Marina M. Vdovenko

FeIII–TAML activator: A potent peroxidase mimic for chemiluminescent determination of hydrogen peroxide

Efforts to replace native peroxidase with its low molecular weight alternatives have stimulated a search for peroxidase mimetics. Herein we describe the oxidation of luminol with hydrogen peroxide catalyzed by commercially available Fe(III)-TAML activator 1a, which was shown to be a more active catalyst than hemin. At Fe(III)-TAML activator 1a use in chemiluminescent assay for H2O2 determination the detection limit value (3σ) of 5×10(-8)M was similar to the detection limit obtained with horseradish peroxidase (1×10(-7)M) and significantly lower than that obtained in the presence of hemin (6×10(-7)M). The linear ranges (R(2)=0.98) of the assay were 6×10(-8)-1×10(-6)M and 6×10(-7)-1×10(-6)M H2O2 for Fe(III)-TAML 1a and hemin, respectively. The CV values for Fe(III)-TAML 1a-based assay measured within the working range varied from 1.0% to 3.7% (n=4), whereas in the case of hemin -5.0% to 9.7% (n=4). Moreover, the sensitivity of Fe(III)-TAML 1a-based method was 56 and 5 times higher than that of hemin- and HRP-based methods, respectively. The obtained results open good perspectives to apply Fe(III)-TAML activator 1a in CL analytical methods instead of hemin, a traditionally used peroxidase mimetic.

Determination of okadaic acid in shellfish by using a novel chemiluminescent enzyme-linked immunosorbent assay method

A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) was developed to determine okadaic acid (OA). Concentrations of the capture monoclonal anti-OA antibodies, conjugate of OA-HRP and a composition of blocking buffers were varied to optimize the assay condition. The values of IC10, IC50 and working range (IC20-IC80) for CL-ELISA were 0.01, 0.07, and 0.03-0.2 ng/mL, respectively. Additionally, the analytical recovery values of CL-ELISA from 3 shellfish spiked samples with OA concentrations of 0.03, 0.1 and 0.2 ng/mL ranged from 86.7% to 111.2%. Closely examining the OA concentrations in 19 various shellfish products performed by CL-ELISA revealed that OA concentrations in 6 of the 19 examined samples was undetected, whereas the 13 samples were contaminated with low levels of OA ranging from 1.2 to 8.0 ng/g.

Fiber-optic immunosensor for detection of Crimean-Congo hemorrhagic fever IgG antibodies in patients.

Crimean-Congo hemorrhagic fever (CCHF) is a severe viral disease with high fatality rate. CCHF virus is endemic in parts of Africa, Asia, the Middle East, and southeastern Europe. Rapid diagnostics of CCHF is vital for appropriate clinical management and prevention of secondary spread from human-to-human. Currently, diagnostics relies on real-time RT-PCR and antibody or antigen detection using ELISA. These methods require trained personnel and expensive equipment and are not appropriate for point-of-care (POC) diagnostics. Furthermore, there are no POC assays available for CCHF. We developed a fiber-optic biosensor for the detection of CCHF IgG antibodies. In order to improve sensitivity, we optimized both the bioreceptor immobilization protocol and the chemiluminescence substrate formulation. The resulting protocol showed a 100-fold greater sensitivity for detection of CCHF antibodies. Finally, we evaluated the fiber-optic biosensor with two CCHF patient sera. We showed that the fiber-optic biosensor is 10-times more sensitive than colorimetric ELISA and is able to detect both patients with high and low levels of IgG antibodies. We believe that the fiber-optic biosensor is a suitable alternative to ELISA as it is much more sensitive and makes it possible to detect a small amount of antibodies at an early stage of infection and can be integrated as a point-of-care diagnostic system of CCHF.

An enhanced chemiluminescence resonance energy transfer system based on target recycling G-guadruplexes/hemin DNAzyme catalysis and its application in ultrasensitive detection of DNA.

An enhanced chemiluminescence resonance energy transfer (CRET) system based on target recycling G-guadruplexes/hemin DNAzyme catalysis was developed for ultrasensitive detection of DNA. CRET system consists of luminol as chemiluminescent donor, and fluorescein isothiocyanate (FITC) as acceptor. The sensitive detection was achieved by using the system consisted of G-riched DNA, blocker DNA, and the Nb.BbvCI biocatalyst. Upon addition of target DNA to the system, target DNA hybridizes with the quasi-circular DNA structure, and forms a DNA duplex. The formation of DNA duplex triggers selective enzymatic cleavage of quasi-circular DNA by Nb.BbvCI, resulting in the release of target DNA and two G-riched DNAzyme segments. Released target DNA then hybridizes with another quasi-circular DNA structure to initiate the cleavage of the quasi-circular DNA structure. Eventually, each target DNA can go through many cycles, resulting in the digestion of many quasi-circular DNA structures, generating many G-riched DNAzyme segments. G-riched DNAzyme segment products assemble with hemin to form stable hemin/G-quadruplexes that exhibit peroxidase-like activity which can catalyze the oxidation of luminol by H2O2 to produce CL signals. In the presence of FITC, CL of luminol can excite FITC molecules, and thus produced CRET between the luminol and FITC. This unique analysis strategy gives a detection limit down to 80 fM, which is at least four orders of magnitude lower than that of unamplified DNA detection methods.

Highly sensitive microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer for the detection of neuron-specific enolase.

A microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer (CRET) was developed for highly sensitive detection of neuron‐specific enolase (NSE). The CRET system consisted of horseradish peroxidase (HRP)/luminol as a light donor and fluorescein isothiocyanate as an acceptor. When fluorescein isothiocyanate‐labeled antibody binds with HRP‐labeled antigen to form immunocomplex, the donor and acceptor are brought close each other and CRET occurs in the immunocomplex. In the MCE, the immunocomplex and excess HRP–NSE were separated, and the chemiluminescense intensity of immunocomplex was used to estimate NSE concentration. The calibration curve showed a linearity in the range of NSE concentrations from 9.0 to 950 pM with a correlation coefficient of 0.9964. Based on a S/N of 3, the detection limit for NSE determination was estimated to be 4.5 pM, which is two‐order magnitude lower than that of without CRET detection. This assay was applied for NSE quantification in human serum. The obtained results demonstrated that the proposed immunoassay may serve as an alternative tool for clinical analysis of NSE.

Development of a chemiluminescent enzyme immunoassay for the determination of dexamethasone in milk

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′-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 IC10, IC50 and working range (IC20–IC80) of the CL-ELISA of DEX were 0.02, 0.9, 0.08–9.3 ng mL−1, 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.

Luminol oxidation by hydrogen peroxide with chemiluminescent signal formation catalyzed by peroxygenase from the fungus Agrocybe aegerita V.Brig.

Conditions of luminol oxidation by hydrogen peroxide in the presence of peroxygenase from the mushroom Agrocybe aegerita V.Brig. have been optimized. The pH value (8.8) at which fungal peroxygenase produces a maximum chemiluminescent signal has been shown to be similar to the pH optimum value of horseradish peroxidase. Luminescence intensity changed when the concentration of Tris-buffer was varied; maximum intensity of chemiluminescence was observed in 40 mM solution. It has been shown that enhancer (p-iodophenol) addition to the substrate mixture containing A. aegerita peroxygenase exerted almost no influence on the intensity of the chemiluminescent signal, similarly to soybean, palm, and sweet potato peroxidases. Detection limit of the enzyme in the reaction of luminol oxidation by hydrogen peroxide was 0.8 pM. High stability combined with high sensitivity make this enzyme a promising analytical reagent.

Chemiluminescent assay of phenol in wastewater using HRP-catalysed luminol oxidation with and without enhancers

Two methods for phenol determination based on the suppression of chemiluminescence (CL) produced upon HRP-catalysed oxidation of luminol were developed. One method used an enhanced CL reaction with 3-(10′-phenothiazinyl)-propane-1-sulfonate and 4-morpholinopyridine as primary and secondary enhancers, respectively, and another one is free of the enhancers. A comparison of the phenol assay with enhancers showed much higher sensitivity and lower LDL value than the assay without enhancers. In addition, the amount of HRP used in the assay with enhancers is 40 times less than in the assay without them. The study of the specificity of the developed assays demonstrated that in the assay without enhancers several phenol compounds, for instance, unsubstituted phenol, suppressed the CL intensity, whereas other phenols, such as 4-chlorophenol and 2,4-dichlorophenol, increased the light output. In contrast to that assay, in the assay with enhancers all tested phenols suppressed the CL intensity. Upon the analysis of phenols in the water plant effluents using the assay without the enhancers, a high matrix effect was observed. On the other hand, the analysis of the same effluents using the assay with enhancers did not show any matrix effect, and the recovery values from the spiked samples were found to be 92–96%.

Phenothiazine derivatives as enhancers of peroxidase-dependent chemiluminescence

Some N-alkyl phenothiazines with different ionic groups were studied as enhancers of chemiluminescence catalyzed by soybean peroxidase. It was shown that under experimental conditions, the compounds with positively charged groups do not exhibit enhancing ability, while the addition of phenothiazines with negatively charged groups to a substrate mixture significantly increased the chemiluminescence intensity. The relationship between the enhancing activity of phenothiazines and their capacity for enzymatic oxidation by hydrogen peroxide was found. The enhancers discovered new opportunities for increasing the sensitivity of determination of analytes by chemiluminescent enzyme immunoassay.

Enzyme immunoassay for the determination of hexestrol in meat

An indirect competitive enzyme-linked immunosorbent assay (ELISA) of hexestrol (HES), an anabolic hormone forbidden for use in livestock farming, has been developed. Conditions of ELISA have been optimized by varying the concentrations of the coating conjugate (HES-ovalbumin), anti-HES antiserum, casein, and Tween 20. In the absence of Tween 20 in the reaction mixture, the detection limit (IC10) equaled 0.01 ng/ml, IC50 equaled 0.17 ng/ml, and the working range (IC20–IC80) equaled 0.03–0.86 ng/ml, while, in the presence of 0.05% Tween 20, these values equaled 0.05, 2.9, and 0.26–32.0 ng/ml, respectively. Standard deviation of the analysis results did not exceed 5.4%. If ELISA was performed in the absence of detergents, the recovery value upon HES determination in spiked beef samples ranged from 74 to 147%.