Quantitative and sensitive detection of rare mutations using droplet-based microfluidics

Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(?) assays and pyrosequencing, while quantitative, cannot detect less than ～1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(?) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(?) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.     

A clinical study of failure in microchannel cooled diodes used in large laser systems

In this paper we investigate the source of failure in commercial, microchannel cooled CW diode bars placed in 12 bar horizontal arrays. The arrays were used to pump Nd:YAG rods in our 10 kW developmental laser. The laser was operated at low duty factor over a period of over 2 years. Experimental evidence indicated that the sudden, catastrophic failure was because of degraded cooling. We used optical microscopes, an X-ray microfocus imager, and a thermal neutron scattering camera to look inside the microcoolers. Our investigations revealed only one possible failure mechanism: cooling flow reduction because of delamination of the Au coating the walls of the microcoolers and the entrapment of Au flakes within the microchannel structures. We observed blisters in the microcoolers under working bars, and flake-like structures in the microcoolers under burnt-out bars (all taken from the laser). We observed no evidence of either massive blockages because of electrochemical deposits, or of corrosion/erosion in the microchannel walls. Integral operation times of the high flow-rate cooling system and of the diodes themselves were too short by one and two orders of magnitude, respectively, to explain the observed failures. Microchannel immersion times in the deionized water were, however, long enough to allow for corrosion of metals that may have been exposed through defects in the Au coatings. Three-dimensional heat flow simulations showed that blockage of multiple microchannels towards the edge of a bar can easily lead to catastrophic temperature increases.     

Minerals of Moscow

By the example of the territory of Moscow, possibilities of studying large cities as independent topomineralogical objects are shown. A cadastre of minerals, their varieties, and mineral formations, consisting of more than 100 names, has been collected for Moscow for the first time on the basis of different sources (publications, geological funds, and museums). Distribution of important discoveries of minerals on the territory of the city is demonstrated in detail and their characteristics are given.     

Electrochemical sensor based on a sepiolite clay nanoparticle-based electrochemical sensor for ascorbic acid detection in real-life samples

Ionics - A new electrode composed of sepiolite clay (SC) carbon paste (CP) was developed and used for the adsorptive stripping differential pulse voltammetric quantification of ascorbic acid (AA)....