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

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

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

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

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