Substrates dioxetanes

C. Dioxetane Substrates for Molecular Biology and Clinical Assays. 1198... 

Nitro-Block enhances light output from the dioxetane substrate in reactions using AMPPD, CSPD, or Lumigen-PPD concentrate. It is required for nitrocellulose and recommended for PVDF membranes. It is not needed for Lumi-Phos 530, AP reactions on nylon membranes, or HRP-based reactions on any type of membrane. Lumi-Phos 530 is not recommended for nitrocellulose membranes. 

Another chemiluminescent enzyme system is based on the use of stabilized dioxetane substrates. Dio-xetanes are intermediates in many chemiluminescent reactions. It s possible to synthesize stabilized dio-xetanes (phosphatase and p-galactose moieties) that do not spontaneously react. When exposed to the right enzyme (alkaline phosphatase and p-galactosidase, respectively) the dioxetane will be destabilized and spontaneously undergo a chemiluminescence reaction. " ... 

Chemiluminescence assays are ultrasensitive (attomole to zeptomole detection limits) and have wide dynamic ranges. They are now widely used in automated immunoassay and DNA probe assay systems, (e.g., acridinium ester and acri-dinium sulfonamide labels and 1,2-dioxetane substrates for alkaline phosphatase labels and the enhanced-luminol reaction for horseradish peroxidase labels [see Chapter 9]). 

Dioxetane substrates for alkaline phosphatase and p-galac-tosidase The four-membered ring peroxides, 1,2-dioxetanes, are too unstable to be used as chemiluminescent substrates due to their low energy of activation (Adam and Cilento, 1983). Substitution of the carbonyls in the dioxetane ring has substantial effects on the rate of decomposition (Wieringa et al., 1972), i.e., half-lives from less than a second for simple dioxetanes to over 21 years for some of the... 

Olesen CEM. Dioxetane substrate for alkaline phosphatase labels. J Clin Ligand Assay 22, 1999 2 129-38. 

Although they have not been used as labels in immunoassays, dioxetane substrates for p-galactosidase have been synthesized and characterized (B18, B23, S9). Current data are sparse, but it would appear that the enzyme is less detectable... 

Infectious Diseases DNA hybridization assays that employ dioxetane substrates for alkaline phosphatase were first briefly reported in 1988, viz., herpes simplex 1 vims (B21) and Chlamydia trachomatis (Cl 7). These two assays were followed by an assay for hepatitis B vims core antigen, HBVc, (B18, B26). Thus, 10 copies of HBVc-containing plasmid DNA were detected in 30 min in a dot-blot assay, whereas a colorimetric test required 10 copies in the same time (B18, B26). Optimization allowed 10 -10 copies to be detected in 2 hr. An in situ hybridization assay for HSV-1 detected its presence after only 5 min (B22). A more detailed account of the Chlamydia assay describes how probes were prepared against two sites on an endogenous 7.4-kb plasmid and how, in a 5-hr assay. 

Moreover, in the same study, the dioxetane substrate was reported to have twice the sensitivity of an enhanced luminol method (Ul). 

One extension of blotting techniques that is likely to be expanded considerably in the near future is DNA fingerprinting for forensic analysis, paternity testing, etc. Already, the dioxetane substrate for alkaline phosphatase has been shown to be a useful tool in this endeavor (G4). 

This is quite inert. It is good for chemiluminescent detection with luminol and 1,2-dioxetane substrates. 

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