Timofey Pylaev, IBPPM RAS, Saratov, Russia
Ekaterina Vanzha,IBPPM RAS, Saratov, Russia
Nikolai G. Khlebtsov IBPPM RAS, Saratov, Russia,
Saratov State University, Saratov, Russia
Polymerase chain reaction (PCR) is one of the great inventions of 20th century widespread in clinical and laboratory nucleic acid research. In spite to extremely high sensitivity, robustness, the specificity of PCR does not match its unparalleled sensitivity even in optimized conditions. A broad list of chemical compounds was used as PCR enhancers. During the last decade few types of nanomaterials were tested to solve the problem. Gold nanoparticles (AuNPs) are one of promising candidates for diagnostic applications, including genosensing, bioimaging and gene therapy owing to their easily tuned physical properties, reproducible synthesis and functionalization protocols and high surface-to-volume ratio. The present study is aimed to reveal the mechanisms by which nanoparticles affect PCR. The enhancing effect on PCR was investigated in sets of experiments with AuNPs in various conditions without changing the typical amplification procedure and concentrations of other PCR components. Optimization steps included the determination of the nanoparticle’s concentration, surface charge, material and morphology to extend significant enhancement of PCR efficiency. All types of AuNPs were tested for two diagnostic models, namely detection of cryptic plasmid genes (MOMP) sequences corresponding to Chlamydia trachomatis and detection of nitrogen fixation (NifD) gene from Azospirillum sp.7 bacterial culture. All experiments had triple repeats at least with all needed positive and negative controls. The post-PCR analysis was carried out by agarose gel electrophoresis with ethidium bromide staining on UV-transilluminator. The optimal concentration (to eliminate the smear and non-specific bands and make specific band more bright) of citrate-stabilized 15-nm negatively charged spherical AuNPs was determined as 0.4 nM. The other types of AuNPs (cationic gold nanorods, Au nanospheres with various cationic surface stabilization agents as CTAB, PDDA) exhibited no enhancing effect on PCR or even inhibit the reaction in three-fold lower concentrations. In contrast, negatively charged 20-nm silica nanoparticles have no impact on PCR efficiency, unless added in the concentration of 100 and 200 nM, which leads to complete PCR inhibition. Therefore, nanoparticle’s material plays sufficient role in PCR enhancement, as well as the type of surface modification, that influence the interaction between the surface of nanoparticles and PCR components.
Dr. Timofey Evgen'evich Pylaev
IBPPM RAS, PhD, research assistant in Lab of Nanobiotechnology, IBPPM RAS
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