Graphene oxide enhances the specificity of the polymerase chain reaction by modifying primer-template matching

Abstract Aiming at improved specificity, nanoparticle assisted polymerase chain reaction (PCR) has been widely studied and shown to improve PCR. However, the reliability and mechanism of this method are still controversial. Here, we demonstrated that 1 μg/mL of graphene oxide (GO) effectively enhances the specificity of the error-prone multi-round PCR. Mismatched primers were designed as interference to produce nonspecific products when the same amounts of matched and mismatched primers were added into semi-multiplex PCR. It was found that GO can enhance specificity by suppressing the amplification of mismatched primers. We monitored the primer-template-polymerase-GO interactions involved in the PCR using a capillary electrophoresis/laser-induced fluorescence polarization (CE-LIFP) assay. The results showed that the addition of GO promoted the formation of a matched primer-template complex, but suppressed the formation of a mismatched primer-template complex during PCR, suggesting that interactions between the primers and GO play an essential role. Furthermore, we successfully amplified the FOXL2 gene from PEGFP-N1 vectors using GO to eliminate the nonspecific products in PCR. Taken together, these results suggest that the GO can be used as an efficient additive for improving the conventional PCR system

Co-exposure of Carboxyl-Functionalized Single-Walled Carbon Nanotubes and 17α-Ethinylestradiol in Cultured Cells: Effects on Bioactivity and Cytotoxicity

17α-Ethinylestradiol (EE2) is the representative of environmental estrogens. Although EE2 can interact with some engineered nanoparticles (NPs), little is known about the bioactivity of NP-associated EE2 in organisms. In this study, we investigated the combined effects of the co-exposed carboxyl-functionalized single-walled carbon nanotubes (cf-SWCNTs) and EE2 in the human breast adenocarcinoma cell line (MCF-7 cells), focusing on the cytotoxicity and bioactivity. There were no significant differences in mitochondrial activity, membrane damage, and cell apoptosis when exposed to cf-SWCNTs with and without adsorbed EE2. However, the bioactivity of adsorbed EE2 on cf-SWCNTs was significantly inhibited. The calculated effective concentration of EE2 in cultured cells showed that less than 0.2% of the total adsorbed EE2 was released, indicating that most EE2 was retained on the cf-SWCNTs during cellular exposure. Furthermore, there were no obvious changes in the bioactivity of adsorbed EE2 in the culture medium containing 5–20% fetal bovine serum (FBS), even up to 10 days of incubation, indicating that the adsorbed EE2 on cf-SWCNTs is highly stable in the cell culture medium. These results mark a promising possibility for EE2 to be adsorbed by cf-SWCNTs in environmentally relevant settings and thereby influenced its toxicity and biological fate. This is also tempting for future studies involving risk assessment ways for association between NPs and contaminants in the environment

Determining the Cytotoxicity of Rare Earth Element Nanoparticles in Macrophages and the Involvement of Membrane Damage

Rare earthelement nanomaterials (REE NPs) hold considerable promise, with high availability and potential applications as superconductors, imaging agents, glass additives, fertilizers additives and feed additives. These results in potential REE NP exposure to humans and the environment through different routes and adverse effects induced by biological application of these materials are becoming an increasing concern. This study investigates the cytotoxicity of REE NPs: nLa₂O₃, nEu₂O₃, nDy₂O₃ and nYb₂O₃ from 2.5 to 80 μg/mL, in macrophages. A significant difference was observed in the extent of cytotoxicity induced in macrophages by differential REE NPs. The high-atomic number materials (i.e., nYb₂O₃) tending to be no toxic whereas low-atomic number materials (nLa₂O₃ and nEu₂O₃ and nDy₂O₃) induced 75.1%, 53.6% and 20.7% dead cells. With nLa₂O₃ as the representative material, we demonstrated that nLa₂O₃ induced cellular membrane permeabilization, through the sequestration of phosphates from membrane. The further mechanistic investigation established that membrane damage induced intracellular calcium increased to 3.0- to 7.3-fold compared to control cells. This caused the sustained overload of mitochondrial calcium by approximately 2.4-fold, which regulated cell necrosis. In addition, the injury of cellular membrane led to the release of cathepsins into cytosol which also contributed to cell death. This detailed investigation of signaling pathways driving REE NP-induced toxicity to macrophages is essential for better understanding of their potential health risks to humans and the environment