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Originally Published IVD Technology April 2001

Detection Technologies

Chemiluminescent detection with 1,2-dioxetane substrates

Substrates for alkaline phosphatase can be variously enhanced so as to provide highly sensitive detection with many assay formats and instrumentation platforms.

Corinne E. M. Olesen, Larry J. Kricka, Brooks Edwards, Ruoh-Rong Juo, John C. Voyta, and Irena Bronstein

The use of 1,2-dioxetane chemiluminescent substrates with alkaline phosphatase (AP) enzyme labels provides highly sensitive detection for numerous immunoassay and nucleic acid detection formats. Current applications include membrane-based detection of proteins and nucleic acids, immunoassays, microplate-based nucleic acid detection and, increasingly, array-based detection.

Dioxetanes are four-membered cyclic peroxides that have been implicated as short-lived, unstable intermediates in oxidation reactions resulting in chemiluminescence.1 The possibility of synthesizing thermally stable 1,2-dioxetane molecules that are actually stabilized intermediates in an oxidation reaction has been demonstrated.

Adamantyl-1,2-dioxetane phosphates that are direct substrates for alkaline phosphatase have been successfully used for bioanalyte and enzyme detection. Hydrolytic dephosphorylation of adamantyl-1,2-dioxetane phosphate substrates by AP results in the formation of a metastable anion, which fragments further to form an excited-state anion that emits light (see Figure 1). The dioxetane phenolate anion decomposes via a chemically initiated electron exchange luminescence (CIEEL) mechanism.2 Charge transfer from the phenolate to the dioxetane ring promotes cleavage of the cyclic peroxide, releasing about 100 kcal to chemically excite one of the resulting carbonyl fragments to a singlet electronic state. This excited species emits light at 477 nm as it reverts to the ground state.

Figure 1. The light-emission mechanism of 1,2-dioxetane AP substrates.

Light emission obtained from the AP-catalyzed dioxetane decomposition reaction is a steady-state glow that makes possible the use of several different imaging platforms for signal detection. These include photomultiplier tube and photodiode-based luminometers, x-ray film, photographic film, phosphor screens, and instrumentation systems using cameras based on low-light-sensitive charge-coupled device (CCD) technology.

A family of 1,2-dioxetane substrates for AP has been synthesized to offer specially enhanced performance characteristics (see Figure 2). The AMPPD, CSPD, CDP, and CDP-Star substrates marketed by Tropix (Bedford, MA) are discussed later in this article. Lumigen PPD, Lumi-Phos, Lumi-Phos 530, and Lumi-Phos Plus, available from Lumigen Inc. (Southfield, MI), are dioxetane formulations incorporating the AMPPD molecule and including enhancers. In addition, 1,2-dioxetane substrates for other hydrolytic enzymes, including ß-galactosidase and other ß-glycosidases, are available.

Figure 2. The chemical structures of some 1,2-dioxetane AP substrates.

The addition of macromolecular enhancers significantly improves the intensity of luminescence produced by 1,2-dioxetanes.3,4 Proton transfer events in aqueous solution generate one thousandth the chemiluminescent intensity obtained in organic solvents. But water-soluble quaternary amine polymers enhance light emission in aqueous reactions approximately 100-fold. Other polymer formulations that contain fluorescein as an energy transfer acceptor further increase the yield of light and shift the maximum wavelength of emission up to 540 nm. A fluorescent micelle enhancer system that is formed from cetyltrimethylammonium bromide and a fluorescein energy transfer acceptor increases chemiluminescence efficiency 400-fold.4

This article discusses 1,2-dioxetane-based detection techniques and the capabilities of the technology.

Immunoassay Detection

The high sensitivity of detection by means of 1,2-dioxetane substrates with AP enzyme labels has been demonstrated for use in quantifying a variety of analytes with chemiluminescent enzyme immunoassay methods, including both sandwich and competitive immunoassay formats. The performance of chemiluminescent 1,2-dioxetane substrates compares favorably with those of colorimetric, radioimmunoassay, and fluorescent methods.5

Several fully automated random-access immunoassay analyzers for clinical analytes utilize 1,2-dioxetane substrates, including the Immulite and Immulite 2000 (Diagnostic Products Corp., Los Angeles), Access (Beckman Coulter Inc.; Fullerton, CA), and Lumipulse 1200 (Fujirebio, Tokyo) instruments. Other luminescence chemistries, such as acridinium ester and acridinium (N-sulfonyl) carboxamide labels, isoluminol, and electrochemiluminescent ruthenium trisbipyridyl labels, also are incorporated into clinical diagnostic assay systems (see Table I).

Company

System

Chemistry

Immunodiagnostics

Abbott

Architect
Prism

Acridinium (N-sulfonyl) carboxamide
Acridinium ester

Bayer Diagnostics

ACS:180
ACS:180Plus
ACS:180 SE

Acridinium ester

Beckman Coulter/Sanofi

Access

1,2-dioxetane

Byk-Sangtec Diagnostica

Liaison

Isoluminol

Diagnostic Products Corp.

Immulite
Immulite 2000

1,2-dioxetane

Fujirebio Lumipulse 1200 1,2-dioxetane
Prionics Prionics Check 1,2-dioxetane (blotting)
Roche Diagnostics Elecsys Electrochemiluminescence (ruthenium salts)
Nucleic Acid Diagnostics
Bayer Diagnostics Bayer System 340 Analyzer 1,2-dioxetane (bDNA)
Digene Hybrid Capture 1,2-dioxetane
Gen-Probe PACE, PACE 2
AccuProbe
Amplified 		Acridinium ester
Table I. Commercialized chemiluminescent diagnostic assays and platforms.

These systems offer a broad test menu, performed with both sandwich and competitive heterogeneous immunoassays. The Immulite and Immulite 2000 systems feature an assay format comprising a proprietary tube design, an antibody-coated polystyrene bead, and a Lumigen PPD substrate. Comparison of chemiluminescent detection with Immulite assays to detection techniques used with immunoradiometric assays and reference assays has shown that the dioxetane-based assay systems offer equivalent or better sensitivity and precision.6,7

The Access immunoassay system incorporates an antibody-coated paramagnetic microparticle solid phase and the Lumi-Phos 530 substrate formulation. Chemiluminescent detection with the Access system has correlated favorably with other immunoassay systems and culture techniques, displaying comparable or higher sensitivity and precision.8 The Lumipulse 1200 system incorporates a coated-ferrite-particle solid phase, magnetic separation, and AMPPD substrate in a variety of competitive and sandwich immunoassay formats.9

CDP-Star substrate is a constituent of the Prionics-Check (Prionics AG; Zürich, Switzerland) assays for bovine spongiform encephalopathy (BSE) and scrapie diagnostics, now used for routine surveillance of BSE in slaughtered animals. The Western immunoblotting assay format provides highly sensitive and accurate detection of the disease-specific prion protein PrPSc, is able to detect subclinical BSE before detectable histopathologic changes occur, and yields results equivalent to those of immunohistochemistry methods in a much shorter time and with less expense.10

Nucleic Acid Detection

Detection methods incorporating alkaline phosphatase labels with 1,2-dioxetane substrates are widely used in nucleic acid hybridization assays. Demand for higher throughput and for integration with automated systems has driven the development of nucleic acid detection assays first in microplate and now primarily in microarray formats. Both in-solution and solid-support capture/hybridization assays can be performed. The benefits of 1,2-dioxetane detection systems that have been observed in membrane-based assays are also achieved in microplate assays. These include high sensitivity and a dynamic range greater than those of colorimetric and radiolabel detection systems.

Several commercial products that employ 1,2-dioxetane detection systems are available for the detection of viral clinical targets. The branched-DNA (bDNA) assay technology, originally developed by Chiron (Emeryville, CA), uses a signal amplification system comprising a bDNA probe system that attaches multiple AP labels to the target nucleic acid, along with Lumi-Phos 530 and Lumi-Phos Plus substrates. This technology is commercialized in nondiagnostic viral load assays for the hepatitis B and C and human immunodeficiency viruses (HBV, HCV, and HIV-1) and as an assay system for messenger RNA quantitation. HBV quantitation using a bDNA assay has been demonstrated.11

Hybrid Capture assay technology (Digene Corp.; Silver Spring, MD) utilizes a signal amplification system incorporating an anti-RNA/DNA hybrid antibody-AP conjugate and CDP-Star substrate. The Hybrid Capture system has demonstrated very sensitive detection of herpes simplex and human papilloma virus.12,13 With both the bDNA and hybrid systems, the combination of signal amplification and 1,2-dioxetane chemiluminescence enables detection of less than one thousand viral targets without nucleic acid amplification.

Detection of viral and other pathogenic sequences has been performed with 1,2-dioxetane AP substrates in microplate formats with several assay methodologies, including polymerase chain reaction (PCR) amplification, quantitative PCR, probe ligation, strand-displacement amplification, and ligase chain reaction.5 Chemiluminescent detection of viral genomes in prepared tissues and cultured cells with in situ hybridization has been accomplished with 1,2-dioxetanes and digital analysis of microscopic images.

Advances in 1,2-Dioxetane Technology

The AMPPD, CSPD, CDP-Star, CDP, and ADP-Star 1,2-dioxetane substrates for AP are all based on a similar derivatized phenyl substituent. The addition and positioning of chlorine groups on both the adamantyl stabilizing substituent and the phenyl group contributes improvements in signal intensity, light-emission kinetics, quantum yield, and the pH requirements. The signal generated by CSPD is more intense than that obtained with AMPPD, and image resolution is superior.14 Of all the substrates in the family, CDP-Star provides the highest-intensity chemiluminescent emission in both solution- and membrane-based applications and the most-rapid emission kinetics for membrane-based applications. It is well suited for rapid film exposures and imaging with low-light-sensitive cameras.15 CDP generates the longest signal half-life.15,16

The family of ß-galactosidase substrates from Tropix, including Galacton, Galacton-Plus, and Galacton-Star, are each related to a counterpart in the AP-substrate series. Following enzymatic deprotection of the ß-galactosidase substrate, the resulting anion breaks down with exactly the same spectral and kinetic performance as displayed by the related AP substrate. Additional substrates developed for quantitation of ß-glycosidase enzymes include Glucuron substrate for ß-glucuronidase and Glucon substrate for ß-glucosidase.17

A 1,2-dioxetane derivative of sialic acid has been developed as a substrate for influenza virus neuraminidase (NA).18 In a chemiluminescence-based assay for NA function in clinical isolates, the substrate provides 67 times the detection sensitivity of the fluorogenic reagent 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (MUN), making possible a detection threshold as low as 3 pM. Its high level of sensitivity enabled NA activity to be detected in 10 of 12 clinical isolates in which NA activity was undetectable with the fluorogenic assay, as reported from a clinical study of the zanamivir antiviral agent. In addition, the chemiluminescence assay provided an increased dynamic range, spread over four orders of magnitude as compared with two orders of magnitude achieved with the MUN substrate. Measurement of NA function is the most reliable and predictable way to monitor in vivo susceptibility to NA inhibitors.

Emerging Applications

The rapid growth in available CCD imaging technology and instrumentation and the desire of IVD manufacturers and pharmaceutical screening groups to develop multiwavelength multiplex detection assay capability have together spurred
the development of new 1,2-dioxetanes with green- and red-shifted emission spectra. (CCD cameras are more spectrally sensitive to green and red wavelengths.) Multiwavelength detection enables multiplexing with numerous applications, including immunoassays, nucleic acid detection, and reporter enzyme quantitation. A multiplex luminescent enzyme immunoassay for potential clinical quantitation of the ratio of pepsinogen I to pepsinogen II has been demonstrated. The assay uses mutant luciferase enzyme labels that provide distinct emission wavelengths.19 Simultaneous chemiluminescent detection of multiple enzymes or labels requires nonabsorbing molecules that provide light emission at discrete wavelengths. Most adamantyl-stabilized 1,2-dioxetane substrates commercialized so far emit inherent chemiluminescence in the range of 475–480 nm.

Most green-emitting systems depend on energy transfer to fluorescein or fluorescein derivatives. Energy transfer systems cannot be used for simultaneous detection of multiple enzymes. A 1,2-dioxetane AP substrate that offers an emission wavelength in the green region of the visible spectrum has recently been described.20

This substrate, BZPD, a benzothiazole dioxetane phosphate, is colorless, nonfluorescent, and stable, and upon cleavage an emission maximum of 550 nm is observed. Using the NorthStar HTS Workstation CCD imaging platform (Applied Biosystems; Bedford, MA) it has been possible to simultaneously measure green light emission from the AP-catalyzed decomposition of BZPD and blue light emission from the ß-galactosidase-catalyzed decomposition of Galacton-Star, which emits at a maximum of 475 nm. Using these substrates, 3.0 ¥ 10–19 moles of AP can be detected in the presence of up to 6.4 ¥ 10–16 moles of ß-galactosidase, and 3.2 ¥ 10–17 moles of ß-galactosidase can be detected in the presence of up to 1.0 ¥ 10–17 moles of AP.

The third-generation Galacton-Star substrate, because of its lowered pKa, allows the use of ß-galactosidase as an enzyme label for membrane blotting applications.21,22 Using ß-galactosidase- and AP-conjugated antibodies with Galacton-Star and an AP substrate, sequential dual detection of two protein antigens on a single membrane blot has been achieved.23 The availability of green-emitting BZPD now makes possible two-color multiplex chemiluminescent detection for membrane-immobilized proteins and nucleic acids.

Recently, 1,2-dioxetane molecules that emit red and yellow light have been synthesized. Chemically induced decomposition of a spiro[1,2-dioxetane-3,6' benzo (c)chromene] with a t-butyldimethylsiloxy trigger at the 2' position results in low-efficiency emission of yellow light.24 Decomposing 3-ethoxy-4,4-diisopropyl-1,2-dioxetanes substituted with a benzo(b)furan-2-yl or a benzo(b)thiophen-2-yl group and having a 5- or 7-position siloxy trigger emit red light in the range of 615–628 nm.25 Chemical decomposition was induced with tetrabutylammonium fluoride in the presence of dimethylsulfoxide. The red-emitting molecules are particularly interesting for their potential application in multichannel biological analysis.

A new class of red-emitting chemiluminescent substrates for alkaline phosphatase has been developed.26 Commercialized as Lumi-Phos CCD (Lumigen Inc.), they provide high-efficiency sustained light emission in either solution-based or membrane-based detection assays. These substrates contain an exocyclic enolphosphate derived from an aromatic thioester of 5,5'-dimethylluciferin. Dephosphorylation generates a transient dioxetanone intermediate that decomposes to produce red-orange light. Linear detection over five logs has been demonstrated using this type of substrate with the Immulite third generation TSH assay kit, and also with membrane-based detection.27

Another class of chemiluminescent AP substrates, marketed as Lumigen APS, are also able to be used in either solution-based or membrane-based detection assays.28,29 Dephosphorylation of these acridan substrates generates a transient dioxetanone intermediate as well. These substrates provide rapid development of peak signal and sustained light emission.

Drug Discovery Applications

The use of 1,2-dioxetane substrates in drug target discovery research and high-throughput pharmaceutical screening is widespread. Also, the substrates are increasingly employed in functional assays for genomics and proteomics efforts. Applications include reporter-gene assays to monitor gene expression, second-messenger quantitation, protein kinase assays, and protein-protein interaction analysis. An immunoassay system for quantitation of cyclic adenosine monophosphate (cAMP) provides detection of 0.06 pmol of cAMP and a dynamic range of 4–5 decades in a competitive immunoassay format.30 A complementation analysis system based on protein-protein-interaction-driven complementation of hybrid fragments of ß-galactosidase, which is quantitated with a chemiluminescent 1,2-dioxetane substrate, can be used for interaction antagonist/agonist screening, functional GTP-binding protein-coupled receptor (GPCR) signaling assays, and protein-protein interaction mapping.31 Reporter-gene applications are myriad, and include quantitation of yeast two-hybrid-system ß-galactosidase expression, which has been used in a functional proteomics application to construct a protein-protein interaction map of Helicobacter pylori.32

Figure 3. The NorthStar HTS Workstation for high-throughput luminescent imaging of microplates (Applied Biosystems; Bedford, MA).


Instrumentation

The light emission of membrane-based assays is usually imaged with standard x-ray film or Polaroid instant photographic film. Acquisition of digitized images, which enables quantitation to be more accurate, is accomplished with a phosphor storage screen and CCD camera instrumentation.

Chemiluminescent signals generated in microplate-based assays are quantified with a variety of commercially available luminometers. Such luminometers are normally instruments based on photomultiplier tube (PMT) technology, which move each well of the microplate directly below the PMT detector or the lens/fiber-optic light-collection interface. Sensitive detection of chemiluminescent signals in 96-well and higher-density microplates and microarrays is also possible with CCD camera instrumentation.33–35

Microscopic CCD imaging of 1,2-dioxetane chemiluminescence for in situ detection has also been demonstrated.36 The NorthStar HTS Workstation has recently been developed to perform high-throughput automated luminescence imaging of 96-, 384-, and 1536-well microplates (see Figure 3).37 This instrument incorporates a cooled CCD camera, integrated liquid and plate handling, data analysis capability, and a filter wheel for multiwavelength multiplex detection.

Conclusion

1,2-dioxetane chemiluminescent substrates provide a robust detection technology that is widely used in diagnostics, research applications, and drug discovery, providing highly sensitive detection in many assay formats and instrumentation platforms. Continued advances in 1,2-dioxetane substrate chemistry will provide additional functionality and versatility for a wide variety of assay formats. Further design of substrate molecules with different distinct emission spectra will enable development of multiplex multichannel chemiluminescent assays for research, diagnostics, drug discovery, and the emerging genome-driven high-throughput biology needs.

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Corinne E. M. Olesen, PhD, is senior manager in R&D at Applied Biosystems, Tropix div. (Bedford, MA) Her research interests include molecular and cell biological approaches to the development of bioluminescence and chemiluminescence-based assay systems for bioanalyte detection, and cell-based detection of cellular signaling events with luminescent sensors. Larry J. Kricka, DPhil, is professor of pathology and laboratory medicine at the University of Pennsylvania and director of the general chemistry laboratory at the Hospital of the University of Pennsylvania (Philadelphia). He is currently president of the American Association for Clinical Chemistry. His research interests include micromachined analytical systems, the analytical applications of bioluminescence and chemiluminescence, nonisotopic immunoassays, and heterophile antibodies. Brooks Edwards, MS, is principal scientist in R&D at Applied Biosystems, Tropix div. He is involved in the synthesis of chemiluminescent molecules for diagnostic applications. His research interests include multistep organic synthesis, organic photochemistry, synthesis of water-soluble polymers, near-infrared absorbing dyes, and photographic chemistry. Rouh-Rong Juo, PhD, is a staff scientist in R&D at Applied Biosystems, Tropix div. He is involved in the synthesis and ongoing development of new functionalities and structures of 1,2-dioxetane chemiluminescent substrates. John C. Voyta, PhD, is the director of science and technology for Applied Biosystems, Tropix div. His research interests include the application of chemiluminescent detection to new analytical detection formats and development of new instrumentation platforms. Irena Bronstein (not pictured) is vice president and general manager at Applied Biosystems, Tropix div. Her research interests include photochemistry, polymer chemistry and development of 1,2-dioxetane chemiluminescent substrate technology, and their analytical application in ultrasensitive luminescent detection of bioanalytes, clinical assays, and cell-based assay systems.

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