Maskless photolithography using UV leads

A UV light emitting diode (LED) with a maximum output of 372 nm was collimated using a pinhole and a small plastic tube and focused using a microscope objective onto a substrate for direct lithographic patterning of the photoresist. Movement of the substrate with a motorised linear stage (syringe pump) allowed lines in SU-8 to be pattered with a width down to 35 lm at a linear velocity of 80 lm s −1 , while in the dry film resist Ordyl SY 330, features as narrow as 17 lm were made at a linear velocity of 245 lm s −1 . At this linear velocity, a 75 mm long feature could be patterned in 5 min. Functional microfluidic devices were made by casting PDMS on a master made by LED lithography. The results show that UV LEDs are a suitable light source for direct writing lithography, offering a budget friendly, and high resolution alternative for rapid prototyping of features smaller than 20 lm

Novel instrument for automated pKa determination by internal standard capillary electrophoresis

The internal standard capillary electrophoresis method (IS-CE) has been implemented in a novel sequential injection−capillary electrophoresis instrument for the high-throughput determination of acidity constants (pKa) regardless of aqueous solubility, number of pKa values, or structure. This instrument comprises a buffer creation system that automatically mixes within a few seconds four reagents for in situ creation of the separation electrolyte with a pH range of 2−13, ionic strength of 10−100 mM and organic solvent content from 0% to 40%. Combined with 1.2 kV/cm and a short effective length (15 cm to the UV detector) fast 20 s electrophoretic separations can be obtained. The low standard deviation of the replicates and the low variation compared to reference values show that this system can accurately determine acidity constants of drugs by IS-CE. A single pKa can be determined in 2 min and a set of 20 measurements in half an hour, allowing rapid, simple, and flexible determination of pKa values of pharmaceutical targets

Time-resolved pharmacological studies using automated, on-line monitoring of five parallel suspension cultures

Early stage pharmacological studies rely on in vitro methodologies for screening and testing compounds. Conventional assays based on endpoint measurements provide limited information because the lack in temporal resolution may not determine the pharmacological effect at its maximum. We developed an on-line, automated system for near real-time monitoring of extracellular content from five parallel suspension cultures, combining cell density measurements with a high-resolution separations every 12 minutes for 4 days. Selector and switching valves provide the fluidic control required to sample from one culture during the analysis of the previous sample from another culture, a time-saving measure that is fundamental to the throughput of the presented system. The system was applied to study the metabolic effects of the drugs rotenone, β-lapachone and clioquinol using lactate as metabolic indicator. For each drug, 96 assays were executed on the extracellular matrix at three concentrations with two controls in parallel, consuming only 5.78 mL of media from each culture over four days, less than 60 μL per analysis. The automated system provides high sample throughput, good temporal resolution and low sample consumption combined with a rugged analytical method with adequate sensitivity, providing a promising new platform for pharmacological and biotechnological studies

Photoinitiated polymerisation of monolithic stationary phases in polyimide coated capillaries using visible region LEDs

The spatially controlled synthesis of poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic stationary phases in polyimide coated fused silica capillaries by visible light induced radical polymerisation using a three-component initiator and a 660 nm light emitting diode (LED) as a light source is presented here

Mild and repetitive very mild axonal stretch injury triggers cystoskeletal mislocalization and growth cone collapse

Diffuse axonal injury is a hallmark pathological consequence of non-penetrative traumatic brain injury (TBI) and yet the axonal responses to stretch injury are not fully understood at the cellular level. Here, we investigated the effects of mild (5%), very mild (0.5%) and repetitive very mild (2×0.5%) axonal stretch injury on primary cortical neurons using a recently developed compartmentalized in vitro model. We found that very mild and mild levels of stretch injury resulted in the formation of smaller growth cones at the tips of axons and a significantly higher number of collapsed structures compared to those present in uninjured cultures, when measured at both 24 h and 72 h post injury. Immunocytochemistry studies revealed that at 72 h following mild injury the axonal growth cones had a significantly higher colocalization of βIII tubulin and F-actin and higher percentage of collapsed morphology than those present following a very mild injury. Interestingly, cultures that received a second very mild stretch injury, 24 h after the first insult, had a further increased proportion of growth cone collapse and increased βIII tubulin and F-actin colocalization, compared with a single very mild injury at 72 h PI. In addition, our results demonstrated that microtubule stabilization of axons using brain penetrant Epothilone D (EpoD) (100 nM) resulted in a significant reduction in the number of fragmented axons following mild injury. Collectively, these results suggest that mild and very mild stretch injury to a very localized region of the cortical axon is able to trigger a degenerative response characterized by growth cone collapse and significant abnormal cytoskeletal rearrangement. Furthermore, repetitive very mild stretch injury significantly exacerbated this response. Results suggest that axonal degeneration following stretch injury involves destabilization of the microtubule cytoskeleton and hence treatment with EpoD reduced fragmentation. Together, these results contribute a better understanding of the pathogenesis of mild and repetitive TBI and highlight the therapeutic effect of microtubule targeted drugs on distal part of neurons using a compartmentalized culturing model

Real-time mass spectrometry monitoring of oak wood toasting: elucidating aroma development relevant to oak-aged wine quality

We introduce a real-time method to monitor the evolution of oak aromas during the oak toasting process. French and American oak wood boards were toasted in an oven at three different temperatures, while the process-gas was continuously transferred to the inlet of a proton-transfer-reaction time-of-flight mass spectrometer for online monitoring. Oak wood aroma compounds important for their sensory contribution to oak-aged wine were tentatively identified based on soft ionization and molecular mass. The time-intensity profiles revealed toasting process dynamics illustrating in real-time how different compounds evolve from the oak wood during toasting. Sufficient sensitivity was achieved to observe spikes in volatile concentrations related to cracking phenomena on the oak wood surface. The polysaccharide-derived compounds exhibited similar profiles; whilst for lignin-derived compounds eugenol formation differed from that of vanillin and guaiacol at lower toasting temperatures. Significant generation of oak lactone from precursors was evident at 225 (o)C. Statistical processing of the real-time aroma data showed similarities and differences between individual oak boards and oak wood sourced from the different origins. This study enriches our understanding of the oak toasting process and demonstrates a new analytical approach for research on wood volatiles

Photolithographic patterning of conducting polyaniline films via flash welding

In this work, two significant advances in photolithographic patterning of polyaniline (PANI) films are reported. Firstly, flash welding was enhanced through the use of polymeric substrates, enabling complete penetration of the welding of PANI films with thicknesses ranging from 5 to over 14 mu m, significantly thicker than reported previously. Masking of parts of the PANI films during flash welding enabled the formation of adjacent conducting and insulating regions as the welding changes the electrical properties of the film. Raman spectroscopy was used to determine the sharpness of these edges, and indicated that the interface between the flash welded and masked regions of the PANI films was typically less than 15 mu m wide. Secondly, using longpass filters, light with a wavelength less than 570 nm was found not to contribute to the welding process. This was confirmed by the use of a 635 nm laser diode for welding the PANI films. This novel approach enabled patterning of PANI films using a direct writing technique with a narrow wavelength light source

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids