858
Anal. Chem. 1981, 5 3 , 658-665
trations are so low that attaining A, = 1.8K in the assay tube from a maximum possible amount of specimen can barely be achieved. After noticing that reactant concentrations which we proposed for measuring least detectable dose and for measuring at high values of A (A, >> K ) agreed with those of Berson and Yalow ( I ) , we repeated the analysis. Using the criterion of maximizing the initial relative slope resulted in the same optimum reactant concentrations as those presented in Figure 2 for all values of A,. Apparently, the criterion of maximizing the initial relative slope is equivalent to that of attaining best normalized sensitivity. Reactant concentrations proposed in this paper, therefore, provide assays characterized by response curves with A, at the midpoint, with best possible normalized sensitivity and with the highest initial relative slope.
LITERATURE CITED Berson, S. A.; Yalow, R. S. Clln. Chlm. Acta 1988, 22, 51-69. Ekins, R. P.; Newman, G. B.; ORiordan, J. L. H. In "Statistics in Endocrinology"; McArthur, J. W., Colton, T., Eds.; MIT Press: Cambridge, MA, 1970; Chapter 19.
Ekins, R. P.; Newman, G. B.; Piyasna, R.; Banks, P.; Slater, J. D. H. J . SteroM Blochem. 1972, 3. 289-304. Rodbard, D.; Lewald, J. E. Acta Endocrnol. (Copenhagen), 1970, SUppl. NO. 747, 79-103. Rodbard, D. In "Principles of Competitive Protein Binding"; Odeli, W. D., Doughaday, W. H., Eds.; Lippincon: Philadelphia, PA, 1971; Chapter 8. Yanaglshita, M.; Rodbard, D. Anal. Blochem. 1978, 88, 1-19. Hales, C. N.; Randie, P. J. Blochem. J. 1983, 88, 137-146. Rodbard, D.; Bridson, W.; Rayford, P. L. J . Clln. Mdocrlnol. Metab. 1968, 28, 770-781. Shaw, W.; Smith, J.; Spierto, F.; Agnese, S. T. Clln. Chlm. Acta 1977, 76, 15-21. Haifman, C. J. Anal. Chem. 1979, 51, 2306-2311. McHugh, R. B.; Meinert, C. L. In "Statistics in Endocrinology";McArthur, J. W., Colton, T., Eds.; MIT Press: Cambridge, MA, 1970; Chapter 22. Schuurman, H. J.; DeLigny, C. L. Anal. Chem. 1979, 51, 2-7.
RECEIVED for review June 23,1980. Accepted December 9, 1980. This work was supported in part by Biomedical Research Support Grants 2-533-730,2-561-730,and 2-582-710, awarded by the Biomedical General Research Support Grant Division of Research Resources, National Institutes of Health, and by the Veterans Administration.
Flavin Adenine Dinucleotide as a Label in Homogeneous Colorimetric Immunoassays David L. Morris," Paul 6. Ellis, Robert J. Carrlco, Fkances M. Yeager, Hartmut R. Schroeder, James P. Albarella, and Robert C. Boguslaskl Ames Research & Development Laboratories and Corporate Chemistry Department, Miles Laboratories, Inc., Elkhart, Indlana 465 15
William
E. Hornby and
Denlse Rawson
Biochemical Products Department, Miles Laboratories Ltd., Stoke Court, Stoke Poges, Bucks, SL2 4 0 0 , United Kingdom
A unique competltlve blndlng method whlch uses a prosthetlc group to label the ligand Is described for the determlnatlon of haptens In solution. The prosthetic group, joined covalently to the ligand, comblnes wlth the appropriate apoenzyme and can be determlned with hlgh sensltlvlty by means of the enzyme actlvlty of the regenerated holoenzyme. Immunoassays are performed wlthout separation of antlbody-bound label from free label since the ability of the prosthetic group resldue to regenerate actlve holoenzyme Is substantlally lnhlblted when the labeled ligand Is complexed wlth Its antlbody. In thls case, the speclflc binding reactlon Is Inltlated, excess apoenzyme Is added, and the resultlng enzyme actlvlty Is related to the amount of unlabeled llgand In the solutlon. This concept has been demonstrated by udng FAD as the prosthetlc group and glucose oxldase as the holoenzyme. Fiavln " 4 2 hydroxy-3-carboxypropyl)adenlne dinucieotlde and flavln N6-(6-amlnohexyl)adenlnedinucleotide have been synthesized and both have been coupled to theophylline resldues. The FAD conjugates were used to demonstrate the concept of the prosthetic group label Immunoassay and to construct a prototype homogeneous colorlmetrlc Immunoassay for theophylline In human serum.
Competitive-binding immunoassays can be classified as either homogeneous or heterogeneous ( I ) . Homogeneous 0003-2700/81/0353-0658$01.25/0
assays have an advantage in that they do not require, at any stage, the physical separation of antibody bound labeled antigen from the unbound form. A wide range of homogeneous immunoassay techniques have been demonstrated which differ from each other basically in the nature of the compound used to label the antigen or hapten. Thus, bacteriophages (2), spin-labeled molecules (3),enzymes ( I ) , fluorescent molecules (4),chemiluminescent molecules (5),enzyme cofactors (6),and enzyme substrates (7) have all been used as labels in homogeneous immunoassays. Here we describe the concept of a homogeneous colorimetric immunoassay in which a prosthetic group, FAD, is used as label. Emphasis is given to the development of reagents, principally FAD conjugates and apoglucose oxidase. However, the utility of the assay for measuring substances at low concentrations in serum is indicated by using an assay for theophylline as a model. Swoboda (8) has shown that glucose oxidase from Aspergillus niger can be dissociated at low pH into FAD and apoglucose oxidase which can then be obtained in stable form after separation from the FAD. He further demonstrated that active glucose oxidase can be rapidly regenerated from the apoenzyme by addition of FAD. In the assay principle envisaged, a conjugate of FAD, covalently bound to a derivative of the ligand to be measured, would be designed so that apoglucose oxidase would be reconstituted to glucose oxidase by the FAD moiety of the conjugate. Moreover, when antibody specific to the ligand is bound to the ligand moiety of the conjugate, it is necessary that the FAD moiety is inhibited 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981 SCHEMATIC ILLUSTRATION OF THE PRINCIPLE
OF THE PROSTHETIC GROUP LABEL IMMUNOASSAY
ANTIBODY
+
LIGAND
ANTIBODY LIGAND
+
-
ANTIEODY LIGAND-FAD
f LIGAND- FAD
APOGLUCOSE OXIDASE
crystals. Recrystallization from 2-propanol with the aid of a Soxhlet extractor gave 96.7 g of white c r y s a n e product. (Anal. Calculated for C6H8C1204SNa:C, 27.20; H, 1.14; C1,26.8; S, 12.1. Found C, 27.27; H, 1.13; C1,26.9; S, 11.85.) The aqueous mother liquor was again reduced to half volume and filtered to give another 57 g of crude white crystals which were recrystallized as above to give 30 g of additional product. (Anal, Found C, 27.53; H, 1.16; C1, 27.27; S, 12.3.) The overall yield of the combined materials was 77 % Methods Used for Measurement of Glucose Oxidase. Activity. Glucose oxidase concentration was determined from the FAD content by measuring the absorbance at 450 nm and using the absorbance coefficient of 1.41 X lo4M-' cm-' per active site
.
(13).
L GLUCOSE i .
GLUCONOLACTONE f
GLUCOSE OXIDASE
02
j
/
2 0 2 PEROXIDASE 8 CHROMOGENS
CHROMOPHORE
Ffgure 1. Schematic illustration of the pn'nciples of the prosthetic goup label immunoassay.
from activating apoglucose oxidase. If a conjugate can be synthesized with those two properties, it was postulated that an assay could be performed by the scheme in Figure 1. In this scheme, ligand-FAD conjugate, not complexed with antibody, is bound by excess apoglucose oxidase added at a suitable time in the assay sequence. The regenerated glucose oxidase activity can be readily followed by monitoring the hydrogen peroxide generated by the enzyme reaction. The absorbance produced by this detection system is then directly related t o the concentration of ligand in the assay. Swoboda (8)has shown that stable apoglucose oxidase could be prepared which binds FAD with high affinity. Also chemically modified FAD has been synthesized which reconstituted active glucose oxidase from the apoprotein. Flavin 1p-ethanoadenine dinucleotide and flavin-8-azido adenine dinucleotide, prepared by Harvey and Damle (9) and KOberstein (IO),respectively, are both active prosthetic groups for glucose oxidase. EXPERIMENTAL SECTION Aspergillus niger glucose oxidase solution (E.C.1.1.3.4; highly purified, low catalase grade; specific activity 198 units/mg), horse radish peroxidase (E.C.1.11.1.7; not less than 60 purpurogallin units/mg), and bovine serum albumin (fraction V and 30% (w/v) solution) were obtained from Miles Laboratories Ltd., Stoke Poges, UK. Lactoperoxidase (E.C.1.11.1.7) and FAD were purchased from Sigma (London) Chemical Co., Poole, Dorset, UK, and used without further purification. Concentrations of solutions of FAD and its derivatives were calculated from absorbances at 450 nm by using the molar extinction coefficient of 1.13 X lo4 M-I cm-' (11).
Sephadex G-10, G-50, and LH-20 were obtained from Pharmacia (GB Ltd.), London, UK. Rabbit antiserum against theophylline was raised according to the method reported by Cook et al. (12). Unless otherwise specified, all reagents used in this study were obtained from BDH Chemicals Ltd., Poole, Dorset, UK, and were AnalaFt grade, where available. Preparation of 2-Hydroxy-3,5-dichlorobenzenesulfonate. 2-Hydroxy-3,5-dichlorobenzenesulfonate required as a chromogenic reagent in the assay for glucose oxidase described below was prepared by hydrolysis of 2-hydroxy-3,5-dichlorobenzenesulfonyl chloride. The sulfonyl chloride (recrystallizedfrom cyclohexane; 163 g, 0.62 mol) was slowly added to an ice cold solution of 110 g (1.31 mol) sodium bicarbonate in 2 L of distilled water. After the evolution of carbon dioxide had ceased, the solution was reduced to half volume in vacuo and filtered to give 102 g of white
650
Glucose oxidase activity was measured by mixing the sample (0-50pL) with 2.0 mL of a solution of 0.1 M sodium phosphate, 0.2 mM 4-aminophenazone, 2.0 mM 3,5-dichloro-2-hydroxybenzenesulfonate, 100 mM glucose, 1% (w/v) bovine qerum albumin, 0.06 mg/mL peroxidase, and l mM EDTA. The reaction mixture was at pH 7.0 and incubated at 20 OC unless otherwise stated. This assay is a modification of the assay reported by Barham and Trinder (14). The activation of apoglucose oxidase by FAD derivatives in a format which allowed simultaneous recombination and measurement of glucose oxidase activity was used in which the FAD derivative and apogluccae oxidase were incorporated into the above assay solution and the absorbance at 520 nm recorded after a fmed time interval. Absorbance measurements were made on a single-beam digital spectrophotometer (CE292-CecilInstruments Ltd., Cambridge, UK) thermostated with a circulating water bath containing a tap-water-cooled coil. Reaction rates were measured with a chart recorder or the absorbance was recorded at a fixed time. The assays were performed in 1-cm light path, disposable, polystyrene cuvettes (Elkay Products, Inc., Worchester, MA). Recordings of progress curves for assay of glucose oxidase provide a linear trace in the absorbance range 0-1.0 at 520 nm. Preparation of '%I-Labeled Glucose Oxidase. A sample of glucose oxidase (250 pg) in 0.04 mL of 0.4 M phosphate buffer, pH 7.0, was mixed with 0.01 mL of Na'=I (1mCi; specific activity 11-17 mCi/pg iodine) obtained from The Radiochemical Centre, Amersham, UK, and 2.5 pL of lactoperoxidase in buffer (0.5 purpurogallin units),in a polystyrene tube (65 X 10 mm; Luckham, Ltd., Sussex, UK). The solution was stirred continuously with a small magnetic flea, and 1 pL of 8.8 mM-H202 in buffer was added. The tube was stoppered, and the solution was stirred for 30 min, after which, 1 mL of buffer containing 5 mg of bovine serum albumin was added. The solution was chromatographed on a column (1X 19 cm) of Sephadex G-50 equilibrated at room temperature with 50 mM phosphate buffer, pH 7.0. The protein peak eluted in the void volume was collected and found to contain 79% of the total radioactivity applied to the column. This material was divided into 0.20-mL samples and stored at -20 OC. It was established that neither the process of iodination nor freezing and thawing affected glucose oxidase activity. Radioactivity was measured with a crystal scintilation counter. Preparation of Apoglucose Oxidase. Mix 14 mL of glucose oxidase solution (about 80 mg of glucose oxidase) with 6 mL of glycerol and add 50 pL of 1261-labeledglucose oxidase. Cool the solution to -5 OC on a salt-ice bath, and add slowly 2.5% (v/v) H$04 to the rapidly stirred solution until the pH is 1.4. Incubate the solution for 2 h on an ice bath, and chromatographon a column (1.6 X 45 cm) of Sephadex G-50 equilibrated with 30% (v/v) glycerol in water adjusted to pH 1.4 with concentrated H$O4 and maintained at 4 OC. Elute the protein peak at a flow rate of 1.3 mL min-', and collect in 4.0 mL of a stirred mixture containing 200 mg of bovine serum albumin and 400 mg of activated charcoal in 0.4 M phosphate, pH 8.0 at 4 OC. The location of the protein peak can be monitored by measuring the radioactivity or UV absorbance of the eluate. Typically,26 mL of eluant was collected. Adjust the pH of the resulting suspension to 7.0 by addition of 2 M NaOH. Stir the suspension on an ice bath for 60 min and then remove the charcoal by successive filtrations through 0.8 pm (Type AA) and 0.22 pm (Type GS) Millipore filters. Finally add 10% (w/v) sodium azide in water to give a final concentration of 0.1% (w/v).
880
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981
Preparation of Flavin I@(6-Aminohexyl)adenine Dinucleotide (Aminohexyl-FAD). p-(Trifluoroacetamidohexy1)adenosine 5'-monophosphate was synthesizedby the method of Trayer et al. (15). Fifty-six milligrams of iP-(trifluoroacetamidohexy1)adenosine5'-monophosphate (0.1 m o l ) was dissolved in about 10 mL of water, and 25 pL of tri-n-butylamine(0.1 mmol) was added. The water was removed under vacuum, and the residue was dissolved in 10 mL of dry dimethylforrnamidewhich was then removed under vacuum. The residue was evaporated from dry dimethylformamidethree more times. The fiial residue was dissolved in 10 mL of dry dimethylformamide , Eighty milligrams of N,N'-carbonyldiimidazole (0.5 mmol) was added and allowed to react for 1.5 h. Then 15 pL of water was added and the solvent was removed under vacuum. The residue of p-(trifluoroacetamidohexy1)adenosine 5'-monophosphate imidazolide was dissolved in 10 mL of dimethylformamide. Forty-seven milligrams of riboflavin 5'-monophosphate (0.1 mmol), purified by the method of Johnson et al. (16),was dissolved in about 10 mL of water and added dropwise to 20 mL of acetone containing 43 pL of tri-n-octylamine (0.1 mmol). A precipiate formed before the addition was complete. The solvent was removed with a rotary evaporator until the riboflavin 5'-mOnOphosphate dissolved. Then 5 mL of acetone and 5-10 mL of dimethylformamide were added, and the mixture was taken to dryness. The residue was dissolved in 15-20 mL of dry dimethylformamide and taken to dryness. This procedure was repeated three times. The residue was dissolved in 5 mL of dimethylformamide and combined with the above-mentioned 10 mL of solution of the imidazolide in dimethylformamide. The reaction mixture was allowed to stand at room temperature overnight and then the solvent was removed. The residue was taken up in 50 mL of water and applied to a 2.5 X 25 cm column of DEAE-cellulose (Whatman DE 23; Whatman, Inc., Clifton, NJ) in the bicarbonate form. The chromatogram was developed with a linear gradient generated with 2 L of water and 2 L of 0.3 M ammonium bicarbonate (23-mL fractions were collected). Thin-layer chromatography on silica gel 60 F254 (E. Merck, Darmstadt, West Germany), using ethanol/l M-triethylammonium bicarbonate, pH 7.8 (7:3, by volume), showed that fractions numbered 68-73 contained major (Rf0.75) and minor (Rf0.36) yellow compounds. These fractions were pooled, and the optical adsorption spectrum had maxima at 267,373, and 450 nm. The solvent was removed from the pooled material, and the residue was dissolved in about 5 mL of water. This solution was adjusted to pH 11.0 with 5 M NaOH and allowed to stand at room temperature for 9 h. Thin-layer chromatography showed that the component with Rf 0.75 disappeared while a new yellow material with Rf 0.37 appeared. The reaction mixture was adjusted to pH 8.0 with hydrochloric acid and applied to'a column (2.5 X 20 cm) of DEAE-cellulose in the bicarbonate form. The chromatogram was developed with a linear gradient of 1L of water and 1 L of 0.2 M ammonium bicarbonate. The yellow effluent from the column was pooled and the solvent was removed. The residue was adsorbed onto 2 g of silica gel which was placed atop a 50-g column of silica gel equilibrated with ethanol/l M triethylammonium bicarbonate, pH 7.8 ( 8 2 by volume). The chromatogram was developed with the same solvent, the yellow component with Rf 0.37 was collected, and the solvent was removed. The yield of aminohexyl-FAD based on absorbance at 450 nm was about 10%. Preparation of Flavin C8-(6-Aminohexy1)aminoadenine Dinucleotide. 8-(Trifluoroamidohexy1)aminoadenosine 5'monophosphate was prepared by the method of Trayer et al. (15). The imidazolide of this compound was prepared and condensed with the tri-n-octylammoniumsalt of riboflavin 5'-monophosphate as described above for the synthesis of aminohexyl-FAD. The resulting flavin was purified by chromatography on DEAE-cellulose to give a yellow material with absorption maxima at 368, 375, and 447 nm. This product had an R of 0.79 when examined by TLC on silica gel using ethanol/l dtriethylammonium bicarbonate, pH 7.8 (82 by volume). The trifluoroacetyl group was removed by adjusting the solution of flavin C8-(trifluoroamidohexy1)aminoadenine dinucleotide to pH 11.0, with 5 M NaOH and allowing the mixture to stand for 13 h at room temperature. A new yellow component with Rf of 0.55 in the above TLC system
appeared. This product gave a positive reaction with ninhydrin. This component was purified by chromatography on DEAEcellulose as described for aminohexyl-FAD. Preparation of Theophylline-FAD (I). 1,3-Dimethyl1,6,7,8tetrahydropyrido[l,2e]pyrine-2,4,9-[3~trione (0.9 mg/3.62 pmol), prepared according to the method of Cook et al. (12),was added to 0.2 mL of dimethyl sulfoxide containing 2.4 pmol of aminohexyl-FAD. After 4 h a further 1.8 mg (7.3 pmol) of the trione was added. The solution was stirred overnight, the solvent was evaporated under vacuum (0.1 mmHg), and the residue was chromatographed on a column (2.5 X 90 cm) of Sephadex LH-20 equilibrated with 0.3 M triethylammonium bicarbonate, pH 7.8. The crude product eluting between 216 and 246 mL of effluent was collected, applied to a 20 cm x 20 cm x 100 pm silica gel plate, and chromatographed by using ethanol/ 1M triethylammonium bicarbonate, pH 7.8 (8:2 by volume). The band containing the desired product (Rf0.77) was scraped from the plate, extracted with 1M triethylammonium bicarbonate buffer, pH 7.8, filtered, and concentrated. Final purification by chromatography on Sephadex LH-20 equilibrated with 0.3 M buffer gave 1.26 pmol of theophylline-FAD (I) as determined by the absorbance at 450 nm, which was a yield of 53%. Preparation of Flavin N6-(2-Hydroxy-3-carboxypropyl)adenine Dinucleotide (Carboxyl-FAD). Carboxyl-FADand flavin-iP-(2-hydroxy-3-carboxylpropyl)adenine dinucleotide were prepared according to the method reported by Zappelli et al. (17). The oxiranacetic acid used to alkylate FAD was prepared by reaction of 3-butenoic acid with 3-chloroperoxybenzoic acid. 3-Butenoic acid (8.6 g, 0.10 mol) was dissolved in 200 mL of methylene chloride. 3-Chloroperoxybenzoic acid (22.4 g, 0.11 mol, 85% (w/w)) obtained from Aldrich Chemical Co., Gillingham, UK, was dissolved in 300 mL of methylene chloride and added dropwise over a 60-min period to the continuously stirred solution of 3-butenoic acid. The reaction mixture was allowed to stand overnight at room temperature. The solvent was removed under reduced pressure to give a solid residue. This residue was stirred with 400 mL of H20 for 10 min to extract the product and then filtered. The filtered precipitate was washed with 50 mL of methylene chloride. The supernatant was then evaporated on a rotary evaporator at 35 "C to give 7 g of a viscous oil which was used without further purification. The oxirane content of the preparation was assessed as follows. The acid content was determined by titration of an aqueous dilution with 0.1 N NaOH using phenolphthalein as indicator. About 1mmol of neutralized oxiranacetic acid solution was added to 2 mL of 1.3 M sodium thiosulfate containing 1 drop of a 1 mg/mL solution of phenolphthalein in ethanol. The reaction of the oxirane group with the thiosulfate produces hydroxide, and the amount of 0.1 M HCl required to maintain neutrality as judged by the indicator color present was used as a measure of the oxirane content. On this basis, oxirane acetic acid, prepared as described above, contained 60-70% oxirane as a percentage of the total acid content. Preparation of Theophylline-FAD (11). Carboxyl-FAD (3.62 mg, 3.1 pmol, calculated as the diammonium salt, dihydrate) and 4.4 mg (18.6 pmol) of 8(3-aminopropyl)theophyllinewere dissolved in a mixture of 0.6 mL of dimethylforrnamide and 0.4 mL of H20. The solution was evaporated under vacuum to remove volatile cations and ensure that the counterions present are the conjugate acid of the aminotheophylline. The complex was dissolved in a mixture of 0.4 mL of dimethylformamideand 0.4 mL of H20and cooled to -10 "C in an ice/methanol bath. l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, 3.5 mg (18.6 pmol), was added; and the reaction mixture allowed to stir and warm to room temperature for 2 h. The reaction mixture was then cooled to -10 "C and a further 18.6 pmol of carbodiimide added. After 2 h of incubation, the reaction solution was evaporated to dryness at 35 "C under vacuum. The yellow residue was dissolved in 2.0 mL of 0.3 M triethylammonium bicarbonate, pH 7.8, and applied to a column (2 X 54 cm) of Sephadex LH-20 equilibrated with the same buffer at 4 "C. Three peaks of yellow material were eluted. Thin-layer chromatography, using isobutyric acid/H20/33% (w/v) NH3 (70291 by volume), indicated the first and major peak consisted of unmodified carboxyl-FAD (Rf0.29); and the second peak, incompletely resolved from the first, consisted of material (Rf0.39) which was identified as theophylline-FAD (11)on the
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981
basis of its activity with apoglucose oxidase and antiserum to theophylline. The fraction of this peak, uncontaminated with carboxyl-FAD, was pooled to give about a 10% yield of theophylline-FAD (11)as calculated from the absorbance at 460 nm. The third peak well separated from the first two was shown by thin-layer chromatography to contain degradation products of FAD. RESULTS AND DISCUSSION Apoglucose Oxidase. Preparation of Apoglucose Oxidase. In the prosthetic group label immunoassay, excess apoglucose oxidase is added a t an appropriate stage of the assay. It was therefore considered important to prepare apoglucose oxidase with not only high recoverable glucose oxidase activity but very low residual glucose oxidase activity so as to minimize background color in the assay. Thus it was anticipated that the quality of the apoglucose oxidase would be important in determining the format, sensitivity, and the reproducibility of the assay. Swoboda (8) described the preparation of apoglucose oxidase by simultaneousdissociation of FAD and precipitation of the apoprotein in acidified ammonium sulfate solution. In our hands this method gave preparations with high apoglucose oxidase activity; but residual glucose oxidase activities could not be reduced below about 1% , despite repeated cycles of dissolution and precipitation of the apoprotein. Moreover, any increase in the scale of the preparation above that used by Swoboda made the problem of residual glucose oxidase more intractable. A considerable effort was therefore applied to the development of a method of apoglucose oxidase preparation which would preserve a high recoverable glucose oxidase activity and yet attain a very low residual activity. This work culminated in the finding that the presence of 30% (v/v) glycerol in an acid dissociation medium has a marked stabilizing effect on the apoglucose oxidase. Thus, it was possible to prolong incubation at low pH to allow almost complete dissociation of the glucose oxidase and separation of the apoprotein from the FAD by gel filtration at low pH. A fiial neutral charcoal treatment was found to be essential; since, although the apoglucose oxidase obtained by gel filtration initially has a very low residual glucose oxidase activity, this activity increases slowly over a period of 24 h to unacceptably high levels. This effect was abolished by a simple incubation with charcoal a t neutral pH. Apoglucose oxidase prepared by the above method had such low residual activity that the PGLIA could be performed by using a much larger excess of apoenzyme than was practical with apoenzyme prepared by other methods. This allowed large improvements to be made in the performance of the immunoassay. Moreover, yields of apoglucose oxidase activity are typically greater than 40% of the total original glucose oxidase activity, and the apoprotein can be prepared reproducibly. During the preparation procedure, the apoglucose oxidase is quite stable at pH 1.4 in 30% (v/v) glycerol in the presence of FAD. However, once isolation of the apoprotein from FAD is started, the protein is unavoidably incubated for a time at pH 1.4 on the Sephadex G-50 column. During this time, the stability of the apoprotein is much reduced. It is therefore important to design the column procedure to take as short a time as is commensurate with good separation from the FAD and to neutralize the apoenzyme solution as it elutes. These observations suggest that there is significant interaction a t pH 1.4 between the apoenzyme and FAD which stabilizes the protein. Characterizationof Apoglucose Oxidase. Apoglucose oxidase activity was determined by measuring the glucose oxidase activity regenerated when about 1.5 pg/mL of apoprotein was incubated a t room temperature in 0.1 M phosphate buffer, pH 7.0, containing 0.1% (w/v) bovine serum albumin, 0.1% (w/v) sodium azide, and 0.1 mM FAD. En-
881
zyme activity was determined by transferring 50 pL of incubation solution to 2 mL of glucose oxidase assay solution and recording the initial rate. The regenerated glucose oxidase activity was expressed as a percentage of the maximum theoretical recoverable activity calculated from the totaloriginal glucose oxidase activity. When the time course of the reactivation was followed, it was typically found that after 1 h, 16h, and 5 days, the recoverable activity was about 50%, 7070, and almost loo%, respectively. In every preparation investigated, full recovery of activity was achieved although some variation in the rate of reactivation was observed. The FAD binding site concentration of apoglucose oxidase was measured by titration with FAD. Apoglucose oxidase was incubated with a range of FAD concentrations in the same medium used to determine apoglucose oxidase activity. After 16 h, the glucose oxidase activity was measured and when plotted vs. FAD concentration resulted in a sharply defined equivalence point. It was established that if the FAD binding site concentration was determined after an incubation time of at least 60 min, it did not change with further incubation despite the fact that up to 5 days total incubation was needed to fully recover glucose oxidase activity. In the development of the PGLIA, the amount of apoglucose oxidase incorporated in the assay was assessed in terms of the FAD binding site concentrations. Although preparations of apoglucose oxidase showed variability in the rate at which glucose oxidase activity was regenerated in the presence of excess FAD, no significant difference was observed due to use of different apoenzyme preparations in the conditions of the PGLIA described later for theophylline. The total glucose oxidase protein in apoenzyme preparations was calculated from the comparison of the total radioactivity of the original glucose oxidase solution and that recovered after charcoal treatment of the apoenzyme. A representative apoenzyme preparation had the following characteristics: a residual glucose oxidase activity of 0.001% of the total recoverable glucose oxidase activity after 5 days activation; a glucose oxidase protein concentration of 2.0 mg/mL; an FAD binding site titration concentration of 21.4 pM. The FAD binding site concentration calculated from the protein concentration and a molecular weight for glucose oxidase of 153000 (8) is 26.7 FM. The specific activity of the original glucose oxidase calculated from the FAD content is 13.4 unita/nmol FAD whereas the specific activity calculated from the FAD titration of the apoglucose oxidase and the maximum recoverable activity is 14.3 units/nmol FAD. The enzyme unit used is defined as the micromoles of hydrogen peroxide produced per minute in the glucose oxidase assay described in the Methods. Stability of Apoglucose Oxidase. The most suitable medium found for storing apoglucose oxidase was 0.1 M phosphate buffer, pH 7.0, containing 30% (v/v) glycerol and 0.1% (w/v) sodium azide. At 4 "C,typical preparations retained about 40% of the original reconstitutable activity after 12 months of storage. The stability of the apoenzyme is pH dependent. In particular, at pH values above 8.0 or below 6.0, the apoenzyme was markedly unstable. FAD Derivatives. The position of derivatization on the FAD molecule was chosen after comparison of the activities of the N6-derivative aminohexyl-FAD and f l a v i n 0 (6aminohexy1)aminoadenine dinucleotide with apoglucose oxidase. When the flavins were incubated in 100-fold molar excess over apoenzyme, the N6-derivative gave maximum glucose oxidase activity after 24 h; whereas, the C8derivative activated much more slowly with a pronounced sigmoidal plot of glucose oxidase activity against time which reached a maximum after 72 h. The maximum activity achieved with the C8derivative, however, was about 80% of that possible
882
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981 NH-R
1.5 -
0
1.0-
R
COMPOUND FAD
H
H-AMINOHEXYL-FAD
-
H-CARBOXYL-FAD
-CH?-
CHe-CH2-CH2'
CH,-
CH2-CH2-NH2
CH-CHp-COOH
I
OH
0
I1
THEOPHYLLINE-FAD I
-
(CHh
-
NHCOICHh
H
I CHJ
0 I1
THEOPHYLLINE-FAD ll
-
CH2 - C H -CH2 -CONH-(CHl)l
I OH
\-jy H
I CH,
FAD
Derivative
nM
Activation of apogiucose oxidase by FAD derivatives (A) FAD, (m) theophylline-FAD (I), (0) theophylline-FAD (II), (0)carboxyl-FAD, and (0)N1-carboxyl-FAD were incubated at the indicated Flgure 4.
concentrations In glucose oxidase assay solution in the presence of 100 nM apoglucose oxidase FAD binding sites at 20 O C .
1
e
- L o g (concentrat~on) M
Activation of apoglucose oxidase by aminohexyi-FAD, theophylline-FAD and FAD. Various concentrations of aminohexyKAD (m), theophylline-FAD (I) (A),and FAD (0)in 0.1 M phosphate buffer, pH 7.0, containing 0.1 % (w/v) bovine serum albumin and 0.1 % (w/v) sodium azide were incubated for 18 h at room temperature In the presence of 40 nM apoglucose oxidase FAD binding sites. The activity of the regenerated glucose oxidase was then measured by mixing 50 pL of incubation solutlon into glucose oxidase assay solutlon at 25 O C and recording the absorbance at 520 nm at 5 min. Flgure 3.
with FAD or aminohexyl-FAD. The binding of the Cs derivative to apoenzyme was apparently much weaker than that of the N6 derivative such that concentrations greater than 1 pM were necessary with the former to give significant activation. Because the aminohexyl-FAD gave stronger binding and faster activation, ligands were coupled through the N6 position. The structures of the FAD derivatives subsequently used in this work are illustrated in Figure 2. The synthetic strategy involved the preparation of FAD derivatives with amino or carboxylic acid functions appended to the N6 position. Derivatives of theophylline containing carboxylic acid or amino groups were then attached to these functional groups by condensation reactions. TheophyllineFAD conjugates were found to be stable for at least 12 months as determined by the reactivation of apoglucose oxidase when stored in 0.1 M phosphate buffer, pH 7.0, containing 0.1% (w/v) sodium azide a t 4 OC in the dark. Activity of Theophylline-FAD with Apoglucose Oxidase. Figure 3 presents the relationship of regenerated glucose oxidase activity to FAD derivative concentration when a constant amount of apoglucose oxidase is incubated with a wide range of flavin concentrations. The modification of the
N6 position of FAD causes a shift of the activation curve to higher flavin concentration. Addition of a theophylline residue to aminohexyl-FAD causes an additional increase in the prosthetic group concentration required to produce a given enzyme activity (Figure 3). Longer incubation of apoenzyme with flavin results in a slow but significant increase in recovered glucose oxidase activity from about 60% of theoretical maximum a t 16 h to about 98% after 5 days, The position of the activation curve relative to flavin concentration does not alter during this period. The immunoassay concept however requires that low FAD label concentrations are detected by addition of excess apoglucose oxidase in order to achieve the maximum possible sensitivity in measurement of free flavin derivative. The effect of apoenzyme concentration on the detection of theophylline-FAD when both were simultaneously mixed in glucose oxidase assay solution was studied. Linear standard curves were obtained of absorbance change at 30 min vs. theophylline-FAD concentration (0-1.0 nM) at different apoglucose oxidase concentrations. FAD binding site concentrations of 32 and 160 nM gave standard curves with absorbance ranges of 0.02-0.40 and 0.20-1.60, respectively. Thus, the sensitivity of detection of the label is greatly increased at the higher apoenzyme concentration. Activities of Theophylline-FAD Derivatives with Apoglucose Oxidase. The most attractive format to use with a homogeneous immunoassay such as the PGLIA concept is one in which all the reagents are mixed without preincubation of any of the interacting species since this procedure is the most rapid and most convenient. The interaction of FAD derivatives with apoglucose oxidase was therefore studied by addition of excess apoglucose oxidase to glucose oxidase assay solution containing an appropriate concentration of flavin derivative. In Figure 4, the activity of the theophylline-FAD conjugates was compared with those of N1-carboxyl-FAD and NG-carboxyl-FAD. As reported by Zappelli et al. N'carboxyl-FAD has little activity with apoglucose oxidase, but considerable activity is obtained following the Dimroth rearrangement to the N6 derivative. It is of interest that condensation of NG-carboxyl-FAD with 8-(3-aminopropy1)theophylline results in a further large increase in activity with
(In,
ANALYTICAL CHEMISTRY, VOL. 53, NO. 4, APRIL 1981
863
Id,
11)
0.9
0.8 E
E In
I
0.4LL2 2.5
5.0
7.5
10.0
07
12.5
Antiserum to Theophylline pI
Flgure 5. Effect of antiserum to theophylline on the activation of apoglucose oxidase by theophylline-FAD (I). Theophylline was incorporated into the assay, by inclusion of 50 pL of 0.1 M phosphate buffer, pH 7.0, containing 1 pL of serum with the following concentrations: (m) 0 pg/mL theophylline; ( 0 )40 g/mL theophylline. apoglucose oxidase. Zappelli et al. (I7) noted a similar increase in activity with apoglucose oxidase when they condensed carboxyl-FAD with polyethyleneimine. It would seem that the binding of FAD to the apoenzyme is inhibited by the presence of a negatively charged group in the vicinity of the N6position of the adenine moiety of FAD. Furthermore, the activity of theophylline-FAD (I)was considerably greater than that of theophylline-FAD (11) which indicates that the hydroxyl group appended to the alkyl chain may also inhibit binding of the derivative to apoglucose oxidase. However, both theophylline-FAD derivatives had sufficient activity with apoglucose oxidase under these assay conditions to be potentially useful labels in an immunoassay. Effect of Antibodies to Theophylline on the Activation of Apoglucose Oxidase by Theophylline-FAD. Theophylline-FAD (I) is more active with apoglucose oxidase than theophylline-FAD (11) so the former label was used in an investigation of the feasibility of a homogeneous PGLIA for theophylline. The effect of antiserum to theophylline on the activation of apoglucose oxidase by this label was first evaluated. The label concentration used was decided by determining the amount needed to give an absorbance change of about 2.0 after a 5-min incubation with 200 nM apoglucose oxidase in the glucose oxidase assay at 25 "C. The assay was performed by mixing 0.10 mL of apoglucose oxidase reagent in 0.1 M phosphate buffer, pH 7.0, with 1.90 mL of glucose oxidase assay reagent containing label. A final label concentration of 20 nM was found to be the most convenient. In order to study the effect of antiserum on this activation it was incorporated at different concentrations into the apoglucose oxidase reagent. This reagent was then combined with label in the presence or absence of theophylline in the glucose oxidase assay according to the following protocol. In one corner of a disposal plastic cuvette 0.10 mL of combined apoglucose oxidase/ antiserum reagent was placed. In the opposite corner was placed 0.05 mL of a diluted human serum with or without theophylline incorporated. The serum was diluted 50-fold with 0.1 M phosphate buffer, pH 7.0. The assay was initiated by rapid addition of 1.90 mL of glucose oxidase assay reagent containing the label. After a 5-min incubation at 25 O C the absorbance at 520 nm was read against glucose oxidase assay reagent. As shown in Figure 6 the activation of apoglucose oxidase by the label is strongly inhibited by antiserum to theophylline in the absence of theophylline. A maximum of about 95% inhibition was effected by the highest concentration of antiserum used. In the presence of theophylline (40 pg/mL serum) a considerable
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1
I
I
I
I
10
20
30
40
Serum Theophylline
Flgure 6.
pg m1-I
Standard curve for theophylline concentration determination
by the prosthetic group label immunoassay.
reversal of the inhibition was observed. The theophylline serum concentration used is the highest concentration which would be used in construction of a standard curve for theophylline. The results shown in Figure 5 demonstrate that the assay reagents exhibit all the prerequisites for a homogeneous PGLIA for theophylline; theophylline-FAD (I) activates apoglucose oxidase, this activation is strongly inhibited by antibody to glucose oxidase and the inhibition is reversed by the presence of theophylline. In similar experiments it was shown that theophylline-FAD (11)also has the properties required to construct a PGLIA for theophylline. Prosthetic Group Label Immunoassay for Theophylline. Assay conditions for the measurement of theophylline in serum were developed on the basis of the results shown in Figure 5. The amount of antiserum incorporated in the assay was selected by comparison of the inhibition in the absence of theophylline with the reversal achieved in the presence of theophylline. The criterion used was the combination of a high inhibition and hence a low background absorbance with an efficient reversal of inhibition. The quantity of antiserum chosen was 3.8 pL and was used to obtain the standard curve shown in Figure 6. The antiserum was incorporated into the apoglucose oxidase reagent and the assay protocol was the same as described for the experiments shown in Figure 5. Standards were prepared with pooled human serum to cover the range 0-40 pg/mL theophylline. Reaction progress curves for this PGLIA are presented i s Figure 7 for selected theophylline concentrations. Each curve has an initial phase during which it is distinctly concave upward. However after about 3 min of incubation they become approximately linear and although the above assay was performed by using single time point absorbance measurements, it would be possible to determine rates from a portion of the curve in the linear phase. The assay conditions were designed to produce a convenient assay for measuring theophylline at therapeutically useful concentrations. The reported therapeutic range for theophylline is relatively narrow, and to provide safe and effective therapy serum concentrations should be maintained between 10 and 20 pg/mL ( 2 4 1 9 ) . Thus, the reagent concentrations were adjusted to obtain sufficient absorbance change in a short time at 25 "C when small serum samples are used. It was necessary to prepare theophylline standards in pooled serum,
664
ANALYTICAL CHEMISTRY, VOL.
53, NO. 4, APRIL 1981
1.4-
351
C
.
1.2-
I
1.0-
25 301
B
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*
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P
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t
..
0.4
A
.
Table I. Precision of Prosthetic Group Label Immunoassay for Theophylline theophylline concn, wg/mL mean Sa cv, %
Intraassay Precision ( n = 30.0
a
2.8 15.8 32.3
S = standard deviation.
tion.
15)
0.4 0.4 0.3
Interassay Precision ( n = 2.8 15.4 25.4
0.4
1.0 1.7
b
6.6 3.4
1.0 20) 13.4 6.4 5.4
CV = coefficient of varia-
since a small but reproducible difference was found between curves obtained by using either aqueous or serum standards. However, when only 1-pL of serum is incorporated into the assay, no difference is evident between different serum samples. The performance of the assay was assessed to a point at which the information collected was sufficient to indicate the system has considerable potential as a method for measuring haptens and possibly antigens such as proteins which are present at low concentrations. Thus, the interassay precision collected by analyzing the samples in triplicate on 15 separate days and the intraassay precision presented in Table I are similar to those observed with a fully automated procedure for an enzyme immunoassay (20)and RIA (21).To determine the reliability of the assay for measuring theophylline in human serum, we analyzed 59 samples from patients receiving therapy by PGLIA and by EMIT theophylline assay (Syva). As shown in Figure 8, the two methods correlate well (correlation coefficient 0.99). The regression line determined by the method of least squares was y = 1.09~- 0.30 with the standard error of estimate equal to 2.0 pg/mL. Effect of Antibody to Theophylline on Glucose Oxidase Reconstituted from Theophylline-FAD (I) and Apoglucose Oxidase. An investigation was carried out to determine if the activity of glucose oxidase reconstituted with theophylline-FAD (I) can be modulated by antibody to theophylline. Thus, glucose oxidase was reconstituted by
Y
'
5
10
= &+A,X
Ao= -0.30
..
/ t/
0
Figure 7. Progress curves for prosthetic group label Immunoassay for with time was monltored for theophylline. The Increase In AblOnm different serum theophylline concentrations: (A) 0 pg/mL; (B) 10 pg/mL; (C) 40 pg/mL.
6.1 11.8 29.9
/I//'
-
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Time minutes
6.0 12.0
: 5
serum concn, pg/inL
:/
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t
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1.09
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I
15 20 EMIT pg mi-'
25
I
I
30
35
Figwe 8. Correlation of results of prosthetlc group label Immunoassay with those of EMIT for theophylline in human serum.
incubating apoglucose oxidase (5.7pM FAD binding sites) and 24.7 pM theophylline-FAD (I) in 0.6 mL of 0.1 M phosphate buffer, pH 7.0, for 16 h at 4 O C in the dark. The reconstituted glucose oxidase was then separated from excess flavin by gel filtration on a column (0.9 X 30 cm) of Sephadex G-50 equilibrated at room temperature with 0.1 M phosphate, pH 7.0, containing 0.1% (w/v) sodium azide. The activity of this enzyme was not affected by excess antiserum to theophylline either when the enzyme was preincubated with antiserum for several hours before assay or when the antiserum was incorporated into the assay medium. It was concluded from these results that, if antibody to theophylline binds to glucose oxidase reconstituted with theophylline-FAD, then it does not inhibit the enzyme activity under the assay conditions used to perform the PGLIA.
CONCLUSIONS The homogeneous assay concept illustrated in Figure 1has been shown to be valid, and methods for preparing highly purified reagents were demonstrated. This technique has a combination of characteristics which gave it a potentially unique advantage over other homogeneous immunoassay systems including those listed in the Introduction (2-7). In the first instance, the reagents used in the PGLIA are welldefined and readily prepared. The FAD labeled ligands can be prepared as pure and well-characterized compounds while apoglucose oxidase can be very reproducibly prepared from commercially available glucose oxidase. Secondly, the assay system has an inherent amplification process due to the glucose oxidase activity generated by the incorporation of a small amount of FAD conjugate into apoglucose oxidase. The results presented here not only show that the reagents demonstrate a new homogeneous assay concept but, by using theophylline as a model, show the potential practical application of the method to the measurement of analytes in biological specimens. In summary, the PGLIA (a) is homogeneous (no separation steps are required to separate antibody bound and free drug conjugate), (b) can be rapid and use a small sample (1pL) when haptens at the therapeutic serum levels of theophylline are analyzed; (c) uses stable nonradioactive reagents; (d) has precision and accuracy similar to other immunoassays; (e) is amenable to total automation; (f) has potentially high sensitivity due to the inherent amplification provided by the enzymic activity of regenerated glucose oxidase and the
Anal. Chem. 1981, 53, 665-676
possibility of the application of extremely sensitive methods of HzOz detection which employ fluorescence or chemiluminescence, and (8) may be applicable to a wide range of biologically important molecules including haptens and antigens. Although the work reported herein illustrates the PGLIA concept by use of a simple haptenic molecule, theophylline, it was also envisaged that the method would be applicable to much larger molecules including protein. Work has thus been in progress which demonstrates that the PGLIA can be used to measure IgG concentration and will be reported in a subsequent paper (22). LITERATURE CITED Rubenstein, K. E.; SchneMer, R. S.; Ullman, E. F. Blochim. Blophys. Res. Commun. 1072, 47, 846-657. Haimovich, J.; Hurwitz, E.; Novik, N.; %!a, M. Biochim. Blophys. Acta 1070, 207, 115-124. Leute, R. K.; Ullman, E. F.; Goldsteln, A.; Herzenberg, L. A. Nature (London) New Biol. 1872, 236, 93-94. Dandliker, W. B.; Schapiro, H. C.; Maduski, J. W.; Alonso, R.; Feigen, G. A.; Hamrick, J. R. Immunochemistry 1884, 1 , 165-191. Schroeder, H. R.; Vogelhut, P. 0.; Carrico, R. J.; Boguslaski, R. C.; Buckler, R. T. Anal. Chem. 1878, 48, 1933-1937. Carrico, R. J.; Christner. J. E.; Boguslaski, R. C.; Yeung, K. K. Anal. Blochem. 1078, 72, 271-282.
885
(7) Burd, J. F.; Wong, R. C.; Feeney, J. E.; Carrlco, R. J.; Boguslaski, R. C. Clin. Chem. ( Winston-Salem, N.C.) 1077, 23, 1402-1408. (8) Swoboda, B. E. P. Biochim. Biophys. Acta 1888, 175, 365-379. (9) Harvey, R. A.; Damle, S. FEBS Lett. 1872, 28, 341-343. (10) Koberstein, R. Eur. J. Blochem. 1878, 87, 223-229. (11) Whitby, L. G. Biochem. J. 1053, 54, 437-492. (12) Cook, C. E.; Twine, M. E.; Meyers, M.; Amerson, E.; Kepler, J. A.; Taylor, G. F. Res. Commun. Pathol. pharmacal. 1078, 13, 487-505. (13) Swoboda, B. E. P.; Massey, V. J. Biol. Chem. 1068, 240. 2209-2215. (14) Barham, D.; Trinder, P. Analyst (London)1872, 97, 142-145. (15) Trayer, I. P.;Trayer, H. R.; Small, D. A. P.; Bottomley, R. C. Blochem. J. 1074, 139, 609-623. (16) Johnson, R. D.; LeJohn, L.: Carrico, R. J. Anal. Blochem. 1078, 88, 526-530. (17) Zappelll, P.; Pappa, R.; Rossodivita, A.; Re, L. J. Eur. Bhhem. 1078, 89, 491-499. (18) Mkenko, P. A.; Ogihrie, R. I. N. Engl. J. Med. 1073, 239, 600-603. (19) Jenne, J. W.; Wyze, E.; Rood, F. S.; MacDonald, F. M. CNn. PharmaCol. Ther. 1872, 13, 349-360. (20) Weldner, N.; McDonald, J. M.; Tlebar, V. L.; Smith, C. H.; Kessler, Q,; Ladenson, J. H.; Dietzler. D. N. Clin. Chim. Acta 1070, 97, 9-17. (21) Forester, R. L.; Wonall, J.; Robertson, W. R.; Watali, L. J.; Wliheim, D. Ther. Drug Monitoring 1070, 1 , 381-385. (22) Yeager, F. M.; Ngo, T. T.; Carrico, R. J.; Morris, D. L.; Boguslaski, R. C.; Hornby. W. E., In preparation.
RECEIVED for review July 1, 1980. Accepted December 22, 1980.
Chemical Derivatization in Electron Spectroscopy for Chemical Analysis of Surface Functional Groups Introduced on Low-Density Polyethylene Film Dennis S. Everhart and Charles N. Reilley' Kenan Laboratories of Chemistry, Universiiy of North Carollna, Chapel Hill, North Carolina 27514
Derlvatlzatlon reagents contalnlng an elemental tag facllltate ESCA analysts of surface functlonal groups Introduced on low-denslty polyethylene film exposed to radlo frequency Inductively coupled N2or Ar plasma. Angulardependent ESCA measurements of polyethylene treated wlth nltrogen plasma Indicate a surface that Is vertically Inhomogeneous In oxygen and nitrogen. Angular-dependent spectra help characterize the reactlons of some of these tagged reagents wlth the plasma-modified polymer. Several tags decompose during ESCA analysis wlth the DuPont 650 B spectrometer, but all derlvatlves are stable to ESCA analysls wlth the PHI 548 spectrometer. Na accelerates decomposltlon. Modest reductlons In anode power can retard sample decomposltlon and help remedy some dire effects whlch sample lnstablllty has on the semlquantltatlve (f15 % ) potentlal of ESCA.
The surface properties of organic polymers are of considerable importance in many industrial, biomedical, and technological applications (1-3). Wettability, adhesion, abrasion resistance, biocompatability, and reverse osmosis character are a few areas where the chemical properties of the surface have a prodigious role. Often modification of polymer surfaces is required while leaving the bulk properties unchanged. For example, surface carbonyl and carboxyl groups are incorporated in low-density polyethylene films upon mild chemical oxidation, and these functionalities are at least in part responsible for the increased wettability of the previously hydrophobic surfaces (4,5). Numerous reports on the identi0003-2700/8 1/0353-0665$01.25/0
fication and chemical modification of functional groups introduced on polyethylene film with chemical oxidation have been made (6-10). Surface modification by exposure to glow discharge (low temperature) plasma has received considerable attention (11-18). The distinguishing feature of surface modification effected by plasma is that the process is experimentally simple and can be selectively controlled to produce drastic alterations in surface properties without affecting the overall quality of the material. A two-component, direct and radiative energy transfer, model has been proposed to characterize argon gas plasma modification of polymers (19). This model predicts that the outermost monolayer cross-links rapidly as a consequence of Hz elimination induced by direct energy transfer from argon ions and metastables. Despite the large amount of effort in this area, the details of plasma-surface interactions are complex and consequently plasma modification of polymers has largely been developed empirically. One technique especially suited for studying surface modification is electron spectroscopy for chemical analysis (ESCA) (20). Clark et al. (21-23) have extensively elaborated on the use of ESCA to study polymers. The utility of ESCA to elucidate changes in structural and chemical features of a modified surface is well substantiated. Unfortunately, ESCA analysis often lacks the resolution required to unambiguously identify a specific functional group. To help overcome this problem, curve fitting procedures are utilized which attempt to decompose the ESCA spectrum into a collection of individual peaks. The binding energies (BE) and areas of the composite spectra are then used to identify the presence of 0 1981 American Chemical Society
