Valeri Pavlov

Lighting up biochemiluminescence by the surface self-assembly of DNA-hemin complexes.

The discovery of catalytic RNAs (ribozymes) has sparked scientific activities directed to the preparation of new biocatalysts and raised the suggestion that these biomolecules participated in the evolutionary process as preprotein catalysts. 2] Analogously, deoxyribozymes, catalytic DNAzymes, are not found in nature but extensive research efforts have demonstrated the successful synthesis of catalytic deoxyribozymes for many chemical transformations. 4] One interesting example of a catalytic DNA that reveals peroxidase-like activity includes a supramolecular complex between hemin and a single-stranded guanine-rich nucleic acid (aptamer). This complex was reported to catalyze the oxidation of 2,2 -azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS) by H2O2, a common reaction used as an assay for peroxidase activity. It was suggested that the supramolecular docking of the guanine-quadruplex layers facilitates the intercalation of hemin into the complex and the formation of the biocatalytically active hemin center. Enzymes and, specifically, horseradish peroxidase (HRP) 9] are used as biocatalytic labels for the amplified detection of DNA-sensing events. The electrochemical amplified detection of DNA has been accomplished in the presence of different enzymes 8] and the chemiluminescent analysis of DNA in the presence of HRP has been reported. The integration of a DNA biocatalyst into DNA-detection schemes could provide a new method for the detection of nucleic acids that might reveal important advantages: 1) The catalytic DNA may substitute the protein-based biocatalysts, and thus eliminate nonspecific binding phenomena; 2) Tailoring of the DNA biocatalyst as part of the labeled nucleic acid might reduce the number of analytical steps for DNA detection. Here we report that two separated nucleic acids that include the segments A and B–constituting the single-stranded peroxidase deoxyribozyme, which forms a layered G-quadruplex structure (see Scheme 1)–self-assemble in the presence of hemin to form a biocatalyst for the generation of chemiluminescence in the presence of H2O2 and luminol. The effect of hybridization with the DNAzyme compounds on the resulting biochemiluminescence is discussed. We also demonstrate the self-assembly of biocatalytic, supramolecular hemin ±nucleic acid complexes on gold electrodes in monolayer configurations, and describe the biocatalytic and bioelectrocatalytic formation of chemiluminescence at the Acknowledgements

Modulated Growth of Nanoparticles. Application for Sensing Nerve Gases

Hydrolysis of acetylthiocholine mediated by acetylcholine esterase yields the thiol-bearing compound thiocholine. At trace concentrations, thiocholine modulates the growth of Au-Ag nanoparticles on seeding gold nanoparticles in the presence of ascorbic acid. Inhibition of the enzyme by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51) or by diethyl p-nitrophenyl phosphate (paraoxon) produces lower yields of thiocholine, promoting the catalytic growth of Au-Ag nanoparticles. Here, we describe the development of a simple and sensitive colorimetric assay for the detection of AChE inhibitors.

Unconventional Application of Conventional Enzymatic Substrate: First Fluorogenic Immunoassay Based on Enzymatic Formation of Quantum Dots

In this study, a simple fluorogenic immunoassay based on in situ formation of semiconductor quantum dots (QDs) is described. We discovered that alkaline phosphatase (ALP), the enzyme broadly used in enzyme-linked immuno-sorbent assay (ELISA), is able to trigger formation of fluorescent CdS QDs. ALP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) leads to the formation of p-nitrophenol and inorganic phosphate. The latter stabilizes CdS QDs produced in situ through interaction of Cd(2+) with S(2-) ions. So, the specific interaction of analyte (antibody) with ALP-labeled antibody can be detected through formation of CdS QDs, monitored by recording emission spectra at λex = 290 nm. The fluorescence intensity showed to be dependent on the concentration of target antibody. This method allowed us to detect as low as 0.4 ng mL(-1) of analyte antibody with a linear range up to 10 ng mL(-1). The sensitivity of this novel assay showed to be 1 order of magnitude better than that of the standard method based on colorimetric p-nitrophenyl phosphate assay.

Enzymatic product-mediated stabilization of CdS quantum dots produced in situ: application for detection of reduced glutathione, NADPH, and glutathione reductase activity.

Glutathione is the most abundant nonprotein molecule in the cell and plays an important role in many biological processes, including the maintenance of intracellular redox states, detoxification, and metabolism. Furthermore, glutathione levels have been linked to several human diseases, such as AIDS, Alzheimer disease, alcoholic liver disease, cardiovascular disease, diabetes mellitus, and cancer. A novel concept in bioanalysis is introduced and applied to the highly sensitive and inexpensive detection of reduced glutathione (GSH), over its oxidized form (GSSG), and glutathione reductase (GR) in human serum. This new fluorogenic bioanalytical system is based on the GSH-mediated stabilization of growing CdS nanoparticles. The sensitivity of this new assay is 5 pM of GR, which is 3 orders of magnitude better than other fluorogenic methods previously reported.

Enzymatic Growth of Quantum Dots: Applications to Probe Glucose Oxidase and Horseradish Peroxidase and Sense Glucose

Three innovative assays are developed for the detection of enzymatic activities of glucose oxidase (GOx) and horseradish peroxidase (HRP) by the generation of CdS quantum dots (QDs) in situ using non-conventional enzymatic reactions. In the first assay, GOx catalyzes the oxidation of 1-thio-β-D-glucose to give 1-thio-β-D-gluconic acid. The latter is spontaneously hydrolyzed to β-D-gluconic acid and H2 S, which in the presence of cadmium nitrate yields fluorescent CdS nanoparticles. In the second assay HRP catalyzes the oxidation of sodium thiosulfate with hydrogen peroxide generating H2 S and consequently CdS QDs. The combination of GOx with HRP, allowed quantification of glucose in plasma by following growth of fluorescent QDs.

Aminoferrocene-based prodrugs and their effects on human normal and cancer cells as well as bacterial cells.

Aminoferrocene-based prodrugs are activated under cancer-specific conditions (high concentration of reactive oxygen species, ROS) with the formation of glutathione scavengers (p-quinone methide) and ROS-generating iron complexes. Herein, we explored three structural modifications of these prodrugs in an attempt to improve their properties: (a) the attachment of a -COOH function to the ferrocene fragment leads to the improvement of water solubility and reactivity in vitro but also decreases cell-membrane permeability and biological activity, (b) the alkylation of the N-benzyl residue does not show any significant affect, and (c) the attachment of the second arylboronic acid fragment improves the toxicity (IC50) of the prodrugs toward human promyelocytic leukemia cells (HL-60) from 52 to 12 μM. Finally, we demonstrated that the prodrugs are active against primary chronic lymphocytic leukemia (CLL) cells, with the best compounds exhibiting an IC50 value of 1.5 μM. The most active compounds were found to not affect mononuclear cells and representative bacterial cells.

Ultrasensitive assay for detection of serum paraoxonase by modulating the growth of fluorescent semiconductor nanoparticles.

Serum paraoxonase (PON1) is an enzyme associated exclusively with high-density lipoproteins and seems to be an antiatherogenic agent that prevents initiation and progression of atherosclerosis. PON1 also hydrolyzes organophosphates, protecting the nervous system from those neurotoxic compounds. Furthermore, PON1 could be a potential indicator for predicting and preventing other diseases, such as coronary artery disease, different kinds of cancers, diabetes mellitus type 2, metabolic syndrome, neurological disorders, liver disorders, etc. Here we report an ultrasensitive assay to measure PON1 arylesterase activity relying on the enzymatic modulation of the growth of fluorescent CdS nanoparticles (NP). The lowest PON1 activity that could be detected by our system was 0.625 mU mL(-1), with a dynamic range up to 5 mU mL(-1). This new system leads to an improvement of the limit of detection by around 15 times, compared to the conventional assays to determine PON1 arylesterase activity. This new system was also applied to determine PON1 arylesterase activity in human serum by the standard addition method. Furthermore, experiments with diluted serum spiked with PON1 demonstrated recovery of PON1 activity near 100%.

Photoelectrochemical detection of enzymatically generated CdS nanoparticles: Application to development of immunoassay

We report an innovative photoelectrochemical process (PEC) based on graphite electrode modified with electroactive polyvinylpyridine bearing osmium complex (Os-PVP). The system relies on the in situ enzymatic generation of CdS quantum dots (QDs). Alkaline phosphatase (ALP) catalyzes the hydrolisis of sodium thiophosphate (TP) to hydrogen sulfide (H2S) which in the presence Cd(2+) ions yields CdS semiconductor nanoparticles (SNPs). Irradiation of SNPs with the standard laboratory UV-illuminator (wavelength of 365 nm) results in photooxidation of 1-thioglycerol (TG) mediated by Os-PVP complex on the surface of graphite electrode at applied potential of 0.31 V vs. Ag/AgCl. A novel immunoassay based on specific enzyme linked immunosorbent assay (ELISA) combined with the PEC methodology was developed. Having selected the affinity interaction between bovine serum albumine (BSA) with anti-BSA antibody (AB) as a model system, we built the PEC immunoassay for AB. The new assay displays a linear range up to 20 ngmL(-1) and a detection limit (DL) of 2 ngmL(-1) (S/N=3) which is lower 5 times that of the traditional chromogenic ELISA test employing p-nitro-phenyl phosphate (pNPP).

Assays for Methionine γ-Lyase and S-Adenosyl-l-homocysteine Hydrolase Based on Enzymatic Formation of CdS Quantum Dots in Situ

S-Adenosyl-L-homocysteine hydrolase (AHCY) hydrolyzes its substrate S-adenosyl-L-homocysteine (AdoHcy) to L-homocysteine (Hcy). Methionine γ-lyase (MGL) catalyzes the decomposition of Hcy to hydrogen sulfide which forms fluorescent CdS nanoparticles in the presence of Cd(NO(3))(2). On the basis of these enzymatic reactions, two new simple and robust fluorogenic enzymatic assays for MGL and AHCY were developed and applied to detection of AHCY inhibitors.

Label free and amplified detection of cancer marker EBNA-1 by DNA probe based biosensors

Epstein-Barr virus (EBV) is a human herpes virus that has been associated with several malignancies as Burkitts lymphoma, nasopharyngeal carcinoma and Hodgkins disease. All EBV associated malignancies showed a distinct viral gene expression pattern, while Epstein-Barr nuclear antigen 1 (EBNA-1) is constitutively expressed in all such disorders. Here, the development of a biosensor to detect EBNA-1 protein is reported, which was based on a nucleic acid bioreceptor and a quartz crystal microbalance with a dissipation monitoring (QCM-D) transducer. The DNA probe for EBNA-1 detection was designed and synthesized to mimic its palindromic target sites in the EBV genome. This DNA probe was immobilized on the Au-surface of a QCM-D electrode, followed by the blocking of the accessible Au-surface with 6-mercapto-1-hexanol (6-MHO). The system showed a limit of detection of 50 ng/mL in direct detection of EBNA-1, however, the sensitivity was improved by 2 orders of magnitude (0.5 ng/mL) when an amplification cascade, employing antibodies labeled with alkaline phosphatase (AP), was applied to the system.