Digital LAMP in a sample self-digitization (SD) chip

This paper describes the realization of digital loop-mediated DNA amplification (dLAMP) in a sample self-digitization (SD) chip. Digital DNA amplification has become an attractive technique to quantify absolute concentrations of DNA in a sample. While digital polymerase chain reaction is still the most widespread implementation, its use in resource-limited settings is impeded by the need for thermal cycling and robust temperature control. In such situations, isothermal protocols that can amplify DNA or RNA without thermal cycling are of great interest. Here, we accomplished the successful amplification of single DNA molecules in a stationary droplet array using isothermal digital loop-mediated DNA amplification. Unlike most (if not all) existing methods for sample discretization, our design allows for automated, loss-less digitization of sample volumes on-chip. We demonstrated accurate quantification of relative and absolute DNA concentrations with sample volumes of less than 2 μl. We assessed the homogeneity of droplet size during sample self-digitization in our device, and verified that the size variation was small enough such that straightforward counting of LAMP-active droplets sufficed for data analysis. We anticipate that the simplicity and robustness of our SD chip make it attractive as an inexpensive and easy-to-operate device for DNA amplification, for example in point-of-care settings.     

Recrystallization and Precipitate Coarsening in Pb-Free Solder Joints During Thermomechanical Fatigue

The recrystallization of β-Sn profoundly affects deformation and failure of Sn-Ag-Cu solder joints in thermomechanical fatigue (TMF) testing. The numerous grain boundaries of recrystallized β-Sn enable grain boundary sliding, which is absent in as-solidified solder joints. Fatigue cracks initiate at, and propagate along, recrystallized grain boundaries, eventually leading to intergranular fracture. The recrystallization behavior of Sn-Ag-Cu solder joints was examined in three different TMF conditions for five different ball grid array component designs. Based on the experimental observations, a TMF damage accumulation model is proposed: (1) strain-enhanced coarsening of secondary precipitates of Ag₃Sn and Cu₆Sn₅ starts at joint corners, eventually allowing recrystallization of the Sn grain there as well; (2) coarsening and recrystallization continue to develop into the interior of the joints, while fatigue crack growth lags behind; (3) fatigue cracks finally progress through the recrystallized region. Independent of the TMF condition, the recrystallization appeared to be essentially complete after somewhat less than 50% of the characteristic life, while it took another 50% to 75% of the lifetime for a fatigue crack to propagate through the recrystallized region.     

Multinuclear solid-state nuclear magnetic resonance and density functional theory characterization of interaction tensors in taurine

A variety of experimental solid-state nuclear magnetic resonance (NMR) techniques has been used to characterize each of the elements in 2-aminoethane sulfonic acid (taurine). A combination of (15)N cross-polarization magic angle spinning (CPMAS), (14)N ultrawideline, and (14)N overtone experiments enabled a determination of the relative orientation of the nitrogen electric field gradient and chemical shift tensors. (17)O spectra recorded from an isotopically enriched taurine sample at multiple magnetic fields allowed the three nonequivalent oxygen sites to be distinguished, and NMR parameters calculated from a neutron diffraction structure using density functional theory allowed the assignment of the (17)O parameters to the correct crystallographic sites. This is the first time that a complete set of (17)O NMR tensors are reported for a sulfonate group. In combination with (1)H and (13)C MAS spectra, as well as a previously reported (33)S NMR study, this provides a very broad set of NMR data for this relatively simple organic molecule, making it a potentially useful structure on which to test DFT calculation methods (particularly for the quadrupolar nuclei (14)N, (17)O, and (33)S) or NMR crystallography approaches.     

Derivations and Dirichlet forms on fractals

We study derivations and Fredholm modules on metric spaces with a local regular conservative Dirichlet form. In particular, on finitely ramified fractals, we show that there is a non-trivial Fredholm module if and only if the fractal is not a tree (i.e. not simply connected). This result relates Fredholm modules and topology, refines and improves known results on p.c.f. fractals. We also discuss weakly summable Fredholm modules and the Dixmier trace in the cases of some finitely and infinitely ramified fractals (including non-self-similar fractals) if the so-called spectral dimension is less than 2. In the finitely ramified self-similar case we relate the p-summability question with estimates of the Lyapunov exponents for harmonic functions and the behavior of the pressure function.     

Synthesis and Structural Characterization of (Z)-3-[(4-Chlorophenylamino) Methylene] Naphthalene-2(3H)-One: An Enol,Keto or Zwitterionic Tautomer?

Abstract  

The structure of the title compound was determined using X-ray crystallography at both 173 and 293 K. The molecular structure and packing did not change significantly with temperature and a disordered structure was identified comprising a keto and enol tautomeric form. Analysis of the bond lengths in the vicinity of the C=O group suggested the keto form was predominantly in its zwitterionic form structure. ¹H NMR spectroscopy showed the presence of a single compound in solution with two diagnostic doublets demonstrating the compound had an NH group next to a CH group resembling the zwitterionic form of the compound.     

Open-access microfluidic patch-clamp array with raised lateral cell trapping sites

A novel open-access microfluidic patch-clamp array chip with lateral cell trapping sites raised above the bottom plane of the chip was developed by combining both a microscale soft-lithography and a macroscale polymer fabrication method. This paper demonstrates the capability of using such an open-access fluidic system for patch-clamp measurements. The surface of the open-access patch-clamp sites prepared by the macroscale hole patterning method of soft-state elastic polydimethylsiloxane (PDMS) is examined; the seal resistances are characterized and correlated with the aperture dimensions. Whole cell patch-clamp measurements are carried out with CHO cells expressing Kv2.1 ion channels. Kv2.1 ion channel blocker (TEA) dosage response is characterized and the binding activity is examined. The results demonstrate that the system is capable of performing whole cell measurements and drug profiling in a more efficient manner than the traditional patch-clamp set-up.     

Dynamic single cell culture array

It is important to quantify the distribution of behavior amongst a population of individual cells to reach a more complete quantitative understanding of cellular processes. Improved high-throughput analysis of single cell behavior requires uniform conditions for individual cells with controllable cell-cell interactions, including diffusible and contact elements. Uniform cell arrays for static culture of adherent cells have previously been constructed using protein micropatterning techniques but lack the ability to control diffusible secretions. Here we present a microfluidic-based dynamic single cell culture array that allows both arrayed culture of individual adherent cells and dynamic control of fluid perfusion with uniform environments for individual cells. In our device no surface modification is required and cell loading is done in less than 30 seconds. The device consists of arrays of physical U-shaped hydrodynamic trapping structures with geometries that are biased to trap only single cells. HeLa cells were shown to adhere at a similar rate in the trapping array as on a control glass substrate. Additionally, rates of cell death and division were comparable to the control experiment. Approximately 100 individual isolated cells were observed growing and adhering in a field of view spanning approximately 1 mm(2) with greater than 85% of cells maintained within the primary trapping site after 24 hours. Also, greater than 90% of cells were adherent and only 5% had undergone apoptosis after 24 hours of perfusion culture within the trapping array. We anticipate uses in single cell analysis of drug toxicity with physiologically relevant perfused dosages as well as investigation of cell signaling pathways and systems biology.     

Pi-complex structure of gaseous benzene-NO cations assayed by IR multiple photon dissociation spectroscopy

[C(6)H(6)NO](+) ions, in two isomeric forms involved as key intermediates in the aromatic nitrosation reaction, have been produced in the gas phase and analyzed by IR multiple photon dissociation (IRMPD) spectroscopy in the 800-2200 cm(-)(1) fingerprint wavenumber range, exploiting the high fluence and wide tunability of a free electron laser (FEL) source. The IRMPD spectra were compared with the IR absorption spectra calculated for the optimized structures of potential isomers, thus allowing structural information on the absorbing species. [C(6)H(6)NO](+) ions were obtained by two routes, taking advantage of the FEL coupling to two different ion traps. In the first one, an FT-ICR mass spectrometer, a sequence of ion-molecule reactions was allowed to occur, ultimately leading to an NO(+) transfer process to benzene. The so-formed ions displayed IRMPD features characteristic of a [benzene,NO](+) pi-complex structure, including a prominent band at 1963 cm(-)(1), within the range for the N-O bond stretching vibration of NO (1876 cm(-)(1)) and NO(+) (2344 cm(-)(1)). A quite distinct species is formed by electrospray ionization (ESI) of a methanol solution of nitrosobenzene. The ions transferred and stored in a Paul ion trap showed the IRMPD features of substituent protonated nitrosobenzene, the most stable among conceivable [C(6)H(6)NO](+) isomers according to computations. It is noteworthy that IRMPD is successful in allowing a discrimination between isomeric [C(6)H(6)NO](+) species, whereas high-energy collision-induced dissociation fails in this task. The [benzene,NO](+) pi-complex is characterized by IRMPD spectroscopy as an exemplary noncovalent ionic adduct between two important biomolecular moieties.     

Degree-independent Sobolev extension on locally uniform domains

We consider the problem of constructing extensions , where is the Sobolev space of functions with k derivatives in Lp and Ω⊂Rn is a domain. In the case of Lipschitz Ω, Calderón gave a family of extension operators depending on k, while Stein later produced a single (k-independent) operator. For the more general class of locally-uniform domains, which includes examples with highly non-rectifiable boundaries, a k-dependent family of operators was constructed by Jones. In this work we produce a k-independent operator for all spaces on a locally uniform domain Ω.     

Local Linear Convergence for Alternating and Averaged Nonconvex Projections

The idea of a finite collection of closed sets having “linearly regular intersection” at a point is crucial in variational analysis. This central theoretical condition also has striking algorithmic consequences: in the case of two sets, one of which satisfies a further regularity condition (convexity or smoothness, for example), we prove that von Neumann’s method of “alternating projections” converges locally to a point in the intersection, at a linear rate associated with a modulus of regularity. As a consequence, in the case of several arbitrary closed sets having linearly regular intersection at some point, the method of “averaged projections” converges locally at a linear rate to a point in the intersection. Inexact versions of both algorithms also converge linearly. Research of A.S. Lewis supported in part by National Science Foundation Grant DMS-0504032. Research of D.R. Luke supported in part by National Science Foundation Grant DMS-0712796.