SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES (SPIONS) MODIFIED WITH SARCOSINE OXIDASE - ENZYMATIC ACTIVITY ANALYSIS BY SDS-PAGE

1 UHLÍŘOVÁ Dagmar
Co-authors:
1 STAŇKOVÁ Martina 1 DOČEKALOVÁ Michaela 2 HOSNEDLOVÁ Božena 3 KEPINSKA Marta 2 RUTTKAY-NEDECKÝ Branislav 1 RŮŽIČKA Josef 4 FERNANDEZ Carlos 3 MILNEROWICZ Halina 1,2,3 KIZEK René
Institutions:
1 Department of Research and Development, Prevention Medicals s.r.o., Tovární 342, 742 13; Studénka-Butovice, Czech Republic, uhlirova@preventionmedicals.cz
2 Department of Human Pharmacology and Toxicology, University of Veterinary and Pharmaceutical Sciences Brno, Palackého třída 1946/1, 612 42 Brno, Czech Republic, kizek@sci.muni.cz
3 Department of Biomedical and Environmental Analyses, Faculty of Pharmacy with Division of Laboratory Diagnostics, Wroclaw Medical University, Borowska 211, 50-556 Wroclaw, Poland, zalewska.m@gmail.com
4 School of Pharmacy and Life Sciences, Robert Gordon University, Garthdee Road, Aberdeen, AB10 7QB, Scotland, United Kingdom, c.fernandez@rgu.ac.uk
Conference:
10th International Conference on Nanomaterials - Research & Application, Hotel Voronez I, Brno, Czech Republic, EU, October 17th - 19th 2018
Proceedings:
Proceedings 10th International Conference on Nanomaterials - Research & Application
Pages:
360-364
ISBN:
978-80-87294-89-5
ISSN:
2694-930X
Published:
28th February 2019
Proceedings of the conference were published in Web of Science and Scopus.
Metrics:
18 views / 7 downloads
Abstract

Sarcosine oxidase (SOX) is an enzyme that catalyzes the oxidative demethylation of sarcosine with the glycine as a product and is physiologically active in human body and other mammals. However prostate cancer cells have a high expression of sarcosine. In this study the superparamagnetic iron oxide nanoparticles (SPIONs) were prepared and their surface was modified with gold nanoparticles (AuNPs). These AuNPs were modified with chitosan (CS) and SOX. Obtained AuNPs were characterized by physicochemical methods, such as dynamic light scattering or spectrophotometry, where the pseudo-peroxidase activity of the AuNPs was used. Hydrogen peroxide decomposes because of the pseudo-peroxidase activity with the appearance of a blue coloration of 3,3’,5,5’-tetramethylbenzidine (TMB). For the analysis of AuNPs enzymatic activity the SDS-PAGE with silver staining has been used. Gels (7.5%) were prepared using acrylamide stock solution 30% (m / V) with bisacrylamide 1% (m / V). Separating gel contained: acrylamide 7.5% (m / V), bisacrylamide 0.5% (m / V), 0.4 M Tris/HCl, 0.1% (m / V) sodium dodecyl sulfate (SDS), pH 8.8. Stacking gel contained: 4.5% acrylamide (m / V), 0.15% bisacrylamide (m / V), 0.1% SDS (m / V), 0.1M Tris/HCl, pH 6.8. Nanoconstructs were diluted 2:1 with a loading buffer (PLB Max). Each well contained 15 µl of the diluted solutions. Electrophoretic measuring conditions were: 120 V, 1.5 hours in a running buffer (24mM Tris, 0.2M glycine and 3mM SDS). After measurement the gel was stained with silver, scanned and evaluated by Colortest in the laboratory system Qinslab. The SPIONs or AuNPs cannot be detected themselves alone using SDS-PAGE, therefore this method served as a confirmation, that all parts of the nanoconstruct are connected and we are able to analyze them and use them for other research or possible diagnostic purposes.

Keywords: superparamagnetic iron oxide nanoparticles, gold nanoparticles, sarcosine oxidase, chitosan, enzymatic activity, SDS-PAGE
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