One-step label-free chemiluminescent assay for determination of exonuclease III activity towards hairpin oligonucleotides.

Fast label-free chemiluminescent assay for determination of exonuclease III (ExoIII) activity measured towards hairpin oligonucleotide substrates was developed. The designed substrates consisted of EAD2 aptamer to hemin which was associated with DNA sequence complementary to 5'-terminus fragment of EAD2. In the presence of ExoIII the associated sequence of the hairpin stem was digested, producing EAD2 aptamer which reacted with hemin with the formation of peroxidase-mimicking DNAzyme (PMDNAzyme). The catalytic activity of the produced PMDNAzyme was measured towards luminol/H2O2. Under the optimized conditions the limit of detection and sensitivity of the one-step chemiluminescent assay of ExoIII were 7.3 nM and 1.7 × 108 M-1, respectively. The coefficient of variation (CV) was lower than 6%.

Microplate chemiluminescent assay for HBV DNA detection using 3-(10'-phenothiazinyl)propionic acid/N-morpholinopyridine pair as enhancer of HRP-catalyzed chemiluminescence.

A sensitive sandwich assay for hepatitis B virus (HBV) DNA detection based on use of commercial CL-ELISA microplates was developed. To reveal the target the covalent conjugate of reporter oligonucleotide and horseradish peroxidase (HRP) was synthesized. An employment of enhanced chemiluminescence reaction, where 3-(10'-phenothiazinyl)propionic acid/N-morpholinopyridine pair was used as enhancer of HRP-catalyzed chemiluminescence, permitted to measure the enzyme activity of the conjugate with high sensitivity. Under the favorable conditions the limit of detection and a linear range of the assay were 3 pM and 0.07-2.0 nM, respectively. The coefficient of variation (CV) for determination of HBV DNA concentrations within the working range was lower than 4%. The obtained results demonstrated that the developed assay had high sensitivity and precision.

Suicide inactivation of covalent peroxidase-mimicking DNAzyme with hydrogen peroxide and its protection by a reductant substrate.

Recently a covalent peroxidase-mimicking DNAzyme (cPMDNAzyme) with the improved catalytic activity was prepared. Here we demonstrate that hydrogen peroxide, the oxidant substrate of cPMDNAzyme is an inactivating agent of this catalyst. Presence of the reductant substrate, 2,2'-azino-bis(3-ethylbenthothiazoline-6-sulfonic acid (ABTS) prevents the inactivation of cPMDNAzyme. The experimental conditions (pH-optimum, concentrations of ABTS and H2O2) for the determination of cPMDNAzyme activity were optimized that allows a construction of the colorimetric cPMDNAzyme-based biosensors and assays with improved sensitivity.

Improved method for chemiluminescent determination of peroxidase-mimicking DNAzyme activity.

The optimization of experimental conditions for the chemiluminescent determination of peroxidase-mimicking DNAzyme (PMDNAzyme) formed at the interaction of hemin and its aptamer EAD2 was performed. The effect of concentrations of hydrogen peroxide and luminol, acidity of the substrate solution, and composition and concentration of the assay buffer was estimated. Under optimized conditions, a value of detection limit for the PMDNAzyme was 350 pM. A comparison of the conditions determined in this work with those reported previously showed that the optimization of the composition of the substrate solution improved the sensitivity of the chemiluminescent determination of the PMDNAzyme. The obtained results open up promising perspectives for using the proposed method to improve the sensitivity of PMDNAzyme-based assays.

3-(10'-Phenothiazinyl)propionic acid is a potent primary enhancer of peroxidase-induced chemiluminescence and its application in sensitive ELISA of methylglyoxal-modified low density lipoprotein.

Using a full factorial design the optimization of experimental conditions of enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP) in the presence of 3-(10'-phenothiazinyl)propionic acid (PPA) as a primary enhancer was performed. The effect of concentrations of PPA, hydrogen peroxide, MORPH, luminol, and Tris on a ratio of peroxidase-catalyzed CL to background was studied. The detection limit value of HRP in ECR with PPA was 0.09 pM. Using PPA the ultra-sensitive chemiluminescent ELISA for determination of methylglyoxal-modified low density lipoprotein was developed. The detection limit value for the developed method was 0.5 ng mL(-1). The obtained results open up very promising perspectives for using PPA to improve the sensitivity of enzyme immunoassay kits.

Development of ultrasensitive direct chemiluminescent enzyme immunoassay for determination of aflatoxin B1 in food products.

A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for determination of aflatoxin B1 (AFB1) was developed. To improve the assay sensitivity, a mixture of 3-(10'-phenothiazinyl)-propane-1-sulfonate and 4-morpholinopyridine previously optimized by a factorial design was used as enhancer of horseradish peroxidase-induced chemiluminescence. Varying the concentrations of the coating anti-AFB1 antibody and conjugate of AFB1 and horseradish peroxidase the conditions of the chemiluminescent assay were optimized. The values of the detection limit value and dynamic working range of CL-ELISA of AFB1 were 0.0015 ng mL(-1) and 0.003-0.03 ng mL(-1), respectively. It was shown that a dilution of rice and mung beans extracts in 5 and 10 times, respectively, prevented a matrix effect of the food products in CL-ELISA. The recovery values from the spiked samples of rice and mung beans were in the range of 90-104% and 102-117%, respectively. Studying 8 rice and 8 mung beans samples purchased in commercial stores the developed CL-ELISA allowed to find 3 samples (1 rice and 2 mung beans) containing AFB1, the content of AFB1 in one sample being higher than the maximum acceptable level established in the European Community.