CIRCULATING TUMOR DNA AS A PROMISING BIOMARKER FOR BREAST CANCER DETECTION: PCR AMPLIFICATION IMPROVED BY USING GOLD NANOPARTICLES

1,2 FERREIRA DA SILVA Andre
Co-authors:
3 UHLÍŘOVÁ Dagmar 1,4 KOLLIOPOULOU Christina 1,4 SEIMENI Anastasia 5 KEPINSKA Marta 1 LOFFELMANN Martin 1 GARGULÁK Michael 2 DOČEKALOVÁ Michaela 1 HOSNEDLOVÁ Božena 1 RUTTKAY-NEDECKÝ Branislav 5 MILNEROWICZ Halina 1,2,4 KIZEK René
Institutions:
1 University of Veterinary and Pharmaceutical Sciences Brno, Pharmaceutical Faculty, Brno, Czech Republic, EU, kizek@sci.muni.cz
2 University of Lisboa, Faculty of Pharmacy Avenida Professor Gama Pinto, 1649-003, Lisboa - Portugal, EU, andre_ferreira.silva@hotmail.com
3 Prevention Medicals, Studenka-Butovice, Czech Republic, EU, uhlirova@preventionmedicals.cz
4 Faculty of Pharmacy, University of Patras, Prefabricated Buildings Area, University Campus, 26504 Rio Patras, Greece, EU, Greecechristinakolliop@gmail.com
5 Department of Biomedical and Environmental Analyses, Faculty of Pharmacy, Wroclaw Medical University, Wroclaw, Poland, EU, zalewska.m@gmail.com
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:
376-379
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:
17 views / 8 downloads
Abstract

Breast cancer is the most commonly diagnosed cancer in women. Circulating tumor DNA (ctDNA) is related to breast cancer development and thus, screening of tumors using ctDNA blood detection is highly sensitive and may significantly improve early-stage breast tumor diagnosis. Recently, a nanomaterial-assisted PCR using gold nanoparticles (AuNPs) has been applied to dramatically improve the specificity and sensitivity of PCR, achieving better gene detection. In this study, we aimed to optimize AuNP-assisted PCR, using a new AuNPs-ODN-ThiC3 nanocomposite. Synthesized AuNPs-ODN-ThiC3 nanocomposites, obtained after 60 min of interaction between AuNPs and ODN-ThiC3, were characterized by differential UV/VIS spectroscopy (AuNPs 35 mg/mL), AuNP peroxidase-like activity (0.5 mM TMB, 60 min, 30 cycles), PAGE 25% gel electrophoresis with silver staining visualization (V = 120 mV, I = 150 mA, P = 20 W) and adsorptive stripping voltammetry (HMDE, from start 0 to end -1.75 V, time of accumulation 240 s). AuNP-ODN-ThiC3-assisted PCR was performed using Elizyme HS Robust Polymerase (5 U/µL), denaturation: 95°C/15 s, annealing: 59°C/15 s, extension: 72°C/15 s, 35 cycles. Analysis of AuNPs modification was achieved with ODN-ThiC3 (in different concentrations) and, therefore, new AuNPs-ODN-ThiC3 nanocomposites were obtained. We observed an increased absorbance signal of AuNPs at 527 nm, with the increase of ODN-ThiC3 concentration (at 260 nm) in the AuNPs-ODN-ThiC3 mixture (y = -0,0397 + 0,0734x, r = 0,99). Mixing ODNs-ThiC3 with AuNPs increased the peroxidase-like activity of AuNPs by 17%. LS voltammograms of cytosine and adenine (CA) reduction (the average potential at -1.34 V) showed a linear dependence (r = 0.99) between peak current and concentration of ODN-ThiC3 in the AuNPs-ODN-ThiC3. A linear dependence (r = 0.99) between bands density and ODN-ThiC3 concentration was also achieved. Analysis of PCR protocols: AuNPs-ODN-ThiC3-assisted PCR (AuNPs 814 µg/mL, ODN-ThiC3 4 µg/mL) showed a better yield (140% of conventional PCR and 120% of AuNP-assisted PCR). Conventional PCR can be optimized by integrating ODN-ThiC3 on the AuNP surface during PCR. This improvement represents an innovative PCR method for breast cancer detection.

Keywords: nanomedicine, gold nanoparticles, anticancer drugs, PCR, breast cancer
Scroll to Top