Microtable arrays for culture and isolation of cell colonies

Cell microarrays with culture sites composed of individually removable microstructures or micropallets have proven benefits for isolation of cells from a mixed population. The laser energy required to selectively remove these micropallets with attached cells from the array depends on the microstructure surface area in contact with the substrate. Laser energies sufficient to release micropallets greater than 100 μm resulted in loss of cell viability. A new three-dimensional culture site similar in appearance to a table was designed and fabricated using a simple process that relied on a differential sensitivity of two photoresists to UV-mediated photopolymerization. With this design, the larger culture area rests on four small supports to minimize the surface area in contact with the substrate. Microtables up to 250 × 250 μm were consistently released with single 10-μJ pulses to each of the four support structures. In contrast, microstructures with a 150 × 150-μm surface area in contact with the substrate could not be reliably released at pulse energies up to 212 μJ. Cassie–Baxter wetting is required to provide a barrier of air to localize and sequester cells to the culture sites. A second asset of the design was an increased retention of this air barrier under conditions of decreased surface tension and after prolonged culture of cells. The improved air retention was due to the hydrophobic cavity created beneath the table and above the substrate which entrapped air when an aqueous solution was added to the array. The microtables proved an efficient method for isolating colonies from the array with 100% of selected colonies competent to expand following release from the array.     

Contact printing of arrayed microstructures

A novel contact printing method utilizing a sacrificial layer of polyacrylic acid (PAA) was developed to selectively modify the upper surfaces of arrayed microstructures. The method was characterized by printing polystyrene onto SU-8 microstructures to create an improved substrate for a cell-based microarray platform. Experiments measuring cell growth on SU-8 arrays modified with polystyrene and fibronectin demonstrated improved growth of NIH 3T3 (93% vs. 38%), HeLa (97% vs. 77%), and HT1080 (76% vs. 20%) cells relative to that for the previously used coating method. In addition, use of the PAA sacrificial layer permitted the printing of functionalized polystyrene, carboxylate polystyrene nanospheres, and silica nanospheres onto the arrays in a facile manner. Finally, a high concentration of extracellular matrix materials (ECM), such as collagen (5 mg/mL) and gelatin (0.1%), was contact-printed onto the array structures using as little as 5 μL of the ECM reagent and without the formation of a continuous film bridge across the microstructures. Murine embryonic stem cells cultured on arrays printed with this gelatin hydrogel remained in an undifferentiated state indicating an adequate surface gelatin layer to maintain these cells over time.     

Automated data reduction for hydrogen/deuterium exchange experiments,enabled by high-resolution fourier transform ion cyclotron resonance mass spectrometry

Mass analysis of proteolytic fragment peptides following hydrogen/deuterium exchange offers a general measure of solvent accessibility/hydrogen bonding (and thus conformation) of solution-phase proteins and their complexes. The primary problem in such mass analyses is reliable and rapid assignment of mass spectral peaks to the correct charge state and degree of deuteration of each fragment peptide, in the presence of substantial overlap between isotopic distributions of target peptides, autolysis products, and other interferant species. Here, we show that at sufficiently high mass resolving power (m/Δm_50% ≥ 100,000), it becomes possible to resolve enough of those overlaps so that automated data reduction becomes possible, based on the actual elemental composition of each peptide without the need to deconvolve isotopic distributions. We demonstrate automated, rapid, reliable assignment of peptide masses from H/D exchange experiments, based on electrospray ionization FT-ICR mass spectra from H/D exchange of solution-phase myoglobin. Combined with previously demonstrated automated data acquisition for such experiments, the present data reduction algorithm enhances automation (and thus expands generality and applicability) for high-resolution mass spectrometry-based analysis of H/D exchange of solution-phase proteins.     

Fast quantitative detection of cocaine in beverages using nanoextractive electrospray ionization tandem mass spectrometry

Without any sample pretreatment, effervescent beverage fluids were manually sprayed into the primary ion plume created by using a nanoelectrospray ionization source for direct ionization, and the analyte ions of interest were guided into an ion trap mass spectrometer for tandem mass analysis. Functional ingredients (e.g., vitamins, taurine, and caffeine, etc.) and spiked impurity (e.g., cocaine) in various beverages, such as Red Bull energy drink, Coco-cola, and Pepsi samples were rapidly identified within 1.5 s. The limit of detection was found to be 7 ∼ 15 fg (S/N = 3) for cocaine in different samples using the characteristic fragment (m/z 150) observed in the MS³ experiments. Typical relative standard deviation and recovery of this method were 6.9% ∼ 8.6% and 104% ∼ 108% for direct analysis of three actual samples, showing that nanoextractive electrospray ionization tandem mass spectrometry is a useful technique for fast screening cocaine presence in beverages.     

Preparation and properties of PU/MCMMT nanocomposites

The cetyltrimethyl ammonium bromide (CTAB) was used as a swelling agent to be intercalated into the galleries of the montmorillonite (MMT) platelets to get the organic MMT (CMMT). Then 4,4′‐diphenylmethane diisocyanate (MDI) were grafted on CMMT by the reaction between hydroxyls in organic MMT platelets and MDI to synthesize the MDI modified CMMT (MCMMT). Polyurethane (PU)/MCMMT composites were prepared by situ polymerization. The MCMMT platelets dispersed in a PU matrix in nanometer scale. The dispersion and intercalation degree of the MCMMT platelets decreased with increase in the content of MCMMT. Under the same content of fillers, the tensile strength and tear strength of PU/MCMMT nanocomposites were higher than those of PU/organic MMT nanocomposites. The reinforcing effect of the MCMMT platelets to the PU was better than that of the organic MMT platelets. With increase in the content of MCMMT, the tensile strength and tear strength of the PU/MCMMT nanocomposites were increased, while the extent of the increase slowed down. Compared with those of PU, the thermal stability of PU/MCMMT nanocomposites was increased. Copyright © 2009 John Wiley & Sons, Ltd.     

Tunable and rapid crystallisation of phase pure Bi2MoO6 (koechlinite) and Bi2Mo3O12 via continuous hydrothermal synthesis

Herein, we report the rapid single step hydrothermal synthesis of phase pure Bi₂MoO₆ (koechlinite) and Bi₂Mo₃O₁₂, via a continuous hydrothermal flow synthesis (CHFS) reactor, which uses supercritical water of 375–450 °C at a pressure of 24.1 MPa as a crystallising medium. The product being obtained as highly crystalline nano-materials with high surface area. Simple variation in synthesis condition and appropriate solution stoichiometry were shown to be sufficient to select the phase of the product. The materials synthesised showed significant photcatalytic activity towards the decolourisation of methylene blue in comparison to a commercial gold standard photocatalyst.     

Benzopentalenonaphthalenones from the intramolecular capture of a merocyanine derived from a naphthopyran

Novel, highly coloured benzopentalenonaphthalenones result from a cascade process initiated by the thermally-induced ring-opening of diarylmethanol substituted 2H-naphtho[1,2-b]pyrans in the presence of acid.     

PXPd-catalyzed borylation of aniline derivatives

Addition of pinacolborane (HBO₂C₂Me₄) to 2-iodoaniline can be catalyzed using a number of palladium complexes, including [(t-Bu)₂PCl]₂PdCl₂ (PXPd), to give the corresponding boronate ester 2-H₂NC₆H₄(BO₂C₂Me₄) in excellent yields. The PXPd system could also be used in the catalyzed borylation of substituted anilines to give the corresponding aminoboronate esters in moderate to high yields.      

Asporyergosterol, a new steroid from an algicolous isolate of Aspergillus oryzae

Asporyergosterol (1), a new steroid with an E double bond between C-17 and C-20, was identified from the culture extracts of Aspergillus oryzae, an endophytic fungus isolated from the marine red alga Heterosiphonia japonica. Moreover, four known steroids including (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one (2), (22E,24R)-3beta-hydroxyergosta-5,8,22-trien-7-one (3), (22E,24R)-ergosta-7,22-dien-3beta,5alpha,6beta-triol (4), and (22E,24R)-5alpha,8alpha-epidioxyergosta-6,22-dien-3beta-ol (5) were isolated. Structures of these compounds were unambiguously established by spectroscopic techniques and by comparison with literature values. All the isolates exhibited low activity to modulate acetylcholinesterase (AChE).