 2) by varying the concentrations of ferricyanide, reduced nicotinamide adenine dinucleotide phosphate (NADPH), or diaphorase. The electron transfer in these two coupling reactions is so fast that an instantaneous jump of the current was seen (instead of a slope of current change). Studies on Optimum Conditions. The maximal activity of CK-MB in the electrochemical assay was found to occur at 37 O C in 0.1 M triethanolamine hydrochloride buffer pH 7.0, containing 0.1 M potassium chloride, Figure 3, 5.4 U/ diaphorase, Figure 4, and 3.4 mM ferricyanide, Figure 5. Preincubation is required for obtaining a stable base line before initiating the electrochemicalreaction; a 2-min period was found to be optimum. Effect of Stirring. The assay solution was stirred by a 2 X 8 mm ministirrer at a constant rate of stirring. The stirring action increases the transport rate of the assay species
192
ANALYTICAL CHEMISTRY, VOL. 53, NO. 2, FEBRUARY 1981
Table I. Study of Precision of Electrochemical Assay normal value normal value Run to Run 8.1 U/L" 6 runs 0.26 U/L 3.3%
av N C
std dev
cv
203.1 U/L'ib 6 runs 5.18 U/L 2.6%
Day to Day 8.07 U/L 204.6 U/L 3 days 3 days 0.25 U/L 7.8 U/L cv 3.1% 3.8% Value of each run was the average of three assays. U/L = units/liter. N = number of runs. av N std dev
3.3
6.6
9.9
-3
FEKY),,
q~
Table 11. Interference Effects of Species in Electrochemical Assay
Flgure 5. The effect of ferricyanide on the relative activity for the electrochemicalassay of CK-MB with immunoinhlbitionunder optimal conditions.
at the electrode, thus increasing the sensitivity. This mode of mass transport is called convection or hydrodynamic transport. Without stirring of the assay solution, the reaction rate is hardly detectable, hence the stirring rate shodd remain constant during the assay. In general, the reaction rate increases with an increase in stirring speed, but the noise level increases significantly and a whirlpool is formed when the stirring speed is 1000 rpm or higher. Therefore, a speed of 800 rpm was used, and satisfactory results were obtained. Effect of Activators. The activator for the creatine kinase, reduced glutathione, is an electroactive species itself (Eo = -0.25 V) (22). At a concentration of 9 X M (Dade kit formula),reduced glutathione reacts with the electron acceptor ferricyanide, thus interfering with the assay. Several other activators, such as cysteine, thioglycerol, 2-mercaptoethanol, n-acetylcysteine (NAC), dithiothreitol (DTT), dithioerythritol (DTE), and 2-aminoethylisothiouonium bromide (AET) (22) were tried as substitutes. Unfortunately similar interferences were obtained. But if the concentration of the activator is reduced to 3 X lo-* M and a preincubation of the CK-MB sample for 2 min is effected, reproducible rates using this electrochemical approach are obtained. Peck et al. (23) successfully used the same approach (i.e., reduction of activator concentration) for the assay of adenosine 5'-phosphosulfate (APS) reductase in the presence of ferricyanide. Effect of Other Trapping Agents for NADPH. 2,6Dichlorophenolindophenol (DCPIP) was investigated as a NADPH trapping agent (instead of using ferricyanide) for the determination of CK-MB in this study. An applied potential of +0.217 V was used. The reproducibility of the results was not as good as that of ferricyanide, probably due to the illdefined redox peak curves in the cyclic voltammogram. Precision. Run-to-run and day-to-day precision studies were performed to evaluate the new method for CK-MB assay. Two levels of pooled blood sera (normal, 8.1 U/L, and abnormal, 203 U/L) were analyzed for three consecutive days. Table I shows the resulta obtained from the proposed method. Excellent precision was observed. Recovery Study. Cardiozyme controls (Dade) were added to the pooled serum to bring the CK-MB activity from 23.0 U/L to values ranging from 35.5 to 313.0 U/L. The average recovery was found to be 97.8%. Specificity. Interferences from adenylate kinase (24,25) and endogenous polyvalent cations (26) acting upon serum creatine kinase have been reported. Several other common interfering substances present in serum were added to the
a
species control serum creatine ascorbic acid uric acid billirubin hemoglobin dL = deciliter.
concn (mg/dLY 34.0 U/L 6.5 6.2 44.0
result
W/Ub
1.8
100.0
34.0 32.4 30.0 42.3 32.6 39.0
discrepancy (U/L)b 0 -1.6 -4.0 t8.3 -1.4 t 5.0
U/L = units/liter.
specimen to study their effect on the accuracy of the assay. The interference effect of all diverse substrates is negligible (Table 11). Comparison Study. Fifteen fresh sera ranging from 8 to 285 CK-ME! U/L were assayed by the proposed electrochemical method and compared with those of the Helena electrophoresis method (from Hotel Dieu Hospital). The linear regression analysis of CK-MB gave a coefficient correlation of 0.999. CONCLUSION Through the coupling of an electroactive pair Fe(CN)6-3/ Fe(CN)6-4to NADH produced from the HK-GPD system, creatine kinase can be determined amperometricallywith the use of a platinum electrode. The method is rapid, simple, and specific, and the linear range is extended to 875 U/L. Positive myocardial infarction cases can be easily determined in 10 min by judging the CK-MB value obtained from the proposed method due to its good sensitivity. This is very helpful to physicians for timely diagnosis of patients with severe cases. Furthermore,this electrochemical technique is free of turbidity interferences encountered in spectrophotometric assays and is easily adaptable to interfacing with other instruments, as well as to the immobilized immunostirrer system developed in this lab for CK-MB assay (27). ACKNOWLEDGMENT We wish to thank both Alfred Hew, Jr. (Hotel Dieu Hospital), and W. T. Wu (Charity Hospital) for their generous supply of analyzed sera. Appreciation is expressed to Joe Giegel at the Dade Corp. (Division of Am Hospital, Miami, FL) for his kind provision of Cardiozyme CK-MB kits and the Merck CK-M antibody. LITERATURE CITED (1) Men, A. S., Prog. Hum. Pathol. 1875, 6, 141-155. (2) . . Roberts. R.: Gowda, K. S.; Ludbrook. P. A.; Sobel, B. E. Am. J . Cardiol. 1875, 36, 433-437. (3) Konttlnen, A.; Somer. H. Br. J . Med. 1873, i,386-389. (4) Wurzburg, U.; Hennrlch, N.; Lang, H. Klln. Wochenschr. 1976, 54, 357-360.
Anal. Chem. 1981, 53, 193-190 (5) Blaedel, Walter; Jenkins, Rodger Anal. Chem. 1875, 47, 1337-1343. (6) Gullbault, George 0.; Cserfalvi, Tamas Anal. Left. 1876, 9 , 277-289. (7) Thomas, Lawrence C.; Christian, Gary D. Anal. Chlm. Acta 1975, 78, 271-276. (8) Christian, Gary D. “Ion and Enzyme Electrodes in Biology and Medicine”; Urban. and Schwarzenberg: Muchen-Berlin-Weln, Germany, 1976. (9) Smith, Marilyn D.; Olson, Carter L. Anal. Chem. 1875, 47,
-
-
i.n 7 . ~. - 1. 0.7,7.
( I O ) Smith, Marilyn D.; Olson, Carter L. Anal. Chem. 1974, 46, 1544-1 547. (1 1) Thomas, Lawrence C.; Christian, Gary D. Anal. Chim. Acta 1876, 82, 265-272. (12) Cheng, Frank S.; Christian, Gary D. Anal. Chem. 1977, 49, 1785-1788. (13) Cheng, Frank S.; Crlstian, Gary D. Clin. Chem. (Winsfon-Salem, N. C.) 1878, 24, 621-626. (14) Gullbault, George G. “Theory, Deslgn and Biomedical Applications of Solid State Chemiail Sensors”; CRC Press: Cleveland, OH, 1978, p 193. (15) Barker, A. S.; Somers, P. J. Top. Enzyme Ferment. Blofechnol. 1978, 2 , 120. (18) Peel, J. L. “Methods In Mlcroblology”; Academic Press: New York, 1972.
193
(17) Purdy, W. C. “Electroanalytical Methods in Blochemisby”; McGraw-Hill: New York, 1965. (18) Thomas, Lawrence C.; Chrlstlan, Gary D. Anal. Chim. Acta 1875, 77, 153-161. (19) Nicholson, Rlchard S.; Shain, Irving Anal. Chem. 1964, 38, 706-723. (20) Adams, Ralph N. “Electrochemlstry at Solid Electrodes”; Marcel Dekker: New York, 1969. (21) Manual of Model 174A, Polarographic Analyzer; Princeton Applied Research: Princeton, NJ, 1977. (22) Reo, P. S.; Evans, R. G.; Mueller, H. S. Biochem. Blophys. Res. Commun. 1977, 78, 648-654. (23) Peck, H. D., Jr.; Deacon, T. E.; DavMson, J. T. Blochim. Blophys. Acta 1865, 96, 429-446. (24) S P S Z , Gabor; Gerhardt, Wlllie; Gruber, Wolfgang; Bernt, Erlch Clin. Chem. (Wlnston-Salem, N.C.) 1976, 22, 1806-1811. (25) Szasa,Gabor; Gruber, Wolfgang; Bernt, Erlch Clin. Chem. (WinsfonSalem, N.C.) 1978, 22, 650-656. (26) Szasz, Galln; Gerhardt, Wlllle; Gruber, Wolfgang Clin. Chem. ( Winston-Salem, N.C.) 1877, 23, 1888-1892. (27) Yuan, C. L.; Kuan, S. S.; Guilbault, G. G. Anal. Chlm. Acta, In press.
Received for review August 14,1980. Accepted October 31, 1980.
Simultaneous Determination of Iron(II), Iron(III), and Total Iron in Sphagnum Moss Peat by Programmable Voltammetry on a Graphite Tubular Electrode F. J. Srydlowskl” and D. L. Dunmire Raltech Scientific Servhces, St. Louis, 900 Checkerboard Square Plaza, St. Louis, Missouri 63 188
E. E. Peck and R. L. Eggers Raiston Purina Company, Health Industries Products Division, Bridgeton, Missouri 63044
W. R. Matson Environmental Sciences Associates, Inc., 45 Wiggins Avenue, Bedford, Massachusetts 0 1730
An integrated step scan voltammetric program was used In conjunction with a pyrolytic graphite tubular electrode to determine the concentration and oxidation state of iron in sphagnum moss peat without Iron( 111) reductive errors due to organic matter. Good correlation was obtained by this method when compared with colorimetric ( f = 0.973) and atomic absorptlon spectrometric ( r = 0.978) procedures; total iron results were compared to the sums of irorr(I1) and iron(111) obtained electrochemically. Total iron recoveries from sphagnum moss peat samples spiked with iron( 11) sulfate ranged from 88% to 109%. Copper interference was not slgnlficant at levels found in thee sphagnum moss peat.
The objective of this study was to develop a reliable iron speciation method for sphagnum moss peat used as a medication carrier for neonatal pigs. Neonatal pigs gain weight rapidly going from 3 to 12 Ibs in 3 weeks. If the pigs do not have access to soil (taken away by modern husbandry techniques) or other sources of iron, anemia can occur causing retarded growth, lower disease resistance, and death (1). Sphagnum moss peat is a complex matrix composed primarily of humus residues and numerous metal complexes (2). Iron’s oxidation state in sphagnum moss peat has been given some attention since animal nutritionista regard the iron(I1) species as the primary biologically usable form of iron (1,3). 0003-2700/81/0353-0193$01.00/0
If the iron(I1) concentration in a sphagnum moss peat product can be accurately determined, a supplemental amount of iron(I1) sulfate could be added to bring the product up to the potency level needed as an anemia-prevention agent. Additionally, iron(II1) salts are toxic, causing lesions in animal’s stomachs (4). If uncomplexed, an organism is able to reduce only a small portion of the iron to its biologically active form. There then is a need for an iron speciation method in the feed industry not only for potency considerations but also for toxicological considerations. A method developed by Sullivan (5) using a,a’-bipyridyl and colorimetric analysis of iron(II), iron(III), and total iron in drugs was used in the preliminary studies of sphagnum moss samples. Due to the complexity of the peat matrix, it was necessary to modify the procedure (5) to permit iron quantitation. Metal is fixed in peat’s humic acids by adjacent phenolics and benzene carboxylic acid groups which must be degraded to release iron for analysis. These additional degradation steps lengthened the procedure more than desirable for routine laboratory use and simpler methods were sought. Degradation also affected iron(II), iron(II1) ratios because of organic matter (6). Various polarographic (7)and electrochemical techniques ( 4 9 ) of determining iron(II)/iron(III) levels were investigated. Since on a graphite electrode, iron ions are reduced from solutions containing the complex elemental ions to the metal, they can be determined by anodic stripping techniques 0 1981 American Chemical Society
