Superparamagnetic iron oxide nanoparticles (spions) modified with sarcosine oxidase-enzymatic activity analysis by sds-page.
Uhlířová, Dagmar; Staňková, Martina; Dočekalová, Michaela; Hosnedlová, Božena; Kepinska, Marta; Ruttkay-Nedecký, Branislav; Růžička, Josef; Fernandez, Carlos; Milnerowicz, Halina; Kizek, René
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 SDSPAGE 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.
|Start Date||Oct 17, 2018|
|Publication Date||Dec 31, 2018|
|Institution Citation||UHLÍŘOVÁ, D., STAŇKOVÁ, M., DOČEKALOVÁ, M., HOSNEDLOVÁ, B., KEPINSKA, M., RUTTKAY-NEDECKÝ, B., RŮŽIČKA, J., FERNANDEZ, C., MILNEROWICZ, H. and KIZEK, R. 2018. Superparamagnetic iron oxide nanoparticles (spions) modified with sarcosine oxidase-enzymatic activity analysis by sds-page. In Proceedings of 10th International nanomaterials conference 2018 (NANOCON 2018): research and application, 17-19 October 2018, Brno, Czech Republic. Ostrava: Tanger[onlin], pages 360-364. Available from: https://www.nanocon.eu/...Proceedings_content.pdf|
UHLIROVA 2018 Superparamagnetic