Papers by Tzonka Godjevargova
Journal of Membrane Science, 1999
Ultra®ltration membrane has been prepared from the copolymer of acrylonitrile±glycidylmethacrylat... more Ultra®ltration membrane has been prepared from the copolymer of acrylonitrile±glycidylmethacrylate and the porosity of the membrane was studied. The asymmetric structure was proved by scanning electron microscopy. The basic characteristics of the membrane were measured ± water permeability, water content, membrane selectivity, etc. The membrane obtained was used as a carrier for immobilization of glucose oxidase. The immobilization was carried out covalently by two methods: direct bonding of the enzyme and indirectly by a spacer (hexamethylenediamine) and cross-linking agent (glutar aldehyde). The amount of bound protein and relative activity of the immobilized glucose oxidase were determined. Temperature optimum, pH optimum and storage stability of the immobilized glucose oxidase were determined. It was proved that glucose oxidase immobilized by the direct method shows better characteristics compared with the indirect method.
Enumeration of the stem cells and leukocytes in peripheral blood and aphaeresis samples was perfo... more Enumeration of the stem cells and leukocytes in peripheral blood and aphaeresis samples was performed by using biomarker expression of CD34+ and CD45+ proteins on their cell surface. The aim of this study was simultaneous counting of live stem cells and leukocytes in aphaeresis samples by using two different fluorescent conjugates. The conjugate between anti-CD34 monoclonal antibody and fluorescent dye DR110 stained stem cells in green color. The conjugate between anti-CD45 monoclonal antibody and fluorescent dye ATTO620 stained leukocytes in red color. The conjugates anti-CD34 antibody-DR110 and anti-CD45 antibody-ATTO620 were prepared by carbodiimide method and purified by gel filtration. The counting was performed by using a new automatic fluorescence microscope EasyCounter BC. The dead cells were counted by monomethyne cyanine dye-Sofia Green. The analyses of six aphaeresis samples were performed. The obtained results by EasyCounter BC were compared with results received by other two standard methodsflow cytometry (Guava easyCyte 8HT) and fluorescent microscopic method (Olympus BX51). The obtained cell counts from EasyCounter BC and the flow cytometer were very similar. The coefficients of variation of the obtained results with EasyCounter BC (3-6%) were lower than those with the flow cytometer (5-8%) and with the Olympus microscope (14-18%).
The Open Biotechnology Journal, Nov 15, 2019
Bioremediation Journal, Feb 28, 2011
The growth and the amino acid composition of the strain Saccharomyces cerevisiae RD1 were studied... more The growth and the amino acid composition of the strain Saccharomyces cerevisiae RD1 were studied in the presence of copper ions. The accumulation of biomass was inhibited with the increase of Cu concentration. It should be noted that the synthesis of aromatic amino acids was promoted at lower Cu concentration (100 mg·L), but at higher concentrations the inhibiting effect was
The Open Biotechnology Journal, Jun 16, 2020
Background: Antibiotic residues are a problem of increasing importance and have direct consequenc... more Background: Antibiotic residues are a problem of increasing importance and have direct consequences for human and animal health. The frequent use of antibiotics in veterinary practice causes their excretion in milk in dairy cattle. This way, they can easily enter the human body through the consumption of milk and dairy products. Objectives: This induces the need for accurate and sensitive methods to monitor antibiotic levels in milk. The aim of this study was to develop a rapid and sensitive magnetic nanoparticle-based fluorescence immunoassay for the simultaneous detection of chloramphenicol and penicillin G in milk. Methods: Magnetic nanoparticles were synthesized and functionalized with (3-aminopropyl)triethoxysilane. Chloramphenicol-Ovalbumin and Chloramphenicol-Ovalbumin-Fluorescein-5-isothiocyanate conjugates were prepared. Penicillin G-ATTO 633 fluorescent conjugate was synthesized. Antibodies against chloramphenicol and penicillin G were immobilized onto the magnetic nanoparticles. The competitive fluorescent immunoassay was developed. The optimal concentration of the antibody-magnetic nanoparticles and the fluorescent conjugates for the assay was determined. The calibration curves for the antibiotics in buffer and milk were plotted. Fluorescent immunoassay for the simultaneous determination of chloramphenicol and penicillin G in milk was developed. Results: The limit of detection by the simultaneous immunoassay of chloramphenicol and penicillin G in milk was 0.85 ng/mL and 1.6 ng/mL, respectively. The recovery of different concentrations of chloramphenicol and penicillin G in milk samples varied from 98% to 106%. Conclusions: A rapid and sensitive magnetic nanoparticle-based immunofluorescent assay for the simultaneous determination of chloramphenicol and penicillin G in milk was developed. The magnetic nanoparticles ensured rapid and easy procedure.
Journal of Applied Polymer Science, Aug 8, 1994
Acrylonitrile copolymer membranes were subjected to partial hydrolysis in order to improve their ... more Acrylonitrile copolymer membranes were subjected to partial hydrolysis in order to improve their dialysis characteristics. The partial hydrolysis time and temperature, as well as the concentration of the hydrolyzing agent, were the variable parameters. The changes in the chemical and physical characteristics of the membrane were proved by infrared spectroscopy, elemental analysis, differential scanning calorimetry, and Mercury intrusion porometry. After each degree of modification, the permeability of the membrane with respect to a solution of vitamin BIZ was determined under static conditions and at temperature of 298 K. The improvement in the permeability at a definite degree of hydrolysis is explained by the conversion of nitrile groups to amide and carboxyl groups, as well as increase of the volume of the pores with average radius. The efficiency of the modified PAN membranes for concentration of biological liquids (diluted serum) was studied. 0 1994 John Wiley & Sons, Inc.
Materialy ... Moskovskogo meždunarodnogo kongressa "Biotehnologiâ: sostoânie i perspektivy razvitiâ", 2020
The purpose of this research was to determine the total cell count and some pathogenic microorgan... more The purpose of this research was to determine the total cell count and some pathogenic microorganisms in 105 milk samples. The somatic cell count (SCC) was significantly higher in mastitic milk samples compared to the uninfected glands and dirty milk samples. The total number of microorganisms (TBC) in dirty and mastitic milk was 5-20 times more than results in healthy milk samples. The influence of the selected pathogenic microorganisms on the number of somatic cells, neutrophils and CD4+ cells was studied. The obtained results were compared with those by a flow cytometer Guava easyCyteTM 8HT. The proportion of different cell types (neutrophils and CD4+ cells) has been shown to be influenced by the type of pathogens.
Food Science and Applied Biotechnology, Mar 18, 2019
The sensitive competitive immunofluorescence method for simultaneous determination of paraoxon an... more The sensitive competitive immunofluorescence method for simultaneous determination of paraoxon and dichlorvos with immobilized multipolyclonal antibody on magnetic nanoparticles was developed. The multi-polyclonal antibody was obtained after the immunization of mixture of two prepared immunogens dichlorvos-cBSA and paraoxon-BSA (1:1). The immunogens dichlorvos-cBSA and paraoxon-BSA were synthesized preliminary. Multi-polyclonal antibody against dichlorvos and paraoxon was covalently coupled on magnetic nanoparticles. The competitive fluorescence conjugates dichlorvos-cBSA-FITC and paraoxon-BSA-ATTO 620 were synthesized. Two typical calibration curves of immunofluorescence assay for determination of dichlorvos and paraoxon in buffer solutions were obtained. The linear interval from 2 to 200 ng.mL-1 for these two pesticides was determined. Then the calibration curves for dichlorvos and paraoxon were obtained in cow milk solutions. The linear range of pesticides in cow milk was determined (from 5 to 300 ng.L-1) and the detection limit for paraoxon (3.5 ng.mL-1) and dichlorvos (4 ng.mL-1) was found. The obtained results for cow milk samples were compare with results in UHT, pasteurized cow milk, sheep and goal milk. Quite different are the results when analyzing paraoxon and dichlorvos in standard solutions prepared in sheep's milk. The linear working range for the two pesticides is between 7 and 300 ng.mL-1 .It is obviously, that there is a shift of all analytical characteristics up to higher values. The reason for this is the high fat content of sheep's milk. The obtained results were showed that the developed method was 2 time more sensitive than method with the results obtained with mixture (1:1) of two separated antibodiesanti-paraoxon and anti-dichlorvos. These results confirmed the potential of the immunoassay for quantitative simultaneous screening of both dichlorvos and paraoxon. Practical applications: The use of immunofluorescence assay based on immobilized multi-polyclonal antibody on magnetic nanoparticles allows us to detect simultaneous paraoxon and dichlorovos in raw milk and other dairy products
Nucleation and Atmospheric Aerosols, 2017
Enzyme-linked immunosorbent assay for determination of aflatoxin M1 based on magnetic nanoparticles
Food Analytical Methods, Apr 12, 2021
A fluorescent immunoassay for separate and simultaneous determination of penicillin (PEN) and sul... more A fluorescent immunoassay for separate and simultaneous determination of penicillin (PEN) and sulphadimethoxine (SDM) in milk was developed. Monoclonal antibodies immobilised on carboxylic magnetic nanoparticles (MNPs) were used. It was proved that the protein A-oriented immobilisation method (OI) provided a 1.5-times more sensitive assay than the random immobilisation one (RI). The activation of MNPs with a mixture of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) ensured a five-times higher sensitivity of immunoassay than the activation using EDC. The linearity of standard curves in milk was: 3/10 ng/mL PEN (OI and RI), 50/250 ng/mL SDM (OI), and 50/500 ng/mL SDM (RI).
Journal of Applied Polymer Science, Jul 25, 2003
A novel, specific sorbent based on polyamide covalently immobilized with dead yeast cells by glut... more A novel, specific sorbent based on polyamide covalently immobilized with dead yeast cells by glutaraldehyde was prepared and characterized. This sorbent exhibits a high capacity for metal complexation based on multifunctional groups of dead cells, as well as a good stability for reuse based on the crosslinking agent, glutaraldehyde. The Cu 2ϩ sorption characteristics of the polyamide modified with immobilized dead cells were studied and compared to those of the polyamide chemically modified without cells. The adsorption capacity of specifically modified polyamide was about 19-fold higher than the chemically modified polymer. The adsorption isotherms of Langmuir and Freundlich for the new specific sorbent were determined. The effect of pH, temperature and co-ions (Zn 2ϩ , Pb 2ϩ , Co 2ϩ , Ca 2ϩ and Mg 2ϩ) on the Cu 2ϩ sorption capacity were studied. The effectiveness of heavy metal desorption and the coefficient of recovery of sorption ability were determined.
Analytical Letters, Oct 20, 2016
Haptens of dichlorvos and paraoxon were conjugated to the carrier proteins of bovine serum albumi... more Haptens of dichlorvos and paraoxon were conjugated to the carrier proteins of bovine serum albumin. The obtained conjugates were characterized by infrared and ultraviolet-visible spectroscopy. The binding ratios of dichlorvos and paraoxon to carrier proteins were also evaluated. The number of hapten molecules per protein molecule of dichlorvos-cationized bovine serum albumin conjugate was higher than for paraoxon-bovine serum albumin conjugate. The sheep polyclonal antibodies were produced against the dichlorvos and paraoxon. New multipolyclonal antibodies were obtained and characterized following the immunization of a 1:1 mixture of the immunogens for the simultaneous determination of dichlorvos and paraoxon by the immunoassay. An indirect enzyme-linked immunosorbent assay was used to characterize the reactivity of the antibodies to hapten conjugates. The multi-antibodies showed lower affinities than the separate antibodies, but their affinities were sufficient for an immunoassay for the simultaneous determination of the analytes. The detection limit and linear range for determination of dichlorvos and paraoxon alone and together were determined. The recovery was characterized to determine dichlorvos and paraoxon fortified in model solutions and milk. These results demonstrate the potential of this immunoassay for the quantitative screening of dichlorvos and paraoxon.
Analytical Letters, Jan 17, 2017
A sensitive and rapid magnetic nanoparticle-based fluorescent immunoassay for the determination o... more A sensitive and rapid magnetic nanoparticle-based fluorescent immunoassay for the determination of aflatoxin M1 in raw milk was developed. Aflatoxin M1 was converted to aflatoxin M1-ocarboxymethyl oxime. The aflatoxin M1-oxime was used for the preparation of aflatoxin M1-oxime-fluoresceinamine conjugate through the carbodiimide reaction. The aflatoxin M1-oxime-fluoresceinamine conjugate was characterized by ultraviolet-visible and infrared spectroscopy. Magnetic nanoparticles (Fe 3 O 4) were synthesized and modified by 3-(aminopropyl)triethoxysilane. The size of initial (139 nm) and functionalized magnetic nanoparticles (147 nm) was determined by particle analysis. The optimal mass of immobilized antibody (25 µg) and optimal concentration of aflatoxin M1-oxime-fluoresceinamine conjugate (15 µg mL −1) for magnetic nanoparticle-based fluorescent immunoassay were determined. The developed immunoassay provided a linear aflatoxin M1 concentration range from 3.0 to 100 pg mL −1 in bovine milk. The detection limit was 2.9 pg mL −1. The results of aflatoxin M1 magnetic nanoparticle-based fluorescent immunoassay in heat-treated milk and phosphate-buffered saline at pH 6.6 were compared. The influence of the somatic cell count, pH, and fat concentration in bovine milk on the aflatoxin M1 immunoassay was investigated. The influence of the milk species on the immunoassay was also characterized. The high fat concentration ovine milk depressed the sensitivity of the aflatoxin M1 immunoassay.
Journal of Membrane Science, Mar 16, 1994
Glucose oxidase was immobilized onto modified acrylonitrile copolymer membranes and showed high a... more Glucose oxidase was immobilized onto modified acrylonitrile copolymer membranes and showed high activity (85.0%) compared to the unmodified membranes (29.8%). The number of amino groups and the degree of hydrophilicity of the support modified the enzyme activity. The insoluble glucose oxidase demonstrated different properties than the free form.
Biotechnology & Biotechnological Equipment, 2013
Two immobilized systems of Aspergillus awamori NRRL3112 and Тrichosporon cutaneum R57 on modified... more Two immobilized systems of Aspergillus awamori NRRL3112 and Тrichosporon cutaneum R57 on modified polyamide beads were obtained. The optimal conditions for immobilization of strains Aspergillus awamori NRRL3112 and Trichosporon cutaneum R57 on polyamide beads were determined. It was found that by combined action of two immobilized systems it is possible to degrade up to 1.5 g•L-1 phenol, while individually immobilized and free cells did not have this capacity (degrade up to 1 g•L-1). The high potential possibility and the synergistic action of both immobilized systems were proved. Biodegradation of different phenolic derivatives was investigated-2 \-chlorophenol (2-CP), 3-chlorophenol (3-CP), 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-DCP), 4-n-nonylphenol (4-NP), 3-methoxyphenol (3-MP), bisphenol A (BPA), 4-chloro-3,5-dimethylphenol (chloroxylenol-ChX), and 2-benzyl-4-chlorophenol (chlorophen-ChP). It was found that the utilization of phenol, BPA, and 2,4-DCP was very fast (16 h); 2-CP, 3-CP, 4-CP, and 3-MP were utilized for 40 h in the range from 44 % to 72 %; NP and ChP were degraded very slowly (about 30 % for 40 h), while ChX remained unchanged for 40 h. The studied phenolic derivatives were listed in the following order, depending on the degree of biodegradation by the two immobilized strains Aspergillus awamori NRRL3112 and Trichosporon cutaneum R57: Phenol > BPA > 2,4-DCP > 3-MP > 3-CP > 2-CP > 4-CP > ChP > NP > ChX. The rate of biodegradation of phenol and some phenolic derivatives with a concentration of 0.1 g•L-1 and 0.3 g•L-1 were determined. The advantages of the two combined immobilized systems for biodegradation of phenol and phenolic derivatives as compared to the separate immobilized systems of the two stains and free cells were proved.
Food Chemistry, Jul 1, 2020
Development of a double immunochromatographic test system for simultaneous determination of linco... more Development of a double immunochromatographic test system for simultaneous determination of lincomycin and tylosin antibiotics in foodstuffs, Food Chemistry (2020), doi:
Journal of Membrane Science, Aug 1, 2004
Microfiltration membranes of polyamide-6 (PA-6) were preliminarily modified by hydrochloric acid ... more Microfiltration membranes of polyamide-6 (PA-6) were preliminarily modified by hydrochloric acid (HCl) and subsequently activated by 1,2-diaminoethane (DAE). They were used as matrix for covalent immobilization of glucose oxidase (GOD) by using glutaraldehyde. Some characteristics of initial and modified membranes were studied: amino groups content, degree of hydrophilicity, water permeability, coefficient of permeability and average pore radius. The amount of bound protein and relative activity of GOD immobilized onto the modified membrane matrices were determined. The highest activity (81.4%) was measured for GOD immobilized onto membrane modified with 6 wt.% hydrochloric acid and 10 wt.% DAE. The properties of GOD immobilized onto modified PA-6 membranes were also determined: pH and temperature optimum, pH and thermal stabilities and storage period of enzyme activity and were compared to those of the native enzyme.
Macromolecular Bioscience, Oct 20, 2004
Food Science, Engineering and Technologies 2009, Plovdiv, Bulgaria. 23-24 October, 2009., 2009
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Papers by Tzonka Godjevargova