Targeted Metabolomics for Biomarker Discovery

Metabolomics is a truly interdisciplinary field of science, which combines analytical chemistry, platform technology, mass spectrometry, and NMR spectroscopy with sophisticated data analysis. Applied to biomarker discovery, it includes aspects of pathobiochemistry, systems biology/medicine, and molecular diagnostics and requires bioinformatics and multivariate statistics. While successfully established in the screening of inborn errors in neonates, metabolomics is now widely used in the characterization and diagnostic research of an ever increasing number of diseases. In this Review we highlight important technical prerequisites as well as recent developments in metabolomics and metabolomics data analysis with special emphasis on their utility in biomarker identification and qualification, as well as targeted metabolomics by employing high‐throughput mass spectrometry.     

Compressibility measurements of quasi-one-dimensional quantum wires

We report measurements of the compressibility of a one-dimensional quantum wire, defined in the upper well of a GaAs/AlGaAs double quantum well heterostructure. A wire defined simultaneously in the lower well probes the ability of the upper wire to screen the electric field from a biased surface gate. The technique is sensitive enough to resolve spin splitting of the subbands in the presence of an in-plane magnetic field. We measure a compressibility signal due to the 0.7 structure and study its evolution with increasing temperature and magnetic field. We see no evidence of the formation of the quasibound state predicted by the Kondo model, instead our data are consistent with theories which predict that the 0.7 structure arises as a result of spontaneous spin polarization.     

Continuous and pulsed ultrasound-assisted extractions of antioxidants from pomegranate peel

There is a great demand for developing efficient extraction methods in order to reduce extraction time and increase the yield and activity of functional antioxidants. The yields, activities, and extraction kinetics of antioxidants from dry peel of pomegranate marc were studied using ultrasound-assisted extraction in continuous and pulsed modes and the results were compared with conventional extraction (CE) at a temperature of 25±2°C and water/peel ratio of 50/1, w/w. The studied factors were intensity level and treatment time for continuous ultrasound-assisted extraction (CUAE), and intensity level, number of pulse repetition, and pulse duration and interval for pulsed ultrasound-assisted extraction (PUAE). The results showed that all factors significantly affected the antioxidant yield, but only treatment time had a significant effect on the antioxidant activity. Compared to CE, PUAE at intensity level of 59.2 W/cm(2), and the 5 and 5s of pulse duration and interval increased the antioxidant yield by 22% and reduced the extraction time by 87%. Similarly, CUAE at the same intensity level increased the antioxidant yield by 24% and reduced the extraction time by 90%. Since PUAE had 50% energy saving compared to CUAE, we recommend using PUAE for the extraction with antioxidant yield of 14.5% and DPPH scavenging activity of 5.8 g/g. A second-order kinetic model was successfully developed for describing the mechanism of ultrasound-assisted extractions under PUAE and CUAE. This research clearly demonstrated the superiority of PUAE for producing antioxidants from peel of pomegranate marc.     

Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented.     

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.     

Microfluidic immunomagnetic multi-target sorting--a model for controlling deflection of paramagnetic beads

We describe a microfluidic system that uses a magnetic field to sort paramagnetic beads by deflecting them in the direction normal to the flow. In the experiments we systematically study the dependence of the beads' deflection on bead size and susceptibility, magnet strength, fluid speed and viscosity, and device geometry. We also develop a design parameter that can aid in the design of microfluidic devices for immunomagnetic multi-target sorting.     

Central recirculation zone analysis in an unconfined tangential swirl burner with varying degrees of premixing

Swirl-stabilised combustion is one of the most widely used techniques for flame stabilisation, uses ranging from gas turbine combustors to pulverised coal-fired power stations. In gas turbines, lean premixed systems are of especial importance, giving the ability to produce low NOx systems coupled with wide stability limits. The common element is the swirl burner, which depends on the generation of an aerodynamically formed central recirculation zone (CRZ) and which serves to recycle heat and active chemical species to the root of the flame as well as providing low-velocity regions where the flame speed can match the local flow velocity. Enhanced mixing in and around the CRZ is another beneficial feature. The structure of the CRZ and hence that of the associated flames, stabilisation and mixing processes have shown to be extremely complex, three-dimensional and time dependent. The characteristics of the CRZ depend very strongly on the level of swirl (swirl number), burner configuration, type of flow expansion, Reynolds number (i.e. flowrate) and equivalence ratio. Although numerical methods have had some success when compared to experimental results, the models still have difficulties at medium to high swirl levels, with complex geometries and varied equivalence ratios. This study thus focuses on experimental results obtained to characterise the CRZ formed under varied combustion conditions with different geometries and some variation of swirl number in a generic swirl burner. CRZ behaviour has similarities to the equivalent isothermal state, but is strongly dependent on equivalence ratio, with interesting effects occurring with a high-velocity fuel injector. Partial premixing and combustion cause more substantive changes to the CRZ than pure diffusive combustion.     

In situ measurement of the thermal and photo-assisted MOVPE of ZnTe using laser reflectometry

In situ laser reflectometry has been used to study the growth kinetics of ZnTe under pyrolytic and photo-assisted conditions from diisopropyltellurium (DIPTe) and dimethylzinc.triethylamine (DMZn.TEN). The growth rate of ZnTe was monitored as a function of VI:II ratio, temperature and laser power density. From the results a model for the growth involving the surface decomposition of DIPTe via a bimolecular reaction with methyl radicals homolytically released from the decomposition of DMZn.TEN is proposed. The activation energy under pyrolytic conditions in the low temperature regime for a 1:1 VI:II ratio was found to be 27.6 kcal/mol and under photo-assisted conditions this activation energy was seen to be lowered to 21.9 kcal/mol. The growth kinetics are explained in terms of two competitive processes. The decomposition of DMZn on the surface will be enhanced with increasing substrate temperature; however, the desorption rate of DIPTe will also be enhanced. The competitive nature of these two processes is seen to be particularly pronounced under laser illumination at high power densities, where a growth rate of 13 a.u./s was observed at 360°C, laser power density 67 mW/cm², whereas at 380°C for a similar power density the growth rate was seen to be reduced to 10.5 a.u./s.