Continuous analysis of dye-loaded, single cells on a microfluidic chip

Continuous analysis of two dyes loaded into single mammalian cells using laser-based lysis combined with electrophoretic separation was developed and characterized on microfluidic chips. The devices employed hydrodynamic flow to transport cells to a junction where they were mechanically lysed by a laser-generated cavitation bubble. An electric field then attracted the analyte into a separation channel while the membranous remnants passed through the intersection towards a waste reservoir. Phosphatidylcholine (PC)-supported bilayer membrane coatings (SBMs) provided a weakly negatively charged surface and prevented cell fouling from interfering with device performance. Cell lysis using a picosecond-pulsed laser on-chip did not interfere with concurrent electrophoretic separations. The effect of device parameters on performance was evaluated. A ratio of 2 : 1 was found to be optimal for the focusing-channel : flow-channel width and 3 : 1 for the flow-channel : separation-channel width. Migration times decreased with increased electric field strengths up to 333 V cm(-1), at which point the field strength was sufficient to move unlysed cells and cellular debris into the electrophoretic channel. The migration time and full width half-maximum (FWHM) of the peaks were independent of cell velocity for velocities between 0.03 and 0.3 mm s(-1). Separation performance was independent of the exact lysis location when lysis was performed near the outlet of the focusing channel. The migration time for cell-derived fluorescein and fluorescein carboxylate was reproducible with <10% RSD. Automated cell detection and lysis were required to reduce peak FWHM variability to 30% RSD. A maximum throughput of 30 cells min(-1) was achieved. Device stability was demonstrated by analyzing 600 single cells over a 2 h time span.     

Kinetic analysis of PI3K reactions with fluorescent PIP<Subscript>2</Subscript> derivatives

Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP₂ derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.     

Three-Dimensional Acceleration Measurement Using Videogrammetry Tracking Data

In order to evaluate the feasibility of multi-point, non-contact, acceleration measurement, a high-speed, precision videogrammetry system has been assembled from commercially-available components and software. Consisting of three synchronized 640 × 480 pixel monochrome progressive scan CCD cameras each operated at 200 frames per second, this system has the capability to provide surface-wide position-versus-time data that are filtered and twice-differentiated to yield the desired acceleration tracking at multiple points on a moving body. The oscillating motion of targets mounted on the shaft of a modal shaker were tracked, and the accelerations calculated using the videogrammetry data were compared directly to conventional accelerometer measurements taken concurrently. Although differentiation is an inherently noisy operation, the results indicate that simple mathematical filters based on the well-known Savitzky and Golay algorithms, implemented using spreadsheet software, remove a significant component of the noise, resulting in videogrammetry-based acceleration measurements that are comparable to those obtained using the accelerometers.     

Continuous-time, stage-structured, multiple-species model with applications to amphibians

Since the 1980s, biologists have noticed a major decline in amphibian population. The reason this is so alarming is because amphibians have been seen as a “Canary in the mine” when it comes to the world’s environmental changes. With global warming and CO₂ emissions all over the news, we have become more aware of how we are impacting our world. If the decline of amphibians is a precursor to what is happening with the environment, then we need to find a good model to give us estimates on what is going to happen in the future. Here we used a predator-prey-competition model to help investigate how three amphibians might interact when confined to the same area.     

The application of gas chromatography/atmospheric pressure chemical ionisation time‐of‐flight mass spectrometry to impurity identification in Pharmaceutical Development

Accurate mass measurement (used to determine elemental formulae) is an essential tool for impurity identification in pharmaceutical development for process understanding. Accurate mass liquid chromatography/mass spectrometry (LC/MS) is used widely for these types of analyses; however, there are still many occasions when gas chromatography (GC)/MS is the appropriate technique. Therefore, the provision of robust technology to provide accurate mass GC/MS (and GC/MS/MS) for this type of activity is essential. In this report we describe the optimisation and application of a newly available atmospheric pressure chemical ionisation (APCI) interface to couple GC to time‐of‐flight (TOF) MS. To fully test the potential of the new interface the APCI source conditions were optimised, using a number of standard compounds, with a variety of structures, as used in synthesis at AstraZeneca. These compounds were subsequently analysed by GC/APCI‐TOF MS. This study was carried out to evaluate the range of compounds that are amenable to analysis using this technique. The range of compounds that can be detected and characterised using the technique was found to be extremely broad and include apolar hydrocarbons such as toluene. Both protonated molecules ([M + H]⁺) and radical cations (M^+.) were observed in the mass spectra produced by APCI, along with additional ion signals such as [M + H + O]⁺. The technique has been successfully applied to the identification of impurities in reaction mixtures from organic synthesis in process development. A typical mass accuracy of 1–2 mm/zunits (m/z 80–500) was achieved allowing the reaction impurities to be identified based on their elemental formulae. These results clearly demonstrate the potential of the technique as a tool for problem solving and process understanding in pharmaceutical development. The reaction mixtures were also analysed by GC/electron ionisation (EI)‐MS and GC/chemical ionisation (CI)‐MS to understand the capability of GC/APCI‐MS relative to these two firmly established techniques. Copyright © 2010 John Wiley & Sons, Ltd.     

On the performance and resonant frequency of electromagnetic induction energy harvesters

This paper investigates the linear response of an archetypal energy harvester that uses electromagnetic induction to convert ambient vibration into electrical energy. In contrast with most prior works, the influence of the circuit inductance is not assumed negligible. Instead, we highlight parameter regimes where the inductance can alter resonance and derive an expression for the resonant frequency.The governing equations consider the case of a vibratory generator directly powering a resistive load. These equations are non-dimensionalized and analytical solutions are obtained for the system's response to single harmonic, periodic, and stochastic environmental excitations. The presented analytical solutions are then used to study the power delivered to an electrical load.     

Proposed performance qualification and calibration method for evaporative light scattering detectors

Summary A method for the performance testing of Sedex 55 Evaporative Light Scattering Detectors (ELSD) has been developed as part of our regulatory compliance programme. The method uses a readily available chromatographic column, caffeine as the test probe and can be completed in less than one hour.     

Caraibical,a new aromatic sesquiterpene from the marine alga laurenciacaraibica

Caraibical, a new aromatic sesquiterpene aldehyde, has been isolated from the marine marine red alga $\text{Laurencia}$$\text{caraibica}$. The structure, which possesses an ether cyclization differing from previous compounds, was defined on the basis of its spectral data.