1.Chemiluminescent detection systems of horseradish peroxidase employing nucleophilic acylation catalyst
Ettore Marzocchi, Stefano Grilli, Leopoldo Della Ciana*. Analytical Biochemistry 377 (2008) 189-194
Synthesis of SPTZ
Because the purity of the enhancer is of fundamental importance in the chemiluminescent process, the two-step literature procedure was replaced by the following improved one-pot synthesis. Sodium hydride (60% dispersion in mineral oil, 1.76 g, 44.2 mmol) was dissolved in dry THF (40 ml) under argon atmosphere. A solution of phenothiazine (8 g, 40 mmol) in dry THF (40 ml) was added, and the mixture was stirred at room temperature for 60 min (a dark orange suspension forms) and then 90 min at 50 oC. After cooling at 0 oC, a solution of 1,3-propanesultone (4.88 g, 40 mmol) in dry THF (40 ml) was added and the resulting mixture was stirred for 30 min. The ice bath was removed, and the mixture was stirred for a further 30 min. The white precipitate was ﬁltered off and subsequently washed with THF and Et2O and further puriﬁed by crystallization from 90% ethanol to give 3-(10’-phenothiazinyl)propane-1-sulfonate (SPTZ) as white crystals (10.0 g, 72% yield).
2. 3-(10’-Phenothiazinyl)propane-1-sulfonate is a potent enhancer of soybean peroxidase-induced chemiluminescence
Marina M. Vdovenko, Leopoldo Della Ciana, Ivan Yu. Sakharov*. Analytical Biochemistry 392 (2009) 5458
Although SbP was a more potent biocatalyst in luminol oxidation than HRP-C itself, in the presence of enhancers HRP-C produced a higher CL intensity than SbP. On the other hand, the addition of HRP-C enhancers such as 4-iodophenol and 4-hydroxy-cinnamic acid to the substrate mixture did not practically increase SbP-induced CL. Therefore, there was a need to ﬁnd enhancers of SbP that could increase the efﬁciency of the luminol oxidation without changing long-term kinetics of SbP-induced CL. The availability of such enhancers would also be expected to increase the detectability of this enzyme, thereby enabling the development of highly sensitive enzyme immunoassay kits. In this article, we describe the use of the sodium salt of 3-(10’-phenothiazinyl)propane-1-sulfonate (SPTZ) as a potent enhancer of SbP-induced CL formed on the enzymatic oxidation of luminol by hydrogen peroxide. We also demonstrate that the simultaneous introduction of SPTZ and 4-morpholinopyridine (MORP) in the substrate mixture results in an additional increase of CL signal as well as a decrease of the lower detection limit (LDL) of SbP. Moreover, comparison of LDL values determined in enhanced CL reaction under favorable conditions for horseradish and soybean peroxidases shows that SbP is a more active biocatalyst in luminol oxidation than traditionally used HRP-C.
3. Development of ultra-sensitive soybean peroxidase-based CL-ELISA for the determination of human thyroglobulin
Marina M. Vdovenko, Alexander V. Zubkov, Galina I. Kuznetsova, Ivan Yu. Sakharov*. Journal of Immunological Methods 362 (2010) 127-130
Recently, we showed that a combination of 3-(10’-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholino-pyridine (MORPH) is potent enhancer system of soybean peroxidase (SbP)-induced CL. A detection limit of SbP/SPTZ/MORPH system (0.03 pM) was 40-fold lower than that of HRP/p-iodophenol (PIP) system traditionally used in CL-ELISA. Moreover, upon luminol oxidation the SbP/SPTZ/MORPH system produced a long-term chemiluminescent signal. Therefore, a replacement of HRP with SbP may increase the sensitivity of CL-ELISA kits simultaneously remaining all advantages of plant peroxidases. In the present work, we describe the use of SbP/SPTZ/MORPH as detection system in ultra-sensitive CL-ELISA for the determination of thyroglobulin in human serum. Comparison of Tg CL-ELISAs with SbP and HRP a label enzymes demonstrated that a sensitivity of the assay was higher with the former enzyme. The results obtained for the measurement of Tg concentration in human serum samples by SbP-based CL-ELISA and commercial AlPh-based kit showed a good correlation.