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We synthesized a series of conjugates of hemin and its aptamer EAD2, named covalent peroxidasemimicking DNAzymes (PMDNAzymes), varying the length, rigidity and 5 0 -/3 0 -position of a linker between the oligonucleotide and hemin. Systemic structure-activity relationship study of these PMDNAzymes showed that covalent PMDNAzyme with hemin bound to the 5 0 -end of EAD2 via T 10 spacer (PMDNAzyme(T 10 )) demonstrated the highest activity in luminol oxidation assay. Its activity was significantly higher in comparison to the non-covalent complex of hemin and aptamer EAD2 (noncovalent PMDNAzyme). Comparison of the detection limit values for the PMDNAzyme(T 10 ) in the reactions of oxidation of luminol and ABTS, which were equal to 0.2 and 1.6 pM, respectively, showed that the chemiluminescent method of PMDNAzyme(T 10 ) detection is preferred over the colorimetric one. Similarity of the detection limit values for the PMDNAzyme(T 10 ) and horseradish peroxidase, whose activity was measured in an enhanced chemiluminescence reaction (0.25 pM), opens up very promising perspectives for the development of highly sensitive PMDNAzyme(T 10 )-based assays and devices.

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An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for the determination of dexamethasone (DEX) was developed using soybean peroxidase (SbP) as an enzyme label. A mixture of 3-(10 0 -phenothiazinyl)-propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) was used as an enhancer of SbP-induced chemiluminescence. Varying the concentrations of the capture antigen (DEX-ovalbumin) and specific anti-DEX antibody, the conditions of the assay were optimized. The values of IC 10 , IC 50 and working range (IC 20 -IC 80 ) of the CL-ELISA of DEX were 0.02, 0.9, 0.08-9.3 ng mL À1 , respectively. It was shown that a pretreatment of cow milk samples by centrifugation and 25% methanol prevented the matrix effect of whole milk. The coefficient of variation (CV) and recovery values from the spiked milk samples estimated by the developed CL-ELISA were in the range of 2.2 to 9.9% and 82 to 142%, respectively.

We developed a homogeneous chemiluminescent DNA assay based on allosteric activation of peroxidase-mimicking DNAzyme.

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