REPORT
il Mary Lynn Grayeski Department 01 ChemisVy Seton Hall University South Orange. N.J. 07079
Chemiluminescence (CL) is observed when light is emitted from a chemical reaction. If the reaction occurs in a living system or is derived from one, the process is called bioluminescence (BL). The intensity of light ( I d is generally ohserved to increase initially and later decrease with time as the reactants are consumed. This can be described by theequation I,,(photons/s) = mcL(photons/moleculesreacted)
X
dCldt(molecu1es reactedlsj where QCI. is the CL efficiency and is equal to the efficiency of production of excited staces (number of excited-state molecules per number of molecules reacted) times the emission efficiency (number of photons emitted per numher of excited-state molecules), and dC1dt is the numher of molecules re-
acting per unit of time. Because the emission intensity is determined by the rate of the chemical reaction, measurement of emission intensity can he used as the basis of quantitation for any species whose concentration determines the rate of the chemical reaction. CL offers several potential advantages for analytical applications. Because of its inherent sensitivity, low detection limits are possible, often in the femtwattomole range. For most analytes, the linear dynamic range is several orders of magnitude wide. In addition, instrumentation is relatively simple, which allows techniques to be developed for on-site analysis when more expensive or cumbersome instrumentation might not be suitable. Many articles describe clinical, biological, and environmental applications using both gas- and solutionphase CL reactions (see Suggested Reading). Although the lack of commercially available instrumentation, reagents, -and methodology has somewhat restricted the widespread use of CL, certain applications are common: the measurement of total microbial cell counts using the firefly reaction and
the determination of oxides of nitrogen with a gas-phase chemiluminescent reaction involving ozone. This REPORT will discuss only a few of the most recent developments in CL using reactions in solution.
Chemilumlneseenceanalysis For the generalized chemiluminescent reaction
-+
A+BC*
C
C' light
any of the components, including the catalyst if one is used, can be measured as the analyte. The reaction conditions are adjusted so that the light measured is a function of the level of analyte to be determined. Because the signal is transient, measurement of ICL is time-dependent. The signal is often recorded at a specific time after mixing or by integration of light during the entire time or during a specific fraction of time when light is emitted. The instrumentation usually involves some means of mixing the reactants and a detector to measure light. The reactants can be mixed directly in
4
0003-2700/87/A359-1243/$0 1.5010 @ 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 21. NOVEMBER 1, 1987
1243A
Flgure 1.
Examples of chemiluminescent measurements
(a) Static. (b) flow system. (c)tw@mse measurement on s o l i surtace. A = analyte; R = chemiluminescent reagent@)mat may be immobilized on wild surface either cwalently, behind a membrane. or by adsorption: L' = luminescent species produced
front of the detector, or an optical fiber can be used to transmit the emitted light. Sometimes other sources of light are removed from the CL signal with a filter that is placed in front of the detector. The measurements reported for the applications described here are performed in one of three ways. Static measurements in solution involve mixing reagents in front of a detector (Figure la). Often, mixing is achieved by the force of injection of a final reagent that is added to a tube containing the other reagents. Flow systems can be used to mix the reagents when the analyte is injected into one or more streams of chemiluminescent reagents (Figure lb). Analytes on solid surfaces such as filter papers can be measured by saturating the surface with CL reagents and recording the light emitted with a microtiter plate reader or by contact printing with photographic detection (Figure IC). From the analyst's point of view, several factors must be considered in developing a chemiluminescent method. Two of the most obvious are the efficiency of the CL reaction, which ultimately affects sensitivity and detection limits, and the reaction kinetics, which determine the precision and sample throughput. In practice, both of these parameters are affected by reaction conditions, including solvents, concentrations, pH, and purity of reagents,
Chemiluminescent reactions Firefly reaction. One of the most efficient and best known chemiluminescent reactions is the firefly system (Figure 2). In the proposed transformation of firefly luciferin to an excited state of luciferin, a dioxetanone is formed by
the action of the luciferase enzyme using oxygen and adenosine triphosphate (ATP). Reaction efficiencies of about 90% have been reported under basic conditions, typically around pH 11.For analytical purposes, ATP, luciferin, or the luciferase enzyme can be directly measured with this reaction. Bacterial BL. The second most commonly studied bioluminescent re-
action is bacterioluminescence from some sea creatures with whom these bacteria live symbiotically. Although differences between strains exist, the basic reaction is believed to involve the formation of an excited state of hydroxylated flavin from the action of an enzyme with oxygen and a long-chain aldehyde on the reduced flavin mononucleotide (FMNH2).
- =w 562nm lax
I Figure 2.
Firefly reaction. PPI = inwganic phosphate (pyrophosphate).
1244A * ANALYTICAL CHEMISTRY, VOL. 59, NO. 21. NOVEMBER 1, 1987
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Although this type of reaction is much less efficient (less than 1%)than the bioluminescent reactions, a number of analytical applications have been investigated with the reaction A competing side reaction is the nonenshown below. It can be used to directly zymatic oxidation of FMNH hy oxymeasure luminol, hydrogen peroxide, gen. metal, or enzyme catalysts. A variety of oxidizing systems have FMNH, 0, FMN H,O, been used in these applications. HyAs with the firefly reaction, highest efdrogen peroxide has been used most ficiencies are reported under basic confrequently and requires addition of a ditions. For analytical purposes, the catalyst or cooxidant. Peroxidase is one use of purified enzymes improves deexample of a catalyst, whereas hemin tection limits as well as enzyme stahilor iron hexacyanoferrate (111) are often ity. Any of the components-the reused as catalyst-cooxidants. duced flavin mononucleotide, the aldePeroxyoxalate reactions. This hyde, the enzyme, or the oxygen-may class of reactions involving the oxidahe measured with bacterial biolumition of oxalate derivatives is among the nescence. most efficient nonenzymatic reactions Luminol. The oxidation of luminol used analytically. They involve the fol(5-amino-2,3-dihydrophthalazine-1,4- lowing steps: dione) is one of the more commonly oxalate derivative H,O, known nonenzymatic chemiluminescent reactions.
+ 0,+ RCHO d FMN + RCOOH + H,O + light
FMNH,
+
-
+
+
-
ro - c =o i
-I b - L o 1-
--
1,2-dioxetanedione (I) (I)+fluorophor fluorophor* LigM
h,,
= 425 nm
fluorophor* t 2 CO, fluorophor light
+
The light is characteristic of the first excited singlet state of the fluorophor. Many analytical applications have measured fluorophors emitting in the
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 21, NOVEMBER 1, 1987
visible light region. Hydrogen peroxide can also he measured with this reaction. Both acid- and base-catalyzed systems compatible with aqueous measurements have been reported. Lucigenin. CL is observed in basic solution from the excited state of N-methylacridone produced hy the oxidation of lucigenin (his-N-methylacridinium nitrate). CH3
,,A
LisM = 470 nm
The oxidation of lucigenin is achieved with several systems. Typically, peroxide and metal catalysts are used, but CL is also observed with a reductant and oxygen. Both mechanisms are proposed to go through a dioxetane intermediate. Because oxidation and reduction steps are involved, it is analytically possible to measure both oxidants and reductants. Hydrogen peroxide and metals have been measured with the first system. Reductants such as ascorbic acid and fructose have been deter-
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mined with the second system. Acridinium esters. The oxidation of acridinium esters by hydrogen peroxide has been extensively studied by McCapra et al. (Reference 9 in Suggested Reading) and is compatible with aqueous solution a t neutral pHs (Figure 3). Both the acridinium ester and hydrogen peroxide have been measured with this reaction. Tetrakis N-alkylaminoethylenes. The reaction of oxygen with tetrakis N-dkylminoethylenes has been demonstrated to be analytically useful for the determination of oxygen in gas-phase analysis.
rn
Rd,
NRz
/
c = c\ R~N'
+o* NRz
-
2 bN\ C =C /
RzN
Light is from the energy transfer hack to aminoethylene.
Categories of measured species For the applications discussed here it is convenient to classify the species quantitated with the CL reaction into one of three categories. (The species mea-
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sured may or may not be the analyte of interest but is related to the analyte, by labeling or with coupled reactions. See Scheme 1.) In the first category the measured species is one of the reagents consumed during the reaction, as is the case with luminol or hydrogen peroxide. There is usually very little background using this approach. In the second category the measured species is not consumed during the reaction. An example is a catalyst such as hemin or metals used with the luminol reaction. This approach should have improved detectability, because theoretically an increased signal can he obtained by adding more reagents. However, there is often a large background. The third category involves coupled reactions in which the measured species is generated by one or more reactions before chemiluminescent reagents are added to produce light. This approach can extend the applicability of CL methods, but it introduces the added concern of compatihility of conditions for all reactions. In the first two categories the measured species may be the analyte of interest, such as the determination of hydrogen peroxide or metals in aqueous samples. The measured species may also be used as a label on the analyte of interest, which is the case in immunoassays when an antigen or antibody is labeled with a luminol derivative. In the third category the analyte is usually a component of one of the coupled reactions. For example, glucose can be determined using the following coupled reaction sequence:
-
glucose-glucose oxidase hydrogen peroxide gluconic acid
+
hydrogen peroxide + CL reagents light
-
. .. -. . ... ... .
SCIEnTIFIC
. I
AcridiniumI chemiluminescent reaction. Figure 3. CIRCLE 127 ON READER SERVICE CARD
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ANALYTICAL CMMISTRY, VOL. 59. NO. 21. NOVEMBER 1. 1987
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Using this approach, one of the components of the coupled reactions may also be used t o label the analyte of interest. Glucose oxidase may be labeled to an antigen and the other reactants added to generate light, which can be measured to quantitate the antigen.
Competitive binding assays Since the development of radioimmunoassays (RIA), many assays that rely on the specificity of the antigen-antibody binding reaction, requiring sensitive, specific detection, have been based on its principles. A typical competitive binding assay for the determination of an antigen (Ag) can be represented by the diagram below, where unlabeled and labeled antigen (Ag*) compete for sites on the antibody (Ab). Ag + Ag* Ab AgAb Ag'Ab After equilibration, the amount of bound and free labeled antigen can be measured, and a calibration curve can be used t o determine the analyte. Many competitive binding assays are limited by binding constants, but much emphasis has been placed on the development of sensitive measurement of the labeled immunogen. The earliest immunoassays used radioactive labels because of the sensitivity of the measurement. Difficulties with these labels, including waste disposal and unstable reagents, have prompted the development of nonradioactive tags, including fluorescent derivatives. However, even methods using fluorescent labels have not provided the low levels of detectability required for most immunoassays. For this reason chemiluminescent tags are attractive alternatives because CL methods are among the few with the required sensitivity and detectability. The CL immunoassay is carried out similarly to RIA using au immunogen labeled with a CL reagent, and the final measurement is made by adding the required reagents and recording the light emitted. Unfortunately, unlike radioactive and even many fluorescent labels, which are relatively unaffected by binding to another species, the
+
1250 A
-
+
chemical binding of a chemiluminescent reagent often significantly affects its ability to chemiluminesce efficiently. Much of the work in this area has been aimed a t developing labels with sufficient CL activity to achieve low detection limits. A variety of reactions have been evaluated and demonstrated to be suitable for use in a CL immunoassav. Indeed. hundreds of reports describing such re: actions have appeared in the literature, and some have recently been made commerciallv available. A few examples are shown in Table I. BL has been used in a number of ways. Labels have been developed using ATP derivatives detected with the firefly reaction ( 1 ) . The activities of firefly and bacterial luciferase labeled to aualytes have been measured using the appropriate bioluminescent reaction (2). Although the use of the en-
,;?Table 1. , , , .,, , ,. ,,,,. .. . ,,.. . :: ,,,,.I(
zymes as labels should result in increased sensitivity, often the enzyme activity is significantly decreased on binding. Nevertheless, detection limits in both cases are comparable to other competitive binding assays. One approach to overcome this limitation is to reactivate the enzyme before the CL measurement is made by removing it from the analyte. This has been demonstrated by Yein et al. in the determination of estriol (3). The luminol reaction can be used in several ways. Isoluminol labels have been used for a variety of analytes, although the CL efficiency of the tagged reagent is not as great as that of the untagged (4). A number of catalysts for the luminol reaction have been investigated as tags, including horseradish peroxidase (HRP) (5)and metal complexes. Both Co(I1) and Fe(II1) complexes were evaluated, and Fe(II1) 4,11,18,25tetracarboxyphthalocyanine [TCPFe(III)] was shown to have the highest catalvtic activitv (6). The peroxyoxilate reaction was used to measure the CL of fluorescent labels, but solvent restrictions significantly affected the precision (7,8). Another approach is to use peroxyoxalate CL to measure the activity of enzymes such as glucose oxidase, which generates hydrogen peroxide (9). Acridinium ester labels have successfully been used to achieve detection limits in the femtomole range. The phenyl carboxylate ester has been attached to the amine group on the anti-
Examples of chemiluminescence (CL) immunoassays
._.,I
Analyte
Label
CL system
Firefly luciferase Mamobexate dinitroDhenoi Firefly luciferase Bacterial luciferase Bacterial luciferase
LuciferinlATP LuciferinlATP FMNH2/RCH0 FMNloxidoreductaselNADH1 decanai .uciferin/ luciferase Luminol/hydrogen peroxide Luminollhydrogen peroxide
DI
microwr-
Acridinium carboxylate ester
* ANALYTiCAL CHEMISTRY, VOL. 59. NO. 21. NOVEMBER 1, 1987
peroxidelbase
Detection llmlt Reference
2.5 10 15 50
pmol
2
DmOl pmOl
pg
3
10 mM
1
25
pg
c
2
pg
I
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gen through an N-succinimide functional group on the ester (10).
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ANALYTICAL CHEMISTRY, VOL. 59,
creatine phosphate + ADP % creatine ATP
+ ATP + luciferin + o2luciferase>
light The sensitivity of the bioluminescent method is more than adequate for the detection of CK activity (14). Blood samples from myocardial infarct patients were analyzed using this method. Although the postcolumn reagents were added in solution form, the possibility of developing reactors using immobilized enzymes has been suggested. Unfortunately, in the case of the firefly luciferase, immobilization results in poor stability. Bacterial BL has been used for de-
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HPLC detection One area of great interest to researchers is the development of detectors for high-performance liquid chromatography (HPLC) and the use of postcolumn reactions to achieve selectivity and sensitivity. CL is particularly attractive in this mode, especially when sensitivity is needed. A variety of reactors have been demonstrated (Table 11) using some form of instrumentation similar to that in Figure lb. The luminol reaction was the first CL reaction demonstrated as a postcolumn reactor when Seitz et al. detected COW)and Cu(I1) separated by ion exchange (11 ) . Luminol and hydrogen peroxide were added postcolumn, but researchers had to ensure the compatibility of separation conditions with postcolumn reaction conditions. Metals are usually separated under acidic conditions using hydrogen chloride but, as previously mentioned, the luminol reaction is most efficient in basic conditions. Therefore lithium chloride was used in the mobile phase. Coupling a photochemical reaction that produces hydrogen peroxide to be detected with luminol CL has been used to determine aliphatic alcohols, aldehydes, ethers, and saccharides. Photooxygenation is induced after separation, and detection limits vary with the analyte (12). The isoluminol label used in immunoassay applications has been adapted as the HPLC derivatizing agent for amines and carboxylic acids. Addition of hydrogen peroxide and metal catalyst postcolumn allows the detection of femtomole amounts of these compounds (13). The isoenzymes of creatine kinase (CK), which is important for muscle contraction, have been separated by ion-exchange chromatography and detected by adding the appropriate firefly reagents postcolumn. The assay is based on the coupled reactions:
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d o n of bile acids separated by chrolatography (15). Bile acids react with icotinamide adenine dinucleotide WD) and 3a-hydroxysteroid-NAD ridoreductase t o generate NADH, hich can be detected by coupling with le bacterial bioluminescent reaction: NADH
+ FMN
-
2NAD
+
F M " 2
IlJCifW~
FMNH,
+ RCHO + 0,+ light
risue et al. also evaluated the use of le peroxyoxalate reaction for detecon of the fluorescent NADH prouced (15). Although the authors reort that sensitivity was not adequate nder the conditions used for the c h i d assays evaluated. the feasibility of aing immobilized enzyme reactors for ostcolumn detection was shown. Peroxyoxalate CL is one of the reac0118 most extensively investigated for catcolumn detection. The attractive sture of this reaction is the ability to etect analytes with native fluores:nee (or compounds derivatized with uorescent labels that are already corniercially available) but that are at levis as much as 2-3 orders of magnitude bwer than those detected with convenonal photoexcitation. Fluctuations nd scattering from the light source, hi& limit d-bility, are eliminated i the chemilumkacmt measurement. The method was fmt demonstrated )r dansylated amino acids where de?&ion limits in the low femtomole lnge are achievable hy the postcolumn ddition of hydrogen peroxide and the
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oxalate ester bis(2,4,6-trichlorophenyl) oxalate (16). Further work has shown that not all fluorophors are efficiently excited by the CL reaction and that therefore an additional degree of selectivity is ohtsined in complex matrices. B i r h et al. detected subpicogram levels of polycyclic aromatic amines in shale and coal oil samples (17).Severalreports of peroxyoxalate CL indicate that detectability is limited by a background CL emitted when no fluorophpr is added. The luclgenm reaction has heen used for the determination of reducing agents and is especially sensitive for ascorbic and dehydroascorbic acids that can be quantitated in the milligram-per-liter range (28).
m-assays DNA hybridization dot assays are also based on the speeifcity of a binding process that of DNA strands for each other. An unknown DNA canbe identitied with the Southern Blot method in which the strands of the analyte are separated and allowed to interact with labeled probe DNA strands on nitrocellulose fdter paper. Unbound strands are washed away, leaving only doublestranded DNA on the fdter paper. If the label on the prohe is detected, the DNA can be identified and, in some cases, quantitated. Again, radioactive tags have been used because of the measurement sensitivity required for quantitation, although these assays often require overnight measurements. Recently, HRP has been used as a DNA probe detected with the luminol
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reaction. Quantitation is possible with photographic detection. The end point is reached rapidly, and levels as low as 1 pg of DNA can be easily detected. This has also been recently developed commercially (19).
Optical sensors Because of the simple instrumentation and sensitivity of CL measurements, CL methods should be advantageous in doing on-site detection, a growing trend in analytical chemistry demonstrated by the current interest in optical sensors. Two sensors based on CL have been reported in the literature. The first was used to detect oxygen in the gas and liquid phases using the tetrakis (dimethy1amino)ethylene oxidation reaction (20). Reagent is held behind a Teflon poly(tetrafluoroethy1ene) membrane through which oxygen diffuses while light is transmitted to the photomultiplier detector. Detection limits of 1 ppm (v/v) in the gas phase should be achievable, and response is similar to that of the oxygen electrode in that the measurement is temperature-sensitive and determines the partial pressure of oxygen. However, the optical probe should not be subject to the same interferences as the electrode, including chlorine and hydrogen sulfide, because these have not been shown to affect the chemiluminescent reaction.
The second sensor determines hydrogen peroxide with horseradish peroxidase immobilized on a photodiode, which is immersed in a basic solution of luminol and analyte. Glucose can also be detected with a bienzyme sensor containing both HRP and glucose oxidase. Hydrogen peroxide concentrations are detectable from 1 to 10 mM; the range reported for glucose is 100 mM-1.5 M (21).
Future trends CL measurements have been demonstrated to be suitable for a wide variety of analytes. Difficulties such as a lack of mechanistic information and the need to modify chemical and instrumental parameters to measure the transient CL signal continue to affect method development. However, probable trends will include further work in DNA hybridization assays, immunoassays, or other applications that would benefit from the low detection limits and simple instrumentation characteristic of CL measurements. References (1) Carrico, R. J.; Johnson, D. R.; and Bo-
guslaski, R. C. Methods in Enzymology; Academic: New York, 1978; Vol. 17, pp.
113-22. (2) Wannlund, J.; DeLuca, M. In Biolumi-
nescence and Chemiluminescence, Basic Chemistry and Analytical Applications; DeLuca, M.; McElroy, W. D., Eds.; Academic: New York, 1981, pp. 693-96.
(3) Yein, F. S.; Marschke, C. K.; Deming,
P. C.; Holznan, T. F.; Satoh, P. S. Anal. Biochem. 1985,149,309-15. (4) Kohen, F.; Kim, J. B.; Lindner, H. R.; Barnard, G. In Bioluminescence and Chemiluminescence, Basic Chemistry and Analytical Applications; DeLuca, M.; McElroy, W. D., Eds.; Academic: New York, 1981, pp. 351-56. (5) Arakawa, H.; Maeda, M.; Tsuji, A.; Kembegawa, A. Steroids 1981,38(4),45364. (6) Hara, T.; Toriyama, M.; Tsubagoshi, K. Bull. Chem. SOC.Jpn. 1983,56,2267-71. ( 7 ) Mahant, V. K.; Miller, J. N.; Thakvar, H.Anal. Chim. Acta 1983,145,203-6. ( 8 ) Grayeski, M. L.; Seitz, W. R. Anal. Biochem. 1984,136,277-84. (9) Arakawa, H.; Maeda, M.; Tsuji, A. Clin. Chem. ( Winston-Salem, N.C.) 1985, 31(3),430-34. (10) Weeks, I.; Woodhead, J. S. Clin Chim. Acta 1984,142,275-80. (11) Neary, M. P.; Seitz, W. R.; Hercules, D. M. Anal. Lett. 1974,7,583-90. ( 1 2 ) Gandelman, M. L.; Birks, J. W. J . Chromatogr. 1982,242, 21-31. (13) Kawasaki, T.; Masako, M.; Tsuji, A. J . Chromatogr. 1985,328,121-26. (14) Bostick, W. P.; Denton, M. S.; Dins-
more, S. R. In Bioluminescence and Chemiluminescence, Instruments and Applications; Van Dyke, K . , Ed.; CRC: Boca Raton, 1985; Vol. 11, pp. 227-46. (15) Arisue, K.; Marui, Y.; Yoshida, T.; Ogawa, Z.; Kohda, K.; Hayashi, C.; Ishidi, Y. Rinsho Byori 1981,29(5), 459-62. (16) Kobayashi, S.; Imai, K. Anal. Chem.
1980,52,424-27. (17) Sigvardson, K. W.; Kennish, J. M.; Birks, J. W. Anal. Chem. 1984, 56(7), 1096-1102. (18) Veazey, R. L.; Nieman, T. A. J . Chromatogr. 1980,200,153-62. (19) Matthews, J. A.; Batki, A.; Hynds, C.; Kricka, L. J. Anal. Biochem. 1985, 151, 205-9. -.. .
1
(20) Freeman, T.M.; Seitz, W.R. Anal. Chem. 1981,53,98-102. (21) Aizawa, M.; Ikariyama, Y.; Kuno, H. Anal. Lett. 1984,17(B7), 555-64.
H
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cence; Kricka, L. J.; Carter, T.J.N., Eds.; Dekker: New York, 1982. (2) Chemi- and Bioluminescence; Burr, J. G., Ed.; Dekker: New York, 1985. (3) Bioluminescence and ChemiEuminescence: Instruments and Applications; Van Dyke, K., Ed.; CRC: Cleveland, 1985; Vols I and 11. (4) Seitz, W. R. CRC Crit. Reu. Anal. Chem.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 21, NOVEMBER 1, 1987
1981,l. (5) Bioluminescence and Chemiluminescence, Methods of Enzymology; Deluca, M., Ed.; Academic: New York, 1978; Vol. 57. (6) Seitz, W. R. Clin. Biochem. Amsterdam 1984.17. 120. ( 7 ) Seitz, W.-R.; Neary, M. P. Anal. Chem. 1974,46,188 A. (8) Isacsson V.: Wettermark. G. Anal. Chirn. Acta 1974,68,339. (9) McCapra, F. Acc. Chem. Res. 1976, 9, 201. (10) Schuster, G. B. Acc. Chem. Res. 1979, 12, 366.
M a r y L y n n G r a y e s k i received h e r Ph.D. in analytical chemistry f r o m t h e University of N e w Hampshire. S h e t h e n joined t h e faculty at S e t o n Hall University, where her research interests include chemiluminescence methods of analysis, HPLC detection, and flow injection analysis.
