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.     

Spin-adaptation in many-body perturbation theory

In this paper we present a method of incorporating spin-adapted configurations in a Many-Body Perturbation Theory (MBPT) framework. It has been demonstrated how the use of doubly excited Serber-type spinadapted configurations leads in a straightforward way to a well-defined MBPT series which in effect involves an infinite summation of orbital-diagonal ladders for all possible spin allocations. The formalism implicitly defines a partition of the hamiltonian in which the diagonal matrix elements of the hamiltonian in the Serber-type configurations figure as the unperturbed part. This may thus be called a spin-adapted Epstein-Nesbet (E-N) type of partition. The formalism has been tested on a selection of 6π-electron conjugated systems where we have calculated their orbital pair-correlation energies, and compared the performance against E-N perturbation results and a variational calculation of the same quantities. The comparison reveals that, with canonical Hartree-Fock orbitals, the E-N partition tends to overestimate the pair-correction energy which the spinadapted formalism tends to compensate, but without involving much additional effort. The results for the localized H-F orbitals are rather less sensitive and no definitive conclusions could be discerned for them. The advantages of using the spin-adapted MBPT formalism have also been delineated.     

Low temperature thermoelectric properties of Cu intercalated TiSe2: a charge density wave material

Y₂O₃ nanoparticles and nanorods have been firstly synthesized in bulk Ti-Y films prepared by magnetron sputtering on Si (100) substrates at different temperatures. X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDS) are used to characterize the structure, morphology, and composition of the as-synthesized nanoparticles and nanorods. The mechanical properties of the sputtered films are investigated using nanoindentation techniques. The results indicate that both the nanoparticles and nanorods have a pure cubic Y₂O₃ structure resulting from the reaction of Y atoms with the residual O₂ in the vacuum chamber, and are free from defects and dislocations with uniform diameters of about 30 nm. The Y₂O₃ nanoparticles mainly distribute at the grain boundaries of the Ti matrix and the nanorods have lengths ranging from 250 nm to more than 1 μm with the growth direction parallel to the (002) plane. As the growth temperature elevates, the nanoparticles turn to be coarsening while more and longer nanorods are inclined to form. Compared with the Ti film, the TiY films have a remarkable increase in hardness, but do not exhibit expected increase in elastic modulus. Finally, the growth mechanism is also briefly discussed.     

Ratiometric fluorescence signalling of fluoride ions by an amidophthalimide derivative

Fluorescence behaviour of 4-benzoylamido-N-methylphthalimide (1), designed and developed for selective detection of fluoride ions, is reported. 1 displays F⁻-induced colour change that allows its detection with the naked eye. The F⁻ specificity of the sensor system is evident from the fact that unlike F⁻, other halides do not affect the absorption characteristics of 1. Apart from the colorimetric response, the fluorescence output of 1 is also modulated by F⁻ in a manner that permits ratiometric fluorescence signalling of F⁻ as well. It is found that the system can detect F⁻ in the concentration range of 10–60 μM. The results of the experiments and theoretical calculations unambiguously suggest that the changes of the electronic absorption and fluorescence behaviour of 1, which have been exploited for signalling purpose, are due to F⁻-induced deprotonation of the 4-amido moiety of the sensor system.     

Stability of charged impurities in a coupled single electron transistor and antidot system

We have fabricated devices on GaAs/AlGaAs heterostructures, containing two-dimensional electron gases, that consist of three point contacts surrounding an etched antidot with an Al/AlO_x/Al single electron transistor. The single electron transistor measurement shows rearrangement of neighboring charged impurities with a characteristic stability time scale of 20 s in one device and greater than 1 h in a second device. We also measured the resistance of the point contact–antidot constriction versus magnetic field. In a device with a 20 s stability time, we see a high noise level and poor reproducibility. In a device with a long stability time, much greater than 1 h, we are able to see reproducible features including Aharonov–Bohm oscillations.     

Thiosemicarbazone complexes of the platinum metals. A story of variable coordination modes

Salicylaldehyde thiosemicarbazone (H₂saltsc) reacts with [M(PPh₃)₃X₂] (M = Ru, Os; X = Cl, Br) to afford complexes of type [M(PPh₃)₂(Hsaltsc)₂], in which the salicylaldehyde thiosemicarbazone ligand is coordinated to the metal as a bidentate N,S-donor forming a four-membered chelate ring. Reaction of benzaldehyde thiosemicarbazones (Hbztsc-R) with [M(PPh₃)₃X₂] also affords complexes of similar type, viz. [M(PPh₃)₂(bztsc-R)₂], in which the benzaldehyde thiosemicarbazones have also been found to coordinate the metal as a bidentate N,S-donor forming a four-membered chelate ring as before. Reaction of the Hbztsc-R ligands has also been carried out with [M(bpy)₂X₂] (M = Ru, Os; X = Cl, Br), which has afforded complexes of type [M(bpy)₂(bztsc-R)]⁺, which have been isolated as perchlorate salts. Coordination mode of bztsc-R has been found to be the same as before. Structure of the Hbztsc-OMe ligand has been determined and some molecular modelling studies have been carried out determine the reason for the observed mode of coordination. Reaction of acetone thiosemicarbazone (Hactsc) has then been carried out with [M(bpy)₂X₂] to afford the [M(bpy)₂(actsc)]ClO₄ complexes, in which the actsc ligand coordinates the metal as a bidentate N,S-donorformingafive-membered chelate ring. Reaction of H₂saltsc has been carried out with [Ru(bpy)₂Cl₂] to prepare the [Ru(bpy)₂(Hsaltsc)]ClO₄ complex, which has then been reacted with one equivalent of nickel perchlorate to afford an octanuclear complex of type [Ru(bpy)₂(saltsc-H)₄Ni₄](ClO₄)₄.     

Hysteresis loss as a measure of metal–rubber adhesion

As in the case of reinforcing filler-induced increase in hysteresis in rubbers, placement of aluminum (A1) foil to the surface of a rubber blend of epichlorohydrin rubber and carboxylated nitrile base induces increased hysteresis of the rubber due to adhesion between Al and the rubber blend. Changes in hysteresis loss due to Al foil can be correlated with the peel strength of Al-rubber-Al joints. © 1995 John Wiley & Sons, Inc.     

Optical Generation of mm-Wave Signal Through Optoelectronic Phase-Locked Loop

In this paper, we propose a scheme for the generation of low phase noise tunable mm-wave signal by beating two lightwaves in a photodiode. These two lightwaves are made phase coherent by an optoelectronic phase locked loop.Calculated mm-wave power at a frequency of 60 GHz is found to be -4 dBm.