Anal. Chem. 1999, 65, 397-402
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Metal Porphyrin Chemiluminescence Reaction and Application to Immunoassay M.Motsenbocker,*J Y. Ichimori, and K. Kondo Takeda Chemical Industries. Ltd., Pharmaceutical Research Division, 17-85 Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532, Japan
A new lumlnol chemiluminescence chemlstry system Is descrlbed whlch does not require preparation or use of an oxldlzer such as hydrogen peroxide. I n the reaction a metal porphyrin catalyzes lumlnol chemllumlnescence In a hlgh pH solution. A comparlson of porphyrln catalysts showed that a metal atom Is needed In the porphyrln, manganese works better than Iron, and substitution of strongly electron withdrawing groups at the para poeltknsof the tetrapyrrole porphlne with no substltutlon of the pyrrole rlngs glves the best activity. The metal porphyrln chemllumlnescencesystem Is enhanced by the presence of unsaturated long-chaln fatty acld In the reaction solution. Sensitivity of detectlon of Mn mes&tetrakls(4-wtfonatophenyl)pphlnewas 120a d . Cwpllng reactbns of a carboxyphenyl derivative to antlbodles were opthnlzed, and a detectlon llmit of 68 amol was obtalned for a metal porphyrln antlbody conlugate. An a-fetoprotelnImmunoassay developed udng the chemiluminescence reaction had a detectlon llmit of 20 pg (0.2 fmol). Good correlatlon was found ( R = 0.99) between Immunoassay resuits and a-fe toproteln In 32 human plasma samples. Because the reactlon solutlon needed for methyl porphyrin chemllumlnescence Is very shnple (lumlnol In NaOHIwater) and has good stability, this chemllumlnescence detection system may have appllcation to automated assays.
INTRODUCTION Chemiluminescencedetection reactions have become popular in analytical chemistry due to the need for greater sensitivity and the availability of photon counting. In many of these reactions luminol is a substrate, hydrogen peroxide or perborate is the oxidant, and iron porphyrin (usually as a prosthetic group of an enzyme)is the catalyst. The oxidation of luminol (5-amin0-2,3-dihydro-l,6phthalazinedione) in these systems results in formation of the aminophthalate ion in an excited state which emits light upon decay. In alkaline aqueous solution, this reaction requires an oxidizing agent and catalyst.'s2 Many oxidizing agents and catalysts have been explored including synthesized porphine derivative^.^!^ Probably the most popular catalyst used for luminol chemiluminescence is horseradish peroxidase which contains an iron porphyrin prosthetic group. By coupling horseradish peroxidase catalyst to an antibody and measuring the label at low concentrations, reasonably sensitive analytical systems based on photon counting have been de~eloped.~ Another chemiluminescence system that uses an iron porphyrin + Present address: Law School, Condon Hall, University of Washington, Seattle, WA 98195. (1) Hummelen, J. C.; Luider, T. M.; Wynberg, H. Pure Appl. Chem. 1987, 59, 639. (2)Schroeder, H. R.; Yeager, F. M. Anal. Chem. 1978,50,1114. (3)Hara, T.: Torivama, H.: Tsukaaoshi, K. Chem. SOC.J m . 1983,56, 2267. (4)Whitehead, T. P.;Thorpe, G. H. G.; Carter, T. J. N.; Groucutt, C.; Kricka, L. J. Nature (London) 1983,305, 158.
catalyst is the microperoxidase (cytochrome heme-peptide) system f i i t extensivelystudied bySchroeder.5 In this system, too, hydrogen peroxide is needed to react with the iron porphyrin. Recently, metal porphyrins themselves (unassociated with protein) have been studied as useful mimics of peroxidaseand catalase. For example,Saitoet al. have studied the peroxidase-like catalyst activities of manganese, cobalt, iron, copper and zinc complexes of some porphyrins.6 These same authors also investigated the catalase-like activities of metal porphyrins.' In each of these cases hydrogen peroxide was used as the oxidant for a chemiluminescenceor colorigenic reaction. Adrawback of using hydrogen peroxide is the inconvenience and hazard of storing and preparing this reagent. Consequently, much effort has gone into finding alternative and simpler oxidation systems. One innovation was to combine oxidant and light emitter in the same compound.8~9In this system, energy from a stable dioxetane released by action of an enzyme enters a lumigenic substituent of the same compound and is released with high quantum efficiency. The use of metal porphyrin to catalyze luminol oxidation in the absence of added oxidizer has not been extensively studied. The closest example of such a system is the ironcatalyzed oxidation of luminol in pH 11solution by dissolved oxygen as described by Seitz and Hercules.10 In that system, the iron had to be kept reduced before reaction with luminol and other metals such as manganese interfered with the reaction. The detection limit was 1.1 X 10-13 mol of iron. In another high pH system for oxidation of luminol in the absence of peroxide, ozone was used as the oxidant." The optimum pH value of this reaction was 10.5, and it was used to measure air ozone concentrations. The present study originated with the finding that certain metal porphyrins being investigated as light-sensitive dye catalysts catalyzed luminol oxidation even in the absence of added oxidant or light energy as shown in eq 1 - 1 2 An luminol+ OH-
-
metal
light
porphyrin
immunoassay was then developed for plasma a-fetoprotein using the metal porphyrin chemiluminescence reaction and a chemiluminescence microtiter plate reader. (5)Schroeder, H. R.; Vogelhut, P. 0.;Carrico, R. J.; Boguslaski, R. C.; Buckler, R. T. Anal. Chern. 1976, 48, 1933. (6) Saito, Y.; Mifune, M.; Nakashima, S.; Nakayama, H.; Odo, J.; Tanaka, Y.; Chikuma, M.; Tanaka, H. Chern.Pharm. BuZl. 1986,7,2885. (7)Saito,Y.;Mifune,M.;Kawaguchi,T.;Odo, J.;Tanaka,Y.;Chikuma, M.; Tanaka, H. Chem. Pharrn. Bull. 1986, 7, 2885. (8) Schaap, A. P.; Chen, T. S.; Handley, R. S.; DeSilva, R.; Giri, B. P. Tetrahedron Lett. 1987, 28, 1155. (9) Bronstein, I.; Edwards, B.; Voyta, J. C. J.Biolurnin. Chemilurnin. 1989, 4, 99. (IO)Seitz, R.W.; Hercules, D. M. Anal. Chem. 1972,44, 2143. (11)Bernanose, H. J.; Rene, M. G. Adu. Chern. Ser. 1959,21, 7. (12)Motsenbocker, M. A.; Sugawara, T.; Shintani, M.; Masuya, H.; Ichimori, Y.; Kondo, K. Anal. Chem., following paper in this issue.
0003-2700/93/0365-0397$04.00/0 0 1993 American Chemlcal Society
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EXPERIMENTAL SECTION Reagents. Luminol was obtained from Aldrich and purified by recrystallization from 1 M NaOH.13 Sodium hydroxide was 99.99% pure semiconductor grade and obtained from Aldrich. Microperoxidase, chlorophyllin sodiumcopper salt, hematin, linoleic acid, and Tween-20 were obtained from Sigma Chemical Co. (St. Louis, Mo). All other porphyrin compounds were obtained from Porphyrin Products (Logan, UT). Water was distilled in glass and further purified by counterion exchange purification (Milli-Q, Millipore Co.). Catalysts were dissolved to l mg/mL in ethanol or 0.001 M NaOH and diluted to 10pg/mL in 0.001 M NaOH. They were further diluted just before use. Anti a-fetoprotein antibodies, a-fetoprotein,and goat serum\nrCreobtained from Tokyo Standard Serum (Tokyo, Japan). Block ace (milk protein solution used to decrease nonspecific binding) was from Snow Brand Milk Products (Sapporo, Japan). All other compounds were obtained from Wako Pure Chemical Co. or from Sigma Chemical Co. and were the purest available. Instrumentation. A fluorometer (Model 650-40 Hitachi, Tokyo, Japan) was used for light spectral determinations. A photon-counting chemiluminometer that accepts 12-mmdiameter by 75-mm high plastic test tubes was used for the remainder of the experiments except for the immunoassay experiments which used a chemiluminescence microtiter plate reader (Model lOOO, Dynatech Laboratories, Chantilly, WV). Luminol Emission Spectrum. Ten microliters that contained 10 pg/mL microperoxidase or 10pg/mL Mn mesotetrakis(4-sulfonatopheny1)porphine was added to 1-mL solutions of 0.05 M NaOH, 1mM luminol. After addition of catalyst, each sample was inserted into the scanning fluorometer. The relative amount of light measured at 10-nm increments between 380 and 600 nm was recorded. Effect of NaOH Concentration, Luminol Concentration, and Temperature. For the NaOH concentration experiment, 100 pg of Mn meso-tetrakis(4-sulfonatopheny1)porphine was added to 1-mL samples of 1 mM luminol solutions that contained various concentrations of NaOH. Chemiluminescence was measured for 1min after adding the catalyst. In the luminol concentration experiment, 0.1 M NaOH was used with various concentrations of luminol from 5to 0.003 mM. One hundred picograms of catalyst were added to 1-mL solutions, and chemiluminescence was measured for 1min. For the temperature studies, 1-mL solutions of 1mM luminol and 0.1 M NaOH were equilibrated to various temperatures in a water bath, and then 10 pL of 100 ng/mL Mn meso-tetrakis(4-sulfonatopheny1)porphinewere added. Light was measured for 5 s immediately after adding catalyst. Comparisonof Different Porphyrins. Each porphyrin was diluted to 1ng/mL in 1mL of 0.2 M NaOH, 1mM luminol solution, and chemiluminescence light was integrated for 1 min. Light measurements were expressed relative to that produced by Mn meso-tetrakis(4-sulfonatopheny1)porphine. EnhancementStudies. One-milliliter solutions of 1mM luminol at pH 12.3 were prepared with 0, 0.003, 0.010, and 0.030% Tween-20. Then, 0.01 mL of 0 pg and of 100 pg of Mn meso-tetrakis(4-sulfonatopheny1)porphinein water were separately added to the solutions, and the resulting light outputs were integrated for 2 s 1 min later. One-milliliter solutions of 0.1 M NaOH and 1mM luminol were prepared with various concentrations of linoleic acid from 0.02 % to 1.25%. Then 10pL of either water or 100pg of Mn meso-terakis(4-sulfonatopheny1)porphinein water were added, and chemiluminescencelight was integrated for 1min. (13) Scott, R. A. W.; Kricka, L. J. In Bioluminescence and Chemiluminescence, New Perspectives; Scholmerich, J., Andreesen, A., Kapp, A.,Ernst, M., Woods, W. G., Eds.; John Wiley and Sons: New York, 1987; p 237.
Metal Porphine Detection Limit. Solutions (0.1 mL) of 1 mM luminol, 0.1 M NaOH, and 0.1% linoleic acid were prepared with 0, 0.25, 0.5, 1.0, 2.0, 4.0, 10, 100, lOOO, and 10 OOO pg of Mn meso-tetrakis(4-sulfonatopheny1)porphine. Light was integrated between 2 and 3 min after adding the catalyst. In order to prevent a high measurement variability due to ambient ozone, a stream of nitrogen gas was directed onto the surface of the samples during light measurement. Synthesis of Mn Porphyrin Protein Conjugates. A succinimide ester of Mn meso-tetrakis(4-cox~henyl)porphine ("Mn porphyrin") was prepared by mixing (1)4 mg of N-hydroxysuccinimide in 1.05 mL of 25 mM sodium phosphate buffer pH 6.0 with (2) 6.3 mg of water-soluble carbodiimide (WSC) in 1.05 mL of 25 mM sodium phosphate buffer pH 6.0 and (3) 3.0 mg of Mn meso-tetrakis(4carboxypheny1)porphine in 2.1 mL of dimethylformamide.14 For optimization experiments the ratio of N-hydroxysuccinimide and WSC to Mn porphyrin was varied from 1:l to more than 30:l. Each mixture was incubated for 1h at room temperature. Then 1.5 mg of anti AFP antibody dissolved in 4.2 mL of 25 mM sodium phosphate buffer pH 6.0 was added to each, and the mixtures were incubated overnight at 6 "C. Derivatized proteins were separated from low molecular weight compounds by Sephadex G-50 gel filtration. Protein was determined by a dye binding method (Sigma Chemical Co. cat. no. 610-A). Porphyrin incorporated into protein was determined by absorbance of the soret band and comparison with a standard solution of the porphyrin. The above Mn porphyrin activation procedure (101N-hydroxysuccinimide and WSC to Mn porphyrin) was also used to prepare activated Mn porphyrin that was mixed at varying ratios with antibody to optimize coupling of activated Mn porphyrin to antibody. The above activation procedure was also used to couple Mn porphyrin to BSA. A 50-mg sample of BSA was dissolved in 25 mL of 0.5% sodium dodecylsulfate, 0.25% mercaptoethanol, and 25 mM sodium phosphate buffer pH 6. This was heated to 90 "C for 10 min. The mixture was cooled and the protein purified by Sephadex G-SO gel filtration in 0.1 M sodium phosphate pH 7.0 buffer. Detergent denatured BSA (1.5 mg) in 0.5 mL of 0.1 M sodium phosphate pH 7.0 buffer was mixed with 3.0 mg of the derivatized Mn porphyrin and incubated overnight at 6 "C. The Mn porphyrin derivatized BSA was purified by Sephadex G-50 gel fitration. A FabBSA-Mn porphyrin conjugate was synthesized using the above BSA-Mn porphyrin preparation. A half milliiam of Fab prepared from type-4 anti AFP antibody was reduced by incubation in 1.0 mL of 2.5 mM EDTA, 1.0 mg of dithiothreitol and 0.1 M sodium phosphate pH 6.0 buffer at 37 OC for 90min. Dithiothreitolwas removed by gel filtration in 0.1 M sodium phosphate buffer pH 7.0. The prepared protein was immediately reacted with 2.0 mg of N,"-bis(3maleimidopropionyl)-2-hydroxy-l,3-propanediamine at 37 "C for 30 min. Excess diamine derivative was removed by gel filtration. Mn porphyrin conjugated BSA protein (1.5 mg) was added to the maleimido Fab' preparation (5.0 mL total volume) and incubated at 6 "C overnight. The prepared FabBSA-Mn porphyrin conjugate was separated from Fab' and BSA-Mn porphyrin by gel filtration. BSA (0.1%) and merthiolate (0.002 5%) were added to the recovered Fab-BSAMn porphyrin conjugate prior to storage at 6 "C. a-Fetoprotein Immunoassay. A one incubation step sandwich type AFP immunoassay was carried out. Wells of a black microtiter plate (Dynatech Laboratories, Inc., Chantilly, WV) were coated by incubating 100pL of 10pg/mL anti AFP antibody type A-295 in 0.1 M carbonate pH 9.5 for 3 h. Wells were washed once with 0.8% NaCl and 0.1 M sodium (14)Roberts, J. C.; Figard, S. D.; Mercer-Smith, J. A.; Svitra, Z.V.; Anderson, W. L.; Lavallee, D. K. J . Zmmunol. Methods 1987,105, 153.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 4, FEBRUARY 15, 1993
phosphate pH 7.0 (PBS) and then blocked with 300 pL of 25% Block ace and 75% PBS. They were washed with PBS and then 100 pL of 0.2% BAS, 75-fold diluted IgGMn porphyrin conjugate or 75-fold diluted Fab-BSA-Mn porphyrin conjugate (approximately 1 pg/mL final concentration), 0.2% partly hydrolyzed algin, and 10% Block ace in PBS solution containing various AFP concentrations were added. After 45 min of incubation with shaking at 6 OC, the plate was washed twice with PBS. A 1.0 mM luminol, 0.1% linoleic acid, and 0.1 M NaOH solution (100 pL per well) was added and chemiluminescence measured. Plasma Studies. The plasma AF'P immunoassay procedure was as described above except the incubation buffer consisted of 105% Block ace, 0.2 % Tween-20,and 0.2 % partly hydrolyzed algin in PBS. To each well was added 180pL of incubation buffer and 20 p L of plasma. Plasma sampleswere contrived by treating freshly drawn adult male whole blood with disodium EDTA and centrifuging to remove blood cells. AFP was added to plasma sampleswhich were between assay studies, and replicates of two were used to determinemeasured AFP concentrations. Thirty-two plasma samples were used for the correlation study. Replicates of 6 and 5 were used for within assay and between assay (day to day) variability determinations,respectively. The calibrator sample consisted of pooled male plasma, and a standard curve (0,20,50,100, 200,400 ng/mL) was determined for each microtiter plate. Chemiluminescence from a 10 ng/mL plasma sample was assayed repeatedly over a 50-min period to investigate the time duration of light output. Metal Porphyrin Detection Reaction Solution Stability. A 50-mL solution of 1.0 mM luminol and 0.1 % linoleic acid in 0.1 M NaOH was prepared. This was stored in a clear plastic tube at room temperature and was exposed to subdued room lighting conditions. At various time intervals two 1.0mL samples were removed and placed into 12-mm-diameter plastic test tubes. Ten microliters of 100 ng/mL metal porphyrin solution or of water were added to these samples which were immediately placed into the chemiluminometer. Light emitted from each tube was integrated between 1and 2 min. Detection Limits. All detection limits were defined as the amount of analyte or catalyst which gave an amount of light equal to twice the mean background level.
RESULTS AND DISCUSSION The wavelength spectrum of light emitted from a metal porphyrin catalyzed chemiluminescence reaction was the same as that reported for luminol chemiluminescence in other reaction systems. This result indicatesthe same light emitting species found in other luminol oxidation systems (the phthalate ion) is the light emitter in metal porphyrin chemiluminescence. Biological sources of porphyrin such as protoporphyrin, hematin, and copper chlorophyllin had little or no activity in the system. However, porphyrins having no substitutions in the pyrrole rings were significantlyactive. Some commercially available porphine8 of this latter group were compared as shown in Table I. Porphyrins having no central metal atom had no chemiluminescence activity and porphyrins that contained manganese had about twice the activity of iron containing porphyrin. Thus, of the metals examined, manganese was the best. It was important to compare the activity of hemin because hemin is a common contaminantof biological fluids, and a low chemiluminescence background is desired for assays that utilize chemiluminescence as a detection system. Hemin activity in this system was approximately 16-fold lower than that from the reference manganese porphyrin compound. There was no evidence of metal
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Table I. Relative Chemiluminescence Activities of Various Porphyrins relative chemicompound meso-tetraphenylporphine Zn meso-tetraphenylporphine Sn meso-tetraphenylporphine Mn meso-tetraphenylporphme meao-tetrakis(4-carboxypheny1)porphme Cu meso-tetrakis(4-carbxypheny1)porphine Co meso-tetrakis(4-carboxypheny1)porphine Sn meso-tetrakis(4-carboxypheny1)phine Fe meso-tetrakis(4-carboxypheny1)porphine Mn meso-tetrakis(4-carboxypheny1)porphine meso-tetrakis(4-sulfonatopheny1)prphine Fe meso-tetrakie(4-sulfonatopheny1)porphine Mn meso-tetrakis(4sulfonatophenyl)porphine
luminescence 0 0 0 241
0 0 0 1 271 524
0 305
803
dissociation from these porphyrins during the reaction with luminol at high pH. The rneso-tetracarboxy-substitutedcompounds gave more catalytic activity than did the unsubstituted compoundsand the rneso-tetrasulfonato-substitutedcompoundsgave higher activity than the rneso-tetracarboxy-subsitutedcompounds. Thus, the presence of electron-withdrawinggroups on the porphine benzine rings improved activity and increasing the electron-withdrawing character of these substituenta apparently increases the activity of the metal porphyrin catalyst. These electron-withdrawinggroups tend to pull the central atom more into the porphine plane, and perhaps this is important to the catalytic activity. The optimum pH for metal porphyrin chemiluminescence was 13.0. Activity dropped off rapidly at lower pH and was less than 10% of the pH 13 level at pH 11.5. The optimum pH in the presence of enhancer was between 12.0 and 13.0. The chemiluminescence activity of 10 pg of catalyst at pH 13.0 was found to rise rapidly with luminol concentration between 0 and 50 pM luminol but to rise more slowly with increasing luminol concentration above this value. The activity was half the maximum at a luminol concentration of about 0.2 mM. The effect of temperature on the reaction was nonlinear. Below 23 "C, there was approximately a 2.2% per degree increase in reaction rate with increasing temperature. This increased to 5% per degree from 23 to 38 OC and was 6 % per degree from 48 to 60 "C. By comparison, most enzyme reactions vary by about 10% per degree C change in temperature. A typical reaction time course for a Mn rneso-tetrakis(4sulfonatopheny1)porphine-catalyzed reaction is shown in Figure 1. The light output from these reactions can be measured for many minutes after mixing the high pH luminol solution with the catalyst. In practice, measurement of light output by integration of photons from 2 to 3 min after starting the reaction was preferred. The Mn rneso-tetr~s(4-s~onatophenyl)porp~e detection limit was 0.12 pg or 120 amol. The light output linearly increased with increasing catalyst concentration up to 10 ng/ mL catalyst concentration. The light drop-off at concentrations above this level limits the use of this catalyst to concentrations below 10 ng/mL. Tween-20 detergent enhancedthe reaction 75 times. When stearic acid, palmitic acid, linoleic acid, and linolenic fatty acids were compared as enhancers, stearic and palmitic acids had no effect but linoleic and linolenic acids were strongly enhancing. Therefore,it is concluded that enhancement may have been due to the presence of unsaturated fatty acids. Figure 2 shows the relationship between concentration of linoleic acid and chemiluminescence light output. The
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 4, FEBRUARY 15, 1993 3
X
1
-'s
I
0
4 20
10
Minutes of Reaction Flgwr 1. Effect of time on the metal porphyrin chemiluminescence reaction. One-mlnute chemllumlnescence measurementswere made from a 1-mL solution of 25 pg of catalyst In 1 mM luminol and 0.1 M
NaOH.
LD
z X
2
n 0
t
. , 0.0
0.2
0.4
0 6
0 8
Percent Linoleic Acid Figure 2. Enhancement of metal porphyrin chemiluminescence by llndelc acid. One-mlnute chemllumlnescence measurements were made from 1-mL solutions of 100 pg of catalyst In 1 mM lumlnol and 0.1 M NaOH.
maximum enhancement here was 10-fold at 1.25% ' linoleic acid. Assuming that the critical micelle concentration for this fatty acid under these conditions is at least 0.05%, it appears there is no relationship between formationof micelles and enhancement. Thus, the enhancement mechanism does not involve micelles. In the stable dioxetane chemiluminescence system, detergent micelles (or hydrophobic pockets in protein) create a hydrophobic environment in which an intermediate compound is stabilized.15 The enhancement mechanism of the present system appears toLbe different because enhancement occurred even at very low fatty acid concentration where micelles are not expected to form. Anionic detergentswere shown to increase the incorporation rate of metals into the porphine ring by interacting with the ring itself.16 It seems likely that long-chain unsaturated fatty acids would similarly bind to the metal porphyrin. The negatively charged acid group would orient near the metal atom and the acyl chain would associate with the porphine ring. This might facilitate the approach of luminol to the metal porphyrin and is suggested as a model for further studies of the enhancement mechanism. Ten ferntomoles of Mn meso-tetrakis(4-sulfonatopheny1)porphine produced 4.9 X 105 counts of photons in 1min. Since the complete oxidation of 3.3 X 10-13 mol of luminol produced 129573 photon counts each catalyst molecule generated chemiluminescence from 11luminol molecules per minute. (15)Schaap, A. P.; Akhavan, H.; Romano,L. J. Clin.Chem. 1989,35, 1863. (16)Lowe, M.B.;Phillips, J. N. Nature 1961, 190, 262.
Table 11. Effect of Varying the Mn Porphyrin Activation Reaction Conditione (Ratio of N-Hydroxysuccinimide and WSC to Mn Porphyrin) on Coupling of Mn Porphyrin to IgG % activity ratio of Mn porphyrin of coupled WSC to Mn coupled porphyrin 2:l 51 101 301 a
per IgG
Mn porphyrin
1.35 2.9 36 120
15
6 49
58
Precipitate formed.
This indicates that metal porphyrin is recycled in the reaction with luminol. At the high pH found to be optimal for the reaction, luminol dianion formation would be favored with a pK of about 13." The luminol dianion may simply react with oxygen as suggested by White et al.1' Metal porphyrin may facilitate this reaction by binding both luminol and oxygen at the same time. This is suggested as a model for further studies. In the present work, attempts were made to remove oxygen by degassing the luminol solutions and purging with nitrogen but failed to decrease the metal porphyrin activity this way. Failure to influence the reaction might reflectjust how low an oxygen concentration is required. There have been many studies on the mechanism of porphyrin reaction with oxygen. It was assumed that the porphyrin iron of horseradish peroxidase and microperoxidase reacts with hydroperoxideto form a complex between trivalent iron and hydroperoxide anion. Later, however, the sameporphyrin oxidation complexes were obtained using nonperoxidatic oxidants, and it was suggested that the primary complex of the heme after oxidation is really a porphyrin &cation radical.18Jg If the active catalyst in the manganese porphyrin system is a porphyrin u-cation radical, then coating of the ring with fatty acids as mentioned above might protect the u-cation radical and prolong its lifetime. This would improve light output by allowing more time for luminol to react with the radical. Application of the metal porphyrin chemiluminescence reaction to immunoassays was carried out. Optimizationsof metal porphine and protein conjugation reactions were first performed to determine the effect of conjugation on the catalytic activity of porphine and the suitability of the conjugate for use in immunoassay. The formation of active esters on Mn meso-tetrakis(4-carboxypheny1)porphine('Mn porphyrin")was optimum when Mn porphyrin waa incubated with water-soluble carbodiimide and succinimide at a ratio of 1Mn porphyrin to 10 of each reagent. At higher ratios the IgG conjugate precipitated, presumably due to activation of more than one carboxyl group per Mn porphyrin molecule. When Mn porphyrin that had been activated by this procedure was incubated with antibody protein, up to 36 Mn porphine5 were incorporated into each antibody, although the protein-boundMn porphyrin chemiluminescenceactivity was lower than that of free Mn porphyrin (Table 11). This table shows that binding of the catalyst to protein interfered with the chemiluminescence reaction but that increasing the binding ratio did not progressively decrease the chemiluminescence activity of the incorporated metal porphyrin. This indicates that bound metal porphyrins did not interfere with each other even though up to 19% of the antibody metal porphyrin conjugate consisted of metal porphyrin. (17)White, E. H.; Zafkiou, 0.; Kagi,H. H.;Hill, J. H.M.J. Am. Chem. Soc. 1964,86,940. (18)George, P.J. J. Biol. Chem. 1963, 201, 413. (19)Dawson, J. H.Science 1988,240,433.
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9
P
w 0
z
W
0
3 z
I .17
-16
-15
-14
-13
-12
Moles of Antibody-Mn-porphine
Fbure 9. Standard curve for metal porphyrin antlbody conjugate. One-mlnute light measurementswere made from 1 mL solutions of 1 mM iuminoi, 0.1% linoleic acid, and 0.1 mM NaOH.
3H w
I 0
+
o
10
20
30
40
50
Reaction Time (min) Flgure 5. Metal porphyrin chemilumlnescence reaction time course. A 10 ng/mL plasma a-fetoprotein sample was repeatedly assayed. Chemiluminescence units are relative “Dynatech” unlts.
Plasma AFP (ng/ml) Flgure 4. Plasma a-fetoprotein standard curve. Each point is the average from three separate assays of 20-pL plasma samples (200pL assay volume). Chemliumlnescenceunits are relative “Dynatech” units.
Figure 3 shows a dose-response curve for chemiluminescence obtained with the best conjugate. The detection limit was 6.8 X lo-” mol. An alternative conjugate synthesis procedure was optimized to couple many metal porphyrins to one antibody binding site via a BSA carrier. This was expected to improve immunoassay sensitivity by coupling many metal porphyrins to one antibody binding site while preserving ita activity. The Fab-BSA-Mn porphyrin conjugate made contained 32 Mn porphyrins and 1 BSA molecule per Fab. Coupling of metal porphyrin to protein by this procedure resulted in 34% loss of ita chemiluminescence activity. When compared by use in the immunoassay the Fab-BSAMn porphyrin conjugate showed a 25 times lower AFP detection limit than the best Mn porphyrin-IgG conjugate (20vs 500pg, respectively). A representative standard curve for blood plasma AFP obtained using the Fab-BSA-Mn porphyrin conjugate is shown in Figure 4. The detection limit in this experiment was 5.2 ng of AFP/mL of plasma. A comparison of 32 plasma samples containing different concentrations of AFP showed good correlation (R= 0.99),within assay variability (CV = 9.3% 1, and between assay variability (CV = 6.3%). These data indicate the metal porphyrin chemiluminescence system can be used for assay of analytes from blood samples down to the femtomole level. Two desirable features of the metal porphyrin chemiluminescence method are the persistance of light emission and the stability of detection solution. A time course for generation of chemiluminescence light from 0.2 ng of AFP obtained by immunoassay is shown in Figure 5. This figure indicates measurements can be made from 1to 20 min after adding the luminol solution. Measurementa were typically made 7 min after adding the detection reaction solution. Because of the high light output from these reactions the PMT voltage had to be adjusted to medium, and black
”
’“1 0
10
20
30
40
Days After Mixing Figurb 6. Stability of detection reaction solution. One nanogram of metal porphyrln was added to 1-mL samples at various times after preparation. microtiter plates were used to prevent overloading the photomultipliertube. The 1.0m M luminol and 0.1% linoleic acid in 0.1 M NaOH reaction solution used for the reaction was unusually stable as can be seen in Figure 6. This data indicates the reaction solution improves with age with a doubling of light output during 40 days of storage. The light produced intheabsenceofMnporphyrincatalyst (background) increased less than 50% during this time, thus the sensitivity (l.Ong/mL Mn porphyrin signallbackground signal) improved somewhat during storage. The reason for the change during storage is unknown. Perhaps a high pH labile inhibitor was slowly removed or there was conversion of enhancer to a more active form. Although luminol solutions were usually prepared weekly, solutions as old as 3 months were occasionally used without a noticeable decrease in performance. Glass containers were not used for the high pH solutions because they leached metals and caused high backgrounds. Also, the quality of water had some effect and we found deionized water to work better than water that had been only distilled.
CONCLUSIONS The metal porphyrin chemiluminescence system had good sensitivity (68 am01 of antibody conjugate). The recommended reaction solution is 1 mM luminol and 0.1 % linoleic acid in 0.1 M NaOH. The mechanism of the catalytic reaction of metal porphyrin with luminol is unknown although the dianion form of luminol is probably important because of the high pH (pH 13) required. It is concluded that manganese porphyrin having meso(4)tetra electron-withdrawinggroups is the best catalyst although many metal porphyrin were not studied and others may be suitable. The mechanism of
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enhancement by long-chain fatty acids and by Tween-20 is unknown although micelles of enhancer are not involved because enhancement occurred at enhancer concentrations lower than the critical micelle concentration. Coupling as many as 36 metal porphyrins to proteins using the procedures developed resulted in up to 50% loss of the metal porphyrin catalytic activity. The reason for the loss in activity upon coupling is unknown. The BSA carrier metal porphyrin synthesis technique is recommended for coupling to proteins such as antibody fragments. No particular problems were experienced upon use of this chemiluminescence technique in a sandwich type immunoassay for a-fetoprotein. Virtually any chemiluminescenceinstrument can be used for light measurement because the light output is
high and sustained for many minutes. Because of the high stability of the reaction solution this chemiluminescence system could be used in analytical procedures where convenience and stability of reagenta are concerns.
ACKNOWLEDGMENT The authors wish to thank Mr. K. Oda for technical assistance, Dr. S. Terau and Dr. T. Sugawara for encouragement, and Dr. M. Nishikawa for permission to publish.
RECEIVED for review June 30, 1992.
18, 1992. Accepted September
