Absolute and Direct MicroRNA Quantification Using DNA–Gold Nanoparticle Probes

DNA–gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA–RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA–AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis

<i>In situ</i> generation of ZnO nanoparticles within a polyethyleneimine matrix for antibacterial zein fibers

In this study the potential of branched polyethyleneimine (PEI) was explored for the in situ generation of ZnO nanoparticles and the electrospinning of zein-based fibers. Zinc acetyl acetonate dihydrate (ZnAcAc·2H2O) was chosen as the precursor for the related sol–gel reactions, leading to the nucleation and growth of wurtzite crystals within the PEI matrix. Control experiments showed that PEI played a vital role in the reaction steps leading to the conversion. Wide angle X-ray diffraction analysis confirmed the crystallographic structure of the ZnO particles formed, while scanning electron microscopy examinations revealed the formation of agglomerates less than 400 nm, made up of much smaller primary nanoparticles. The obtained ZnO/PEI microsuspensions in ethanol were mixed in an ethanol/water solution of zein and used for electrospinning of different types of zein-modified fibers. Differential scanning calorimetry analysis showed that PEI acted as a plasticizer for zein, causing also a broadening of the glass transition, while Fourier-transform infrared spectroscopy indicated that the amine groups in PEI interacted with the surface groups of ZnO crystals. The results provide useful insights for formulating fibers, films, and coatings exhibiting antibacterial characteristics, as well as higher mechanical flexibility and toughness relative to plain zein.</div

Gold-Nanoparticle-Based Colorimetric Discrimination of Cancer-Related Point Mutations with Picomolar Sensitivity

Point mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene are being increasingly recognized as important diagnostic and prognostic markers in cancer. In this work, we describe a rapid and low-cost method for the naked-eye detection of cancer-related point mutations in KRAS based on gold nanoparticles. This simple colorimetric assay is sensitive (limit of detection in the low picomolar range), instrument-free, and employs nonstringent room temperature conditions due to a combination of DNA-conjugated gold nanoparticles, a probe design which exploits cooperative hybridization for increased binding affinity, and signal enhancement on the surface of magnetic beads. Additionally, the scheme is suitable for point-of-care applications, as it combines naked-eye detection, small sample volumes, and isothermal (PCR-free) amplification

Lifespan curves of <i>Drosophila</i> flies nurtured with AuNPs treated food (5, 15, 40, and 80 nm) compared to two populations bred with normal food (CTRL) or supernatant treated food (SN).

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029980#pone-0029980-g001" target="_blank">Fig. 1</a>, top and bottom, are relative to TES and TNN approach, respectively. Experimental points represent the average from 5 independent experiments (the standard deviations are reported as the curve symbols size). The lifespan curves of both TES and TNN experiments were validated by the non-parametric log-rank (Mantel-Cox) test (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029980#pone.0029980.s006" target="_blank">Table S3</a>).</p

Label-Free Isothermal Amplification Assay for Specific and Highly Sensitive Colorimetric miRNA Detection

We describe a new method for the detection of miRNA in biological samples. This technology is based on the isothermal nicking enzyme amplification reaction and subsequent hybridization of the amplification product with gold nanoparticles and magnetic microparticles (barcode system) to achieve naked-eye colorimetric detection. This platform was used to detect a specific miRNA (miRNA-10b) associated with breast cancer, and attomolar sensitivity was demonstrated. The assay was validated in cell culture lysates from breast cancer cells and in serum from a mouse model of breast cancer

mRNA expression level analyzed by RT-qPCR of <i>Drosophila</i> treated with TES (top) and TNN (bottom) approaches.

<p>All data relative to RT-qPCR experiments were analyzed by statistical software to evaluate the significant difference with respect to the control (ns  =  non significant, i.e. p-value >0.05; *p-value <0.05; **p-value <0.01 ***p-value <0.001).</p

Male (left) and female (right) fertility tests relative to TES (top) and TNN experiments (bottom).

<p>Experimental points represent the average from 10 independent experiments and the error bars indicate the standard deviation (ns  =  non significant, i.e. p-value >0.05; **p-value <0.01; ***p-value <0.001).</p

ROS measurements by DCF assay on TES and TNN treatments (top and bottom, respectively).

<p>Data are reported as relative fluorescence intensity normalized to the control (ns  =  non significant, i.e. p-value >0.05; ***p-value <0.001). Error bars  =  SD.</p

Representative confocal microscopy images of Drosophila midgut in flies obtained from TES treatment.

<p>Nuclei are stained with Hoechst 33342 (blue) while cells containing DNA strand nicks are detected by TUNEL assay and fluoresce red (highlighted by the white arrows).</p

Sputtering-Enabled Intracellular X‑ray Photoelectron Spectroscopy: A Versatile Method To Analyze the Biological Fate of Metal Nanoparticles

The investigation of the toxicological profile and biomedical potential of nanoparticles (NPs) requires a deep understanding of their intracellular fate. Various techniques are usually employed to characterize NPs upon cellular internalization, including high-resolution optical and electron microscopies. Here, we show a versatile method, named sputtering-enabled intracellular X-ray photoelectron spectroscopy, proving that it is able to provide valuable information about the behavior of metallic NPs in culture media as well as within cells, directly measuring their internalization, stability/degradation, and oxidation state, without any preparative steps. The technique can also provide nanoscale vertical resolution along with semiquantitative information about the cellular internalization of the metallic species. The proposed approach is easy-to-use and can become a standard technique in nanotoxicology/nanomedicine and in the rational design of metallic NPs. Two model cases were investigated: silver nanoparticles (AgNPs) and platinum nanoparticles (PtNPs) with the same size and coating. We observed that, after 48 h incubation, intracellular AgNPs were almost completely dissolved, forming nanoclusters as well as AgO, AgS, and AgCl complexes. On the other hand, PtNPs were resistant to the harsh endolysosomal environment, and only some surface oxidation was detected after 48 h