Efficient and accurate KRAS genotyping using digital PCR combined with melting curve analysis for ctDNA from pancreatic cancer patients.

A highly sensitive and highly multiplexed quantification technique for nucleic acids is necessary to predict and evaluate cancer treatment by liquid biopsy. Digital PCR (dPCR) is a highly sensitive quantification technique, but conventional dPCR discriminates multiple targets by the color of the fluorescent dye of the probe, which limits multiplexing beyond the number of colors of fluorescent dyes. We previously developed a highly multiplexed dPCR technique combined with melting curve analysis. Herein, we improved the detection efficiency and accuracy of multiplexed dPCR with melting curve analysis to detect KRAS mutations in circulating tumor DNA (ctDNA) prepared from clinical samples. The mutation detection efficiency was increased from 25.9% of the input DNA to 45.2% by shortening the amplicon size. The limit of detection of mutation was improved from 0.41 to 0.06% by changing the mutation type determination algorithm for G12A, resulting in a limit of detection of less than 0.2% for all the target mutations. Then, ctDNA in plasma from pancreatic cancer patients was measured and genotyped. The measured mutation frequencies correlated well with those measured by conventional dPCR, which can measure only the total frequency of KRAS mutants. KRAS mutations were detected in 82.3% of patients with liver or lung metastasis, which was consistent with other reports. Accordingly, this study demonstrated the clinical utility of multiplex dPCR with melting curve analysis to detect and genotype ctDNA from plasma with sufficient sensitivity.

Photoluminescence from Carbon Dot-Gold Nanoparticle Composites Enhanced by Photonic and Plasmonic Double-Resonant Effects.

Carbon dots (CDs) exhibit chemical stability and low toxicity, so they are promising for biomedical and imaging applications. The quantum yield of the photoluminescence is typically 10-20%, which limits practical applications. We fabricate carbon dot-gold nanoparticle photonic crystals (CD-GNP PCs) and demonstrate enhanced photoluminescence intensity from the carbon dots using the photonic and plasmonic double-resonant effects. A severalfold enhancement was obtained compared to the neat CD. The method developed in this study provides a universal scheme to enhance light-emitting materials, which is promising for the development of ultrahigh molecular sensing and bioimaging techniques.

Space-Selective Fabrication of Light-Emitting Carbon Dots in Polymer Films Using Electron-Beam-Induced Chemical Reactions.

Nanocarbon-based materials have excellent properties, including high electrical conductivity as well as charity-dependent optical absorption and luminescence; therefore, the materials are promising in applications for nanoelectric devices, nanophotonics, and so on. Carbon dots (CDs) are one of the carbon materials recently fabricated. Optical properties of CDs have been reported to be similar to those of polycyclic aromatic hydrocarbons (PAHs). For this reason, the CDs are considered to be composed of PAH. Synthesis of CDs has previously been accomplished through hydrothermal synthesis and microwave irradiation. These methods require a long synthesis time, and the processes involve multiple steps. In this study, we developed a fabrication method of CDs in simple and spatially selective ways, by using radical reactions in an organic polymer film with focused electron-beam irradiation. We investigated various organic polymers as reaction materials and found that polystyrene has a higher efficiency for CD formation than other organic polymers investigated. Absorption, photoluminescence, and Raman scattering properties of the electron-beam-irradiated sample were in good agreement with those reported for the CDs. The technique developed in this study is promising for fabricating light-emitting CDs and photonic crystals in a simple and flexible manner.