The horseradish peroxidase (HRP)-catalyzed chemiluminescent oxidation of luminol is widely used in many molecular biology-based assays, such as Western blots, dot blots, and enzyme-linked immunosorbent assays (ELISAs), as well as in immunohistochemistry. Much effort has been made to improve the efficiency and analytical performance of this reaction. In particular, the addition of enhancers to the reaction substrate greatly increases light output and duration kinetics. Molecules with enhancer properties are essentially redox mediators capable of exchanging electrons between the peroxidase enzyme and luminol. Enhancers include substituted phenols, substituted boronic acids, indophenols, and N-alkyl phenothiazine derivatives.
Synthesis of SPTZ (PTZ-343)
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, PTZ-343) as white crystals (10.0 g, 72% yield).
Effect of nucleophilic acylation catalysts on SPTZ-enhanced substrates
The working solution was freshly prepared using the following concentrations: 5 mM luminol sodium salt, 1.5 mM SPTZ (PTZ-343), and 4 mM sodiumperborate in 0.125 MTris–HCl (pH 9.0). This solution was split into portions, and the following compounds were added to each portion to a final concentration of 1.5 mM: (a) pyridine, (b) 4-picoline, (c) PPY, (d) DMAP, (e) MORP, (f) triethanolamine (TEA), and (g) reference (no addition of acylation catalyst). The same enzyme addition protocol described above was used. Measurements were carried out with the fluorescence spectrophotometer(15 min total acquisition time).
Light emission from working solutions containing MORP: Effect of pH
The working solutions NoMORP and MORP were freshly prepared. The NoMORP working solution contained 5 mM luminol, 1.5 mM SPTZ, and 4 mM sodium perborate in 0.125 M Tris–HCl (pH 9.0). The MORP working solution contained 5 mM luminol, 1.5 mM SPTZ (PTZ-343), 1.5 mM MORP, and 4 mM sodium perborate in 0.125 M Tris–HCl (pH 9.0). The pH for each working solution was adjusted using negligible amounts of HCl (5 M) or NaOH (5 M) in the pH 8 to 10 interval. The same enzyme addition protocol described above was used. Measurements were carried out with the fluorescence spectrophotometer (10 min total acquisition time).
N-Alkyl-substituted phenothiazine enhancers were first described by Sugiyama in the patent literature. A water-soluble derivative, SPTZ (PTZ-343), was employed most often in these studies. However, no major improvement over the performance of phenolic enhancers was demonstrated. A few years later, much better results in terms of luminescent intensity and duration were reported by using a carefully purified sample of SPTZ (PTZ-343). In fact, the author found that the two-step, low-yield method used for the synthesis of SPTZ (PTZ-343) is largely responsible for the presence of impurities, especially unreacted phenothiazine, that quench L– and drastically reduce light output. Unfortunately, these impurities are not easily removed by conventional purification methods. For these reasons, the author developed a new one-step synthesis of SPTZ (PTZ-343).
Considering now the system in more detail, it is worth observing that the reduction potential of SPTZ (SPTZ+/SPTZ) is 0.83 V versus NHE, nearly identical to the value reported for luminol and close to the optimal value observed for phenolic enhancers. On the other hand, phenothiazine itself, with a reduction potential (PTZ+/PTZ) of 0.62 V versus NHE, is much easier to oxidize than is SPTZ (PTZ-343) and effectively quenches the production of luminescence even when present in trace amount.
Reference：
Ettore Marzocchi, Analytical Biochemistry 377 (2008) 189-194