1114
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
Chemiluminescence Yields and Detection Limits of Some Isoluminol Derivatives in Various Oxidation Systems Hartmut R. Schroeder" and Frances M. Yeager Ames Research and Development Laboratory, Miles Laboratories, Inc., Elkhart. Indiana 465 14
A number of chemiluminescent compounds were evaluated as potentlal nonisotopic labels for monitoring competltive protein-binding reactions. I n a novel approach, several detection reactions were used to measure such chemiluminescent labels at picomolar and subpicomolar levels. The concentration of chemilurnlnescent compounds was llnearly related to peak light intensity and to total light emitted. Their light yields were reduced 20- to 50-fold after covalent attachment of thyroxine. I n contrast, a similar biotin conjugate produced at least one half the chemiluminescence. The latter was detectable at a concentration of IO-" M, when oxidlred by H202and microperoxidase at pH 12.6. The corresponding thyroxine conjugate was not detectable below IO-" M. The most efflclent thyroxine conjugate, naphthalhydrazide 27,was M. Quenching of chemiluminescence detected at 2 X by thyroxine was apparently due to the iodine atoms, and thus should be absent in other noniodinated conjugates. The H20,-microperoxidase system was the most sensitive, versatile, and reproducible detection system explored. Similar sensltivity was obtained with reactions at pH 8.6 to 13.0. The average peak light intensity from reactlons performed in trlplicate was reproducible to f 5 %.
T h e oxidation of luminol (5-amino-2,3-dihydrophthalazine-l,&dione) leads to formation of aminophthalate ions in the excited state which emit light upon decay. In aprotic solvents, such as dimethyl sulfoxide and dimethylformamide, only oxygen and base are required for the reaction, whereas in alkaline aqueous systems an oxidizing agent and catalyst are necessary ( I ) . Many oxidizing agents and metal ion catalysts have been utilized under aqueous conditions (2-4) and tetrabutylammonium hydroxide and potassium tertbutoxide ( 5 )have been most commonly used with nonaqueous systems. Furthermore, hydrogen peroxide and peroxidase a t neutral pH (6, 7) and superoxide anion generated by xanthine oxidase at p H 10 ( 8 , 9 ) have been used to oxidize luminol in aqueous systems. The quantum yield of the chemiluminescent reaction depends on both the structure of the phthalhydrazide derivatives and the oxidation reaction conditions. For luminol, the yield is about 5% in dimethyl sulfoxide (1)and about 1.5% in aqueous systems (10). Electron donating substituents a t positions 5 and 8 of phthalhydrazide increase light yield more than a t positions 6 and 7 and electron withdrawing substituents on the benzene ring decrease chemiluminescence efficiency (11,12). Substitution on the heterocyclic ring leads t o complete loss of light production (13, 14). T h e relatively high quantum efficiency of the aminophthalates suggested their possible use as labels to monitor competitive protein binding reactions in assays currently employing radiolabels to determine hormones, drugs, and metabolites a t low levels in body fluids. We decided to attach ligands of interest through an alkyl bridging group to the amino function of isoluminol (6-amino-2,3-dihydrophthalazine-l,.l-dione), Although isoluminol has only about 10% the efficiency of luminol, alkylation a t this position leads to 0003-2700/78/0350-1114$01 .OO/O
several-fold increase in chemiluminescence capability, whereas similar substitution on luminol leads t o steric hindrance resulting in a large decrease (10, 15, 16). In a earlier study, a biotin-isoluminol conjugate was synthesized which was detectable a t 5 nM concentration (17). The present objective was to synthesize more efficient chemiluminescent labels and to investigate the effect of attachment of various bridging groups and ligands on light yields. Whereas, previous chemiluminescent systems were designed to detect the catalyst or oxidant of the reaction, the measurement of the chemiluminescent compound itself represents a novel application. T o measure these labels in the nano- and picomolar range, numerous oxidation systems were investigated and those of merit are presented.
EXPERIMENTAL Light Measurements. Chemiluminescent reactions were carried out in 6 X 50 mm test tubes mounted in a DuPont 760 Luminescence Biometer, with a sensitivity setting of 820. Each tube contained a chemiluminescent compound in a reaction volume of 150 pL. The reagents varied according to the oxidation system employed. A 10-pL aliquot of the designated oxidant or substrate was hand-injected from a 25-pL Hamilton syringe with an unbeveled needle. The peak light intensity (PLI) occurring during the reaction was measured. All reactions were done in triplicate and reported values represent the average. Near the end of this study, a custom-built solenoid pump equipped with a sapphire piston in a Teflon cylinder was used t o add HzOz in order to improve reproducibility of chemiluminescence measurements. The flow of liquid to and from the pump was regulated by Teflon valves (Series 2, Model 1 solenoid valve, General Valve Corp., East Hanover, N.J.) which were operated by solenoids and coordinated electronically. Teflon tubing (0.95-mm i.d. and 0.3-mm wall thickness) carried the oxidant to a glass orifice (0.78-mm id.) which was positioned over the reaction tube in front of the photomultiplier. The pump delivered 10.1 f 0.06 pL of solution in 30 ms. Total light measurements were made by recording the light output with a Hewlett-Packard recorder (Model 7100B-19-22)and integrating the areas under the curve with a planimeter. Later measurements were made with a custom-built microprocessor connected to the Biometer. This instrument monitored the signal output of the Biometer at 1-or 10-msintervals continuously over the chosen run time. Peak light intensity, total light, and time to peak values were obtained from the print out. Solutions of Chemiluminescent Compounds. Stock solutions of luminol, isoluminol, and various isoluminol derivatives were made in glass vials at about 1mM in 0.1 M NaZCO3,pH 10.5, and kept at 4 "C. Lower solubility of the aminonaphthalhydrazides required solution to be made in 0.1 M Na2C03-0.15 M NaOH, pH 12.6. These stock solutions were usually diluted in polystyrene tubes to the desired concentrations in water just prior to an experiment. Later in the study, dilute solutions of the thyroxine-isoluminol conjugates were made in 75 mM barbital (5,5-diethyl barbiturate) buffer, pH 8.6, and glass tubes because this resulted in greater stability. Solutions of Heme Catalysts. A stock solution was prepared in a glass vial by dissolving 1 mg of microperoxidase (M 6756, Sigma Chemical Corp.) in 2.5 mL of 10 mM Tris-HC1, pH 7.4. This 200 pM solution was stable at 4 "C for at least 1 month. The stock solution was diluted in glass tubes t o 2 pM concentration with 75 mM barbital buffer, pH 8.6, just prior to use, although diluted solutions were stable for 2 h or more. 0 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, JULY 1078
A catalase (Worthington Biochem. Corp.) solution at 456 000 units/mL (68000 units/mg) was diluted 100-fold in 10 mM Tris-HC1, pH 7.4, just prior to use. Deuterohemin (a gift from Winslow Caughey, Department of Biochemistry, Colorado State University) stock solution was prepared a t 345 pM concentration in 0.1 M NaOH and diluted to 0.35 pM with water just prior to use. This solution gave variable results after several days, and stock solutions prepared in buffers at pH 7.4 or 10.5 decreased in catalytic activity even more rapidly, probably due to aggregation phenomenon. A 1mM hematin (Sigma Chemical Corp.) stock solution in 0.1 M Na2C03,pH 10.5,4 OC, was allowed to age at least 1 week to reduce variability when used for the assay. After 1 week, autooxidation or other aging effects of hematin continued more slowly and a concurrent gradual decrease in its activity in the chemiluminescent reaction was observed. However, stock solutions were usable for at least 2 months by increasing the amount used for the chemiluminescent reactions two- to three-fold as required. Stock solutions were diluted to 1pM with water just prior to use. Oxidation Systems. Hz02-Microperoxidase. Reaction mixtures (150 pL) of the following composition were assembled: 50 mM NaOH, 57.5 mM barbital (preadjusted t o pH 8.6), 0.27 pM microperoxidase (penultimate addition), and chemiluminescent compound at indicated concentrations. Final pH of the solution was pH 12.6. After a 10-min incubation, a 10-pL aliquot of 90 mM HzOz in 10 mM Tris-HC1, pH 7.4, was injected to produce chemiluminescence. Later in the study, the assay was simplified by premixing NaOH and microperoxidase 10 min prior to use and adding a 55-yL aliquot to the chemiluminescent compound in 95 pL of 7 5 mM barbital buffer, pH 8.6, attaining the same final concentrations. In an alternate procedure, the chemiluminescent reaction was carried out at pH 8.6. The composition of the reactant solution was: 75 mM barbital, pH 8.6, 0.27 pM microperoxidase and chemiluminescent compound at indicated concentrations. After a 10-min incubation, a 10-pL aliquot of 3 mM HzOzin 10 mM Tris-HC1, pH 7.4, was injected into the reactant solution to produce chemiluminescence. HzOz-Catalase. Reaction mixtures (150 pL) were of the following composition: 50 mM NaOH, 57.5 mM barbital (preadjusted to pH 8.6), 304 units of catalase (added last), and chemiluminescent compound (final pH 12.6). After 10 min, a 10-pL aliquot of 9 mM HzOzwas injected to produce chemiluminescence. H20z-Deuterohemin. Reaction mixtures (150 p L ) containing chemiluminescent compound in 50 mM NaOH with deuterohemin (penultimate addition) at 0.04 pM concentration were incubated for 10 min and then injected with 10 pL of 9 mM HzOzto produce light. HzOz-Hematin. Reaction mixtures (150 p L ) containing chemiluminescent compound in 50 mM NaOH with hematin (penultimate addition) at 0.07 pM concentration were incubated for 10 min and then injected with 10 pL of 90 mM HzOzto produce light. HypochEorite-CoClz. A solution (150 pL) of chemiluminescent compound was made 333 pM in CoC1, and 0.05 M in NaOH and 10 p L of 10 mM NaOCl was injected t o initiate light production. Persulfate. A solution (150 pL) of chemiluminescent compound in 0.05 M NaOH was injected with 10 p L of 0.2 M KzSzOs (pH 4.7) to initiate chemiluminescence. KOz. Chemiluminescent compounds were dissolved in 140 p L of (a) dry dimethylformamide, (b) 0.1 M NaZCO3,pH 10.5, or (c) 0.1 M Tris-HC1, pH 8.0. Ten microliters of dry dimethylformamide containing 50 mM KOz and 100 mM 1,4,7,10,13,16hexaoxacyclooctadecane(to solubilize KOz) was injected to initiate light production. NaZO,. Ten microliters of 50 mM NaI04 was added to a solution (150 wL)of chemiluminescentcompound in 50 mM NaOH to produce light. Hz0z-K3Fe(CN)6. A solution (150 pL) of chemiluminescent comDound in 50 mM Na,CO,. DH 10.5. was iniected with 10 WL of a'l mM HZOz-5 pMi(,fe(CN), solution i o produce light. HzOz-Lactoperoxidase. A solution (150 pL) of chemiluminescent compound and 10 pg lactoperoxidase in 0.1 M Tris-HC1 (a) pH 8.8 or (b) pH 7.4 was incubated a t 25 "C for 2 min. Then 10 pL of 1 mM HzO2in 10 mM Tris-HC1, pH 7.4, was injected
1116
t o initiate the light yielding reaction, Hypoxanthine-Xanthine Oxidase. A solution (140 p L ) of chemiluminescent compound, EDTA (previously neutralized) at 0.89 mM, containing 9.1 X IU of purified xanthine oxidase in 0.11 M Na2C03,pH 9.5, was incubated for 15 s after enzyme addition. Twenty microliters of 5 mM hypoxanthine previously adjusted with base to pH 7.5 was injected to initiate the chemiluminescent reaction. The xanthine oxidase (Sigma Chemical Co., St. Louis, Mo.) was purified on a Sephadex (2-25 column to remove (NH4)&304 from the suspension, since the salt interfered with the chemiluminescence. IO]-Potassium tert-Butoxide. A solution (150 pL) of luminol in dimethylsulfoxide was injected with 10 pL of 0.2 M potassium tert-butoxide in dimethyl sulfoxide. General Methods. The water used throughout this study was charcoal filtered and glass distilled. All glassware was soaked in concentrated nitric acid overnight and rinsed extensively with distilled water. Polystyrene tubes and bacterial culture tubes were used directly as obtained from the supplier. All dilutions were done with disposable glass pipets (untreated) and pneumatic pipettors with disposable plastic tips. Synthesis of Chemiluminescent Compounds. The synthesis of various aminophthalhydrazides and aminonaphthalhydrazide and their conjugation to thyroxine are shown schematically in Figures 1-3 and reported elsewhere (18). Compounds utilizing the 6-aminohexyl bridging group were synthesized in a similar fashion as those with the 4-aminobutyl group (18). RESULTS AND DISCUSSION Evaluation of Chemiluminescent Detection Reactions. In screening various oxidation systems capable of producing chemiluminescence, both enzymatic and nonenzymatic catalysts were evaluated according to the following rationale. Reactions were conducted at several p H values between 7 and 13 in each system. Light production was initiated by addition of oxidant or substrate t o otherwise complete reaction mixtures containing chemiluminescent compound. With luminol (Table I), the peak light intensity was generally attained within 2 s and light output returned to background levels in 1 to 2 min. All reactions produced some background light signal in the absence of chemiluminescent compounds. The signal t o background ratio was optimized to maximize sensitivity. Therefore reagents were not necessarily a t saturation levels. The concentration of chemiluminescent compounds was linearly related to both the peak light intensity and total light produced by the reaction, as previously reported (17) and illustrated in Figure 4 with 26 and 27. In most cases, we utilized the more rapid and convenient peak light intensity measurement. The sensitivity of various oxidation systems for detection of luminol is shown in Table I. The potassium tert-butoxide-dimethylsulfoxide system allowed measurement of luminol a t a lower limit of 4 X M. T h e reason for the lack of greater sensitivity, expected because of the high quantum efficiency of luminol in this nonaqueous system (IO), is not known. With KOz as an oxidant, tenfold greater sensitivity was achieved in dimethylformamide. Furthermore, KO2 oxidized luminol in aqueous solutions as well. However, water had to be rigorously excluded from the oxidant prior to injection in order to prevent its rapid decomposition. At higher p H (10.5), in carbonate buffer, generation of longerlived carbonate radical intermediates (19) may have enhanced the superoxide systems sensitivity. T h e popular ferricyanide-catalyzed system, involving one electron oxidation of the luminol dianion (20), was of intermediate sensitivity (Table I). We also used a number of metal ions, as catalysts with hydrogen peroxide or hypochlorite as oxidant. A cobalt-hydrogen peroxide complex has been postulated as the oxidant in one system (21). We found that cobalt also enhanced chemiluminescence with hypochlorite (22)as the oxidant and were able to detect luminol at 1x 10-l'
1116
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
/
?
HO
0
CH2CHCOOCOOC2Hg
HO+O
NHCOCF3
I'
I 6
i
I 7
9;R 1U;R
-
CH2CH3 H
Flgure 1. Synth sis of 6-(N-ethyl-N-[2-hydroxy-3-(thyroxinylamido)propyl]amino)-2,3-dihydrophthalazine-1,4-dione. a Synth sized as in 17
&WCHz-W
17, n = 4 12, n = 6
0
13; n = 4 14: n = 6
0
i
1
NHzNH2
NHzNH2
17' n
14. n
8
15; n = 4 16, n = 6
-
19; n = 4 20; n = 5
4 = 6
21; n 22, n
--
4
0
6
Flgure 2. Synthesis of 6-{N-ethyl-N-[6-(thyroxlnylamido)aIkyl]amlno)-2,3-dlhydrophthalazlne-l ,441one
M (Table I). The persulfate system, for which a radical mechanism is suggested (9),gave similar sensitivity. A maximum light signal was noted in less than 1 s, the signal decreased sharply and a gradual second peak was produced by 15-20 min, and the reaction was complete by about 30-45 min. The almost instantaneous first peak was abolished by the presence of chelating agents such as 8-hydroxyquinoline or o-phenanthrolene, whereas the second peak was unaffected. This system suffered from variability due to extraction of metal ions (which catalyzed the first peak) from the syringe used to add persulfate and the second peak was too slow for routine total light measurements. Oxidation with ferricinium tetrachloroferrate, sodium perborate, and 2,5-dimethyl2,8bis(hydroxyperoxyl)hexane permitted detection of luminol
in alkaline solution at 0.1, 1.0, and 2.0 nM, respectively (data not shown). The enzymatically catalyzed reactions were less sensitive than nonenzymatic reactions, but had the advantage of being active a t neutral to moderately alkaline pH. With lactoperoxidase at optimum pH of 8.8 for chemiluminescence, luminol was detectable to as low as 5 X lo-" M (Table I). Slow generation of superoxide anion (8) in the xanthine oxidase reaction produced light intensity with a maximum in about 1 min, returning to background within 2-3 min. Thus, accurate total light measurements were feasible. The alkaline oxidation systems with hydrogen peroxide and heme containing catalysts gave the greatest sensitivity, 1 x M luminol (Table I). However, autooxidation (23)of the
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
HO
1117
2%
Figure 3. Synthesis of 7- [ N-ethyl-N-(4-thyroxlnylamldo)butyl]amlnonaphthalene-l,2-dicarboxyllc acid hydrazide
Table I. Sensitivity of Detection of Luminol in Various Oxidation Systems a Detection limit,b Oxidation system PH PM 8.6 1 H,O, -Microperoxidase 12.6 1 H, 0 ,-Microperoxidase 12.6 1 H,O ,-Catalase 13.0 1 H, 0,-Deuterohemin 13.0 1 H,O -Hematin 13.0 10 Hy pochlorite-CoC1, 13.0 10 Per sulf ate 10.5 10 KO, 10 KO, 13.0 20 NaIb, 10.5 40 H,O,-K,Fe(CN), KO, 8.0 400 H,0,-Lactoperoxidase 8.8 50 H,O, -Lactoperoxidase 7.4 300 9.5 250 Hy poxanthine-Xan thine Oxidase" [ 01-Potassium tert-butoxide? 400 a Luminol at various levels was oxidized in each system as described in the Experimental. Chemiluminescence measurements related PLI to concentration, The lower limit was defined as about 1.5-2 times background. Concentrations shown are those in the 160-pL reaction prior to injection of oxidant or substrate. KO, was solubilized in anhydrous dimethylformamide by making solutions 100 mM in 1,4,7,10,13,16-hexaoxacyclooctadecane (18-crown-6). Solutions were kept in rubber capped vials to protect against decomposition of KO, by atmospheric moisture. A 1 mM H,O, solution containing 5 p M K,Fe(CN), was injected. e Twenty pL of 5 mM hypoxanthine was injected into 140 pL of reaction mixture. f A 10-pL sample of 0.2 M potassium tert-butoxide was injected into 150 pL of dimethylsulfoxide containing luminol at various levels. vinyl side chains on hematin as well as aggregation (24) also observed with deuterohemin in stock solutions made them less appealing, although they worked well as catalysts during assay periods. In contrast, catalase and microperoxidase solutions were quite stable and reproducible, perhaps because the protein portion stabilized the heme *moiety. Furthermore, microperoxidase (a hydrolysis product of cytochrome c
consisting of heme attached to a 11 amino acid chain) was exceptional in that it alone catalyzed luminol oxidation equally well at pH 8.6 as at 12.6. In control reactions, addition of HzOZ produced no light signal with luminol in the absence of microperoxidase. Sensitivity, stability, and versatility make the HzOzmicroperoxidase system the method of choice for detection of chemiluminescent compounds. Similar peak light intensity values were obtained in reactions with luminol at p H 8.6 in barbital, glycine, phosphate, borate, and 2-amino-2methyl-1,3-propanediol buffer, but values were one-third in Tris-HC1 and bicine prevented all light emission. However, lower pH drastically reduced light yields, while equally sensitive systems (results not shown) were possible at pH from 8.6 to 13.0. Relative Chemiluminescence of Labels. Several chemiluminescent labels were tested in five oxidation systems. The peak light intensity was measured as a function of derivative concentration and expressed as a percent of that observed with luminol. Since relative light yields depend somewhat on the oxidation system employed, comparisons between the derivatives shown in Table I1 can be made only within one oxidation system. Alkylation of the amino function of isoluminol generally increased chemiluminescence (Table 11),as has been reported with quantum measurements (10). The 4-aminobutyl side group increased chemiluminescence considerably more than the 3-amino-2-hydroxypropyl group. Ethylation of these derivatives introduced a second alkyl group and resulted in two- to fivefold light yields, but exceptions were observed.
6-[N-(4-Aminobutyl-N-ethylamino]-2,3-dihydrophthalazine-l,Cdione, 19, produced less chemiluminescence than the unethylated derivative, 17, in the enzymatic and persulfate systems, but more in the remaining systems. Electronic interaction or steric influences of the attached groups are probably responsible for lower light yields with those particular oxidation mechanisms. The relative efficiencies of the oxidation systems themselves are seen by comparing the lower limit of detection for luminol for each (Table 11). Also, relative light yields of isoluminol derivatives are nearly the same in the hypoxanthine-xanthine oxidase system whether evaluated by peak light intensity or total light measurements. Similar results have been reported for relative quantum efficiency of a series of substituted
1118
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1878
Table 11. Relative Light Yields of Chemiluminescent Derivatives in Various Oxidation Systems HypochloriteHzOaHypoxanthine-Xanthine Oxidation oxidase COCl, Persulfate system" lactoperoxidase Light measurement Compounde Luminol 17 19
Peak,b %
Limit,c PM
100
30
50
100
Peak,b Total,d %
%
100 96 29 29 24 4.5
100 109 27 28 26 6
Limit: PM
Peak,b Limit,c Peak,* % PM % 10 50
100 9 31 5.5 2.5 2.5
250 250 500
100 31
Limit,c PM
H,O,-hematin Peak,* Limit,c % PM
10
100
50 50 250 1000
14
1 20 2 5 20
84 27 30 100 22 46 4.5 100 500 8.3 250 4 10.4 1.5 100 5 00 5.1 1000 5 4.6 30 1.3 1000 100 2-4000 1000 1.4 Isoluminol a Oxidation reactions with various compounds and chemiluminescence measurements were performed as in the Experimental. (The H,O,-Lactoperoxidase system was used at pH 8.8.) Calculated as a ratio of the change in peak light intensity per unit change in concentration over the range of isoluminol derivative, to luminol, times 100. Approximate lower limit (pM) for certain detection. Concentrations shown are those in the 1 5 0 - p L reactions prior to injection of Total light produced, (%) of luminol as in b. e Chemiluminescent compounds at 7-11 X lo-, M oxidant or substrate. in 0.1 M Na,CO,, pH 10.5, were diluted in water just prior to use.
Table 111. Relative Light Yields of Isoluminol Derivatives and Their Ligand Conjugates in Heme Catalyzed Reactions A,minophthalhydrazidesa
H,O,-hematin systemC ChemiDetection limit, luminescence b PM
NH
0
Luminol Isohminole Diethylisoluminolf 5 4 10 9 17 19 21 18 20
...
R,
-H -CHaCH, -H -CH,CH, -H -CH,CH2 -H -CH,CH, -CH,CH, -H -CH,CH,
...
R,
-H -CH,CH, -CH,CH(OH)CH,NH, -CH,CH( OH)CH,NH, -CH,CH( OH)CH,NHCO-T, -CH,CH( OH)CH,NHCO-T,
,"
-(CH2)4NH2
-(CH, ),", -(CH,),NHCO-T, -(CH,),", -(CH,),",
100
1
5 100
30
10
20 5 200 100 20 2 100
46
1 2 14 84 2 17 44
1
10 5
H,O,-microperoxidase systemC Luminol 20 22 5
Biotinyl-58
...
...
-CH,CH, -CH,CH, -H -H
-(CH,),", -(CH,),NHCO-T, -CH,CH(OH)CH,NH,
100
-CH,CH(OH)CH,NHCO-biotin
78 1 5 5
1 2 200
10 10
Aminonaphthalhydrazidesh
0.1 (0.5),' 420 (120),i -CH,CH, -(CH,),", 20 (3.2)' 2.0 (l0)Z -CH,CH, -(CH,),NHCO-T, a Synthesis outlined in Figures 1 and 2. Peak light intensity is expressed as a percent of that observed with luminol (100%)as in Table 11. Reactions were performed at pH 12.6 as described in the Experimental. T, refers to thyroxine (L-tetraiodothyronine)linked through the carboxyl group, e Synthesized as described in ref. 25. Synthesized according to Gundermann et al., ref. 15. Synthesis is described in ref. 17. Synthesis outlined in Figure 3. IReactions were conducted with the H,O,-microperoxidase system at pH 8.6. 26 27
phthalhydrazide derivatives, although total light yields were slightly higher (15, 17). Therefore, the relative peak light intensity values in any one oxidation system probably closely reflect relative quantum efficiency of the derivatives. Relative Light Yields of Label Conjugates. A number of the isoluminol derivatives were covalently linked to a ligand of special interest to us. Several labels, NJ-diethylisoluminol (15),4, 19, and 20, produced chemiluminescence with nearly as great efficiency as luminol and were detectable at as low as 1 to 5 picomolar in our most sensitive detection systems
(Table 111). However, covalent attachment of these labels to thyroxine led to compounds 9, 21, and 22, which had 1 to 2 % the chemiluminescence capability, reducing sensitivity to 100 to 200 picomolar (Table 111). Increasing the length of the bridging arm between thyroxine and the chemiluminescent label (structures 9 vs. 21 and 22) did not reduce this loss in quantum yield. Furthermore, the chemiluminescence enhancing effect of the second alkyl group was decreased (structure 10 vs. 9). Thyroxine itself had no effect on the peak light intensity produced with 19, even when present in
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
0 Compound A
26
Compound 27
(10”’M) (lo-” M)
Figure 4. Quantitation of chemiluminescent compounds by peak light intensity produced durlng oxidatlon. Reaction mixtures (150 pL) contained the (0)arninonaphthylhydrazide 26 or its (A) thyroxine conjugate 27 at indicated concentrations, 0.27 pM microperoxidase, 57.5 rnM barbital and 50 mM NaOH (final pH 12.6). Manual addition of 10 pL of 90 rnM H202initiated light production. Values are averages from reactions performed in triplicate
1000-fold molar excess, which indicated the absence of an intermolecular effect. The quenching effect was not overcome by changing polarity, viscosity, and temperature of the chemiluminescent reaction mixture. Thus, the loss of quantum efficiency brought about by conjugating the label to T4was undoubtedly due to the presence of the rather bulky iodine atoms, which are known to be excellent fluorescence quenchers (26,27). In contrast, the label 5 attached to biotin produced identical light yields in the conjugate biotinyl-5 in the HzOz-lactoperoxidase system (17), whether compared by peak light intensity or total light measurements in either system. However, 5 and biotinyl-5 were 5% as efficient as luminol when judged by peak light intensity and 15% as efficient by total light yield in the HzOz-microperoxidase system. At any rate, the biotin conjugate biotinyl-5, was detectable at 1 X M whereas the thyroxine conjugate 10, being less efficient, was detectable at only 1 X M. These results lead to the conclusion that isoluminol derivatives conjugated to ligands other than thyroxine should lose little of their quantum efficiency in chemiluminescent reactions. The aminophthalhydrazides with three- to fourfold greater quantum efficiency as luminol (28) presented a further opportunity to increase sensitivity. Indeed, the label 26 was measured a t levels as low as 0.1 pM (0.015 femtomole) with the H&-microperoxidase system, pH 12.6 (Table I11 and Figure 4). Less efficient detection at pH 8.6, 0.5 pM 26, may be related to lower solubility a t less alkaline pH. Detection of the thyroxine-naphthylhydrazide conjugate 27 was possible to a lower limit of 2 pM, whereas a similar aminophthalhydrazide conjugate 21 was limited to 100 pM a t pH 12.6. Thus, greater efficiency of the label 26 and lower quenching by the thyroxine moiety in this conjugate resulted in a substantial increase in sensitivity. Chemiluminescence Measurements. Improvements in instrumentation constituted another approach to enhance sensitivity as well as reproducibility of chemiluminescence measurements. Consequently, several methods of initiation of the light reaction and measurements were compared in the best oxidation system with various chemiluminescence derivatives. The reproducibility of chemiluminescence measurements was demonstrated in the H20z-microperoxidase system since it was the most versatile and sensitive oxidation system tested. The mean PLI produced with 15 identical reaction mixtures at p H 12.6 containing the thyroxine conjugate 21 (1.67 nM) had a coefficient of variation of f18%. By evaluating these reactions in triplicate, this variability was reduced to *5%. However with highly efficient compounds such as luminol(11.3
1119
pM) or N,N-diethylisoluminol, the mean P L I for triplicate reactions still varied by k12.7%. Perhaps more rapid light production with efficient compounds increased variability in the PLI values. Indeed, peak light emission with diethylisoluminol, determined with the microprocessor, was attained nearly twice as fast (about 0.7 s) as with the thyroxine conjugate at pH 12.6 (about 1.3 s), but similar (0.2 to 0.3 s) in the more rapid reaction at p H 8.6. A custom-built automatic injection device, which allowed the peroxide to contact only Teflon and glass, reduced the coefficient of variation of the mean PLI of triplicate reactions to h 5 % or better at either pH 12.6 or 8.6. This device also circumvented the problem of interaction of the HzOz with metal in the manual method which required use of a new Hamilton syringe when variability in PLI became high. The stability of reaction mixtures and reproducibility of their assembly were determined with batch reactions containing 51.4 pM N,N-diethylisoluminol, using this device. The respective PLI values (n = 28) a t 10 min and 1 h were 40.7 f 3.1 and 40.4 f 2.2, indicating complete stability of the reaction mixture for this time period. Batch reaction mixtures assembled the next day produced a mean PLI ( n = 28) of 43.5 f 2.7, which amounted to 6% difference between only two sets. Since peak light intensity values are sensitive to mixing speed and kinetics, we hoped to improve reproducibility with total light measurements. Light emission decayed from the peak value to one half in 4.5 s at pH 12.6 and in 0.5 s a t p H 8.6 with either N,N-diethylisoluminol or conjugate 21. Therefore rapid total light measurements were feasible, especially at pH 8.6. However, we were unable to improve either sensitivity or reproducibility with total light measurements. Perhaps air bubbles trapped in the reaction mixture during the forceful injection of peroxide resulted in variable light scatter which would affect reproducibility of either peak light intensity or total light measurements. Nonetheless, some of the other detection systems might benefit from such measurements. To summarize, chemiluminescent conjugates are readily synthesized, stable and detectable with great sensitivity (picoto nanomolar). They may be used as labels to monitor competitive protein-binding reactions, as demonstrated in assays to determine biotin (17)and thyroxine (18). Rapid peak light measurements with the HzOz-microperoxidase system at pH 12.6 and 8.6 are compatible with monitoring competitive protein-binding reactions in a heterogeneous and homogeneous manner, respectively. Improved mechanical injection or utilization of a flow system should increase precision and complete automation of the detection reaction is possible. ACKNOWLEDGMENT We thank R. T. Buckler and E. 0. Snoke for the synthesis of chemiluminescent compounds which made this study possible. We are also grateful to P. 0. Vogelhut and R. Rogers for invaluable suggestions, as well as design and assembly of instrumentation. Helpful discussions with R. C. Boguslaski and R. J. Carrico and the technical assistance of F. Marmarinos is also appreciated. LITERATURE C I T E D E. H. White, 0. Zafriou, H. H. Kagi, and J. H. M. Hill, J. Am. Chem. Soc., 66. 940-941 (1964). W. R. Seitz and ’ D. M. Hercules in “Chemiluminescence and Bioluminescence”, M. J. Cormier, D. M. Hercules and J. Lee, Ed., Plenum, New York, N.Y., 1973, pp 427-449. U. Isacsson and G. Wettermark. Anal. Chim. Acta, 68, 339-362 (1974). W. R. Seitz and M . P. Neary, Anal. Chem., 46, 188A-202A (1974). P.D. Wildes and E. H. White, J. Am. Chem. Soc., 95, 2610-2617 (1973). M. J. Cormier and P. M. Prichard, J . Bo/. Chem., 243, 4706-4714 (1968). P. M Prichard and M. J. Cormier, Biochem. Siophys. Res. Commun., 31, 131-136 (1968). G. M. Oyarnburo, C. E. Rego, E. Prodanov, and H. Soto, Biochm. Blophys. Acta, 205, 190-195 (1970). ~
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(9) E. K. Hodgson and I. Friedovich, Phofochem. Phofobiol., 18, 451-455 (1973). (10) R. B. Brundrett, D. F. Roswell, and E. H. White, J . Am. Chem. Soc., 94, 7536-7545 (1972). (11) H. D. K. Drew and F. H. Pearman, J . Chem. SOC.,566-592 (1937). (12) A. Spruit-Van Der Burg, Recuel, 8g, 1536-1544 (1950). (13) H. D. K. Drew and R. F. Garwood, J . Chem. Soc., 1841-1846 (1937). 64, 2644-2649 (14) E. H. Huntress and J. V. K. Gladding, J. Am. Chem. Soc., (1942). (15) K.-D. Gundermann and M. Drawert, Chem. Bet‘., 95, 2018-2026 (1962). (16)R. B. Brundrett and E. H. White, J . Am. Chem. Soc., 96, 7497-7502 (1974). (17) H. R. Schroeder, P. 0. Vogelhut, R. J. Carrico, R. C. Boguslaski, and R. T. Buckler, Anal. Chem., 48, 1933-1937 (1976). (18) H. R. Schroeder, R. C. Boguslaski, R. J. Carrico, and R. T. Buckler, in “Methods in Enzymology”, VoI. 57, M. DeLuca, Ed., Academic Press, New York, N.Y., 1978. (19) K. Puget and A. M. Michelson, Photmhem. Photobbl.,24, 499-501 (1976).
(20) P. B. Shevlin and H. A. Neufeld, J . Org. Chem., 35, 2178-2182 (1970). (21) T. G. Burdo and W. R. Seitz, Anal. Chem , 47, 1639-1643 (1975). (22) W. R. Seitz, J . Phys. Chem., 79, 101-106 (1975). (23) S.B. Brown, P. Jones, and A. Suggett, Trans. Faraday Soc.,64,986-993 (1968). (24) N. A. Brown, R. F. G. J. King, M. E. Shillcock, and S. B. Brown, Biochem. J., 137, 135-137 (1974). (25) H. D. K. Drew and F. H. Pearman, J . Chem. Soc., 26-33 (1937). (26) G.K. Radda, in “Methods in Membrane Biology”, Vol. 4,E. D. Koru, Ed., Plenum Press, New York, N.Y., 1975,p 140. (27) G. R. Fleming, A. W. E. Knight, J. M. Morris, R. J. S. Morrison, and G. W. Robinson, J . Am. Chem. Soc., 99, 4306-4311 (1977). (28) K.-D. Gundermann, W. Horstmann, and G. Bergmann, Ann. Chem., 684, 127-141 (1965).
RECEIVED for review February 23, 1978. Accepted April 10, 1978.
Proton Induced y-Ray Analysis of Atmospheric Aerosols for Carbon, Nitrogen, and Sulfur Composition Edward S. Macias” and C. David Radcliffe Department of Chemistry, Washington University, St. Louis, Missouri 63 130
Charles W. Lewis and Carole R. Sawicki Environmental Sciences Research Laboratory, U S . Environmental Protection Agency, Research Triangle Park, North Carolina 277 1 1
A technique for the simultaneous quantitative determination of carbon, nitrogen, and sulfur using in-beam y-ray spectrometry has been developed for use with atmospheric aerosol samples. Samples are collected on quartz filters, and the aerosol composltlon is determined by analyzing the y rays emitted following the inelastic scattering of 7.0-MeV protons. Samples are nondestructively irradiated for 1000 s in a helium atmosphere, are not subjected to reduced pressures, and can be used for subsequent analysis. Detection limits for atmospheric samples are in the pg/cm* range with a precision of 5 % . The technique is compared with several more conventional methods of analysis.
Many of the adverse effects of air pollution are due to atmospheric suspended particulate material (aerosols). For this reason much work has been done in the past few years to improve the techniques of analyzing these particles with the ultimate goals of understanding the complex processes leading to aerosol formation in the atmosphere and of assessing the effects. Instrumental elemental analysis methods such as neutron activation analysis ( I ) and x-ray fluorescence (2) are currently being used to analyze for many of the chemical elements with atomic numbers between those of sodium and lead. Except for sulfur, these heavier elements are present in only trace quantities in typical atmospheric aerosol samples. The four light elements, carbon, nitrogen, oxygen, and sulfur, account for most of the fine-particle mass ( 3 ) . At present, a convenient, fast, nondestructive and inexpensive technique to measure these abundant light elements is not in general use. These data are essential to determine the mass balance of atmospheric particulate matter and to understand the chemistry of these particles. In this paper, we report on the development of a new nondestructive method for the determination of carbon, nitrogen, and sulfur in atmospheric aerosols. The method is based on y-ray emission following the inelastic scattering of 0003-2700/78/0350-1120$01.00/0
protons. An earlier paper by Shabason and Cohen ( 4 ) also describes this technique. The method takes advantage of the large cross section of many light elements for excitation to a low-lying nuclear state. The resultant rapid y-ray emission is, in general, unique to a particular nuclide and thus can be used as a signature for the chemical element. This analytical method is rapid because the y-ray spectrum is recorded inbeam during a short irradiation. Filters on which aerosol samples have been collected are irradiated without pretreatment, which avoids errors introduced by sample dissolution required for the more conventional chemical analysis. The method measures the total elemental abundance in a sample, not just the fraction that dissolves in a particular solvent. Furthermore, the method analyzes the bulk sample rather than just the surface as measured in techniques such as ESCA. The y rays emitted from carbon, nitrogen, and sulfur are all of energy greater than 2 MeV (half thickness >16 g cm-2);therefore, no y sample absorption corrections are necessary. The proton beam passes through the sample losing less than 0.4 MeV in the entire filter and aerosol deposit, thus no proton absorption correction is necessary. Preliminary reports of the use of this method for atmospheric aerosols have appeared previously (5-10). This paper contains a detailed description of the method and a comparison with other analytical techniques.
EXPERIMENTAL Sample Collection. Samples of atmospheric aerosols used in this work were collected with manual dichotomous virtual impactor samplers (1.2). Such samplers fractionate the aerosol into two size classes, a “fine” fraction consisting of particles having aerodynamic diameters less than 3.5 pm, and a “course” fraction including particles between 3.5 and approximately 20 gm. This classification corresponds approximately to respirable and nonrespirableparticles. The two classes of particles are uniformly deposited on separate filters in each sample. Five samplers were operated simultaneously side by side. The use of replicate samplers generated matched samples which were used in extensive comparison between the y-ray and alternative analytical methods. Two samplers employed 37-mm diameter 0 1978 American
Chemical Society
