CLINICAL CHEMISTRY
Chemiluminescence and Bioluminescence L a r r y J. Kricka Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 The time period covered by this review is the Chemical Abstracts citation dates January 1, 1991 to October 1992. Chemiluminescence (CL) is the light emission produced in a chemical reaction from the decay of chemiexcited species to the electronic ground state. Similar reactions occur in nature (‘living light” or bioluminescence, BL) and involve a luciferin substrate in conjunction with either a luciferase enzyme or a photoprotein. Several books (RI-R3) and numerous reviews (R4-R25) describe recent progress and the range of applications of CL and BL in clinical analysis. This survey is limited to analysis of human fluids and tissues. The application of CL and BL in immunoassay and other types of ligandbinder assay (nucleic acid hybridization, blotting) is covered in another section of this review issue. New Reagents. Enzyme-triggerable adamantyl-12-dioxetanes have provided a new direction in CL enzyme assays (e.g., alkaline phosphatase, &galactosidase) ( R 2 6 R 3 0 ) . Dephosphorylation of an adamantyl-1,2-dioxetane aryl phosphate byalkalinephosphatase produces anunstable phenolate intermediate that decomposes to produce a long-lived light emission. The assay is extremely sensitive and zeptomole amounts (10-21 mol) of this enzyme can be detected. Other CL RSSRVR alkaline have. ,~ o~~~~~~~~~~~~~~~~~~~ , for~ DhosDhatase ~ ~and B-Palactosidase been devised based on 5. hrdmo-4-chlorc-3-indolyl phosphate and galactoside (detection limits 100 rmol) (R31).Diaiolu. minomelanin (R321,chemilwninescent pyridopvridazines t hat show great promise as alternatives to luminol (R33). and various acridinium ester derivatives (R34) have also been evaluated as reagents. Generally, CL reactions are inefficient (low CL quantum yield); hence there is scope for improvement (enhancement) of these reactions. Several new types of enhancers for CL reactions have been discovered including polymers [e.g., polyvinylbenzyl(benzyldimethylammoNum) chloride] for adamantyl-1,2-dioxetane reactions (R30) and hydroxytluorenones (R35), oxazole, thiazole, and imidazole derivatives for the CL horseradish peroxidase-catalyzed oxidation of luminol (R36). A number of analytically useful bioluminescent proteins have now been cloned and they provide an alternative and reproducible source of assay reagents. e.g., aequorin, Renilla luciferase (R37). Fusion proteins, e.g., protein A hacterial luciferase, have also been prepared for use as conjugates in immunological assays (R38). Immobilized reagents offer advantages of analytical convenience and improved stability, and recombinant firefly luciferase has been immobilized for use in a range of BL assays (R39). Cellular CL a n d Luminol- and Lucigenin-Enhanced CL. Cellular CLcontinues to beaveryactiveareaofresearch and accounts for the majority of the annual CL literature. Luminol and lucigenin enhance cellular CL intensity thus facilitatin its measurement. Luminol- orlucigenin-enhanced cellular C!l is used to measure active oxygen species enerated duringhagocytosis and serves as a convenient ancfsensitive meth for investigatingfactors influencingphagocyte biology or immunology, e.g., the effect of anti-asthma drugs on superoxide generation by polymorphonuclear leukocytes (PMNL) (R40),neutrophil-endothelialcell interaction (R41), the effect of bi hosphonate drugs (clodronate, etidronate) on neutrophil 8 L (R42), and the role of platelet activating factor in superoxide production in PMNLs (R43). A commercial system is now available for the assessment of inflammation based on the cellular CL assay that determines opsonin receptor expression on the surface of phagocytes (R44). Alternatives to luminol and lucieenin as reactive oxveen probes have been developed. These include CL probes, e.g., 7-dimethylnaphthalin-1,2-dicarboxylic hydrazide, which has a 3-fold higher CL efficiency compared with luminol (R45), and BL probes, e.g., coelenterate luciferin derivatives (R46R49). In addition the long-lived oxidant taurinechloramine can be detected in cellular supernatants by means of pholasin, the luciferin from the mollusk Pholas dactylus (R50). ~~
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ANALYTICAL CHEMISTRY. VOL. 65. NO. 12. JUNE 15. 1993
Larry J. KrlCka isa PTofesscfof P a m O ~ and Laboratory Medicine at Uw Unlversily of PennsylvaniaandDireclorof lh8Genaral Chemistry Laboratory. at me Hospita of hUnNerskyof Penns)nvanla. Hereceived his B.A. and 0.Phil. degrees from York UnNersW. England. His research interests ’, Indude Uw anawcal applicaUons of chsfnC ’ c kminescence a d bidunhescence.r w m b , , +;, . toDic immunoassavs and nucleic acid .ecs.&,.. hybridization assays. h u m a n antimouse antibodies.and mlcromhiaturizedanalytical systems. He is a member of the editorial board of Analytical 8iochemlstry, Journal ! of Immunoassay, and Talanta. and is the ediior-inchief of me Journal 01 Bioluminescence and Ctmrnlhminescence. Or. Kricka is a fellow of me Royal College of Paltwmlsts and lhe Royal Society 01 Chemistry and a member of the Assoclation of Clinical Biochemistsandthe Academy of Cllnlcal Laboratory Physlclans and Scientists.
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Rapid Microbiology. ATP is present in all living cells and thus cells can be detected and enumerated via assay for ATP using the BL firefly luciferase-luciferin reaction (R52R53). Improvements in the luciferase-luciferin reagent have permited detection of 80 fmoliL ATP and measurement of ATP from less than 10 hacterial cells. This BL assay has broad applications, e.g., antimicrobial susceptibility testing (R15,R54-R56), determination of the kinetics of bactericidal activity (R57), evaluation of microbial growth (e.g., Pneumocystis carinii) (R5.8).studyingcell chemotaxis (R59),sperm viability (R60),and antitumor chemosensitivitytesting (R61). This type of assay has been automated using a microtiter plate luminometer (R53). Both somatic and microbial cell ATP can be determined in a sample usin a sequential analytical process involving a detergent, l i p d t o protect the BL reagents, a hydrolytic enzyme, and a bactericide (R62). Cell counts can also be determined with a CL method using a menadioneFe-EDTA-luminol reagent (detection limit for yeast cells 8.8 X 10‘) (R63). CL Assays. Any component of a reaction that involves hydrogen peroxide is amenable to CL analysis. Usually peroxide production as a consequence of the presence of the analyte is detected using the luminol or a peroxyoxalate reaction. New assays based on this assay principle continue to appear, e.g., platelet-activating factor (R64).oxalate (R65R66), phosphatidylcholine hydroperoxides in blood plasma (R67j, choline-containing hospholipids (2 pmol/test) (R68), and acetylcholine (R22). 8ephalosporin antibiotics intensify and prolong the light emission from the Co(II)-luminolperoxide reaction and form the basis of a simple assay for compounds such as cephalothin (range 0.4-400rgimL) (R69). Antioxidants inhibit light emission from the 4-iodophenolenhanced horseradish peroxidase-catalyzed luminol-peroxide reaction,and this has beenused toassay antioxidantcapacity of biological fluids. The antioxidant produces a lag in light emission and the delay in light production is a measure of antioxidant concentration (R70). BL Assays. Dehydrogenases and kinases,and components of reactions catalyzed by these enzymes, can be measured using the NAD(P)H-dependentBL marine bacterial reaction and the ATP-dependent firefly luciferase reaction, res ec tively. The sensitivity of this type of reaction has mate i i particularly well-suited toanalysis ofmicrosamples, e.g.,ATP and phosphocreatine in single muscle fibers (R71).GGPDH in mucosal biopsies as a marker of malignancy in the large intestine (R72), intracellular inorganic pyrophosphate in lymphocytes (detection limit
