Louis W. Bezuidenhout

Ultrathin layer chromatography on nanostructured thin films

Ultrathin layer chromatography (UTLC) is a relatively new variant of thin layer chromatography, with a 10mum thick monolithic silica sorbent layer that gives faster separations with lower limits of detection and reduced analyte and solvent volumes. We have produced UTLC plates with controllable nanostructure and thickness, and show that the layer separation characteristics depends on the film nanostructure. We also show that layers made with in-plane anisotropic nanostructures will exhibit a decoupling effect, where the analyte spots do not develop in the same direction as the solvent front movement. The added layer morphology and material selection adds a degree of freedom to UTLC, and may have applications in multi-dimensional TLC.

Engineered Anisotropic Microstructures for Ultrathin-Layer Chromatography

The strong dependence of separation behavior on ultrathin-layer chromatography (UTLC) stationary phase microstructure motivates continued UTLC plate design optimization efforts. We fabricated 4.6-5.3 mum thick normal phase silica UTLC stationary phases with several types of in-plane macropore anisotropies using the glancing angle deposition (GLAD) approach to engineering nanostructured thin films. The separation behaviors of two new media, isotropic vertical posts and anisotropic bladelike films, were compared to that of anisotropic chevron media. Channel-like structures within the anisotropic media introduced preferential mobile phase flow directions that could be exploited to give separation tracks diagonal to the development direction. Extraction of chromatograms from these angled tracks required the development of a new analytical approach that involved a commercial flatbed film scanner and custom numerical image analysis software. GLAD stationary phase performance was quantified using the Dimethyl Yellow dye separated from a lipophilic dye mixture over migration distances less than approximately 10 mm. The limits of detection were 10 +/- 4 ng for the vertical posts and 11 +/- 3 ng for the bladelike media. We obtained theoretical plate heights that varied with film microstructure between 12 and 28 mum. Unoptimized separation performance was comparable to that of other planar chromatography media. Macropore anisotropies engineered by GLAD may expand the capabilities of future UTLC stationary phases.

Miniaturized Planar Chromatography Using Office Peripherals

High-performance thin-layer chromatography is a separation technique commonly used to identify and quantify components in chemical mixtures. Sophisticated analytical tools are required to extract the full analytical power from this technique and especially for miniaturized planar chromatography its utility has not been harnessed. A new approach uses an elegant, simplified system assembled from ordinary consumer printers and scanners to perform separations on monolithic and nanostructured ultrathin-layer phases. This system is shown to outperform existing planar chromatographic tools for analysis on miniaturized plates. Analysis can be completed in a manner of minutes, running numerous samples in parallel at a reduced cost, with very low sample and reagent volumes, all using a familiar computer interface with common office peripherals.

Control of the principal refractive indices in biaxial metal oxide films

We provide both an extensive experimental characterization and a model for metal oxide, slanted columnar thin films fabricated using glancing angle deposition. The model is applicable to slanted posts of any type, deposited at a constant deposition angle, with variable azimuthal substrate rotation. The model is capable of predicting the column tilt, principal refractive indices, and in-plane birefringence under a single unified framework, given knowledge of common material parameters. This paper also establishes a number of additional important results, including the occurrence of negative in-plane birefringence and the occurrence of uniaxial films with nonzero columnar tilt.

Matrix‐free laser desorption/ionization mass spectrometry using silicon glancing angle deposition (GLAD) films

Glancing angle deposition (GLAD) was used to fabricate nanostructured silicon (Si) thin films with highly controlled morphology for use in laser desorption/ionization mass spectrometry (DIOS-MS). Peptides, drugs and metabolites in the mass range of 150-2500 Da were readily analyzed. The best performance was obtained with 500 nm thick films deposited at a deposition angle of 85 degrees . Low background mass spectra and attomole detection limits were observed with DIOS-MS for various peptides. Films used after three months of dry storage in ambient conditions produced mass spectra with negligible low-mass noise following a 15 min UV-ozone treatment. The performance of the Si GLAD films was as good as or better than that reported for electrochemically etched porous silicon and related materials, and was superior to matrix-assisted laser desorption/ionization (MALDI)-MS for analysis of mixtures of small molecules between 150-2500 Da in terms of background chemical noise, detection limits and spot-to-spot reproducibility. The spot-to-spot reproducibility of signal intensities (100 shots/spectrum) from 21 different Si GLAD film targets was +/-13% relative standard deviation (RSD). The single shot-to-shot reproducibility of signals on a single target was +/-19% RSD (n = 7), with no indication of sweet spots or mute spots.

Effect of interleukin-1β treatment on co-cultures of human meniscus cells and bone marrow mesenchymal stromal cells

BackgroundInterleukin-1β (IL-1β) is a major mediator of local inflammation present in injured joints. In this study, we aimed at comparing the effect of IL-1β on engineered tissues from MCs, BMSCs and co-cultured MCs and BMSCs.MethodsWe compared the effect of IL-1β in 3 groups: (1) MCs, (2) BMSCs and, (3) co-cultures of MCs and BMSCs. We selected 1 to 3 ratio of MCs to BMSCs for the co-cultures. Passage two (P2) human BMSCs were obtained from two donors. Human MCs were isolated from menisci of 4 donors. Mono-cultures of MCs and BMSCs, and co-cultures of MCs and BMSCs were cultured in chondrogenic medium with TGFβ3, as cell pellets for 14 days. Thereafter, pellets were cultured for 3 more days in same medium as before with or without IL-1β (500 pg/ml). Pellets were assessed histologically, biochemically and by RT-PCR for gene expression of aggrecan, sox9, MMP-1, collagens I and II. Statistics was performed using one-way ANOVA with Tukey’s post-tests.ResultsCo-cultured pellets were the most intensely stained with safranin O and collagen II. Co-cultured pellets had the highest expression of sox9, collagen I and II. IL-1β treatment slightly reduced the GAG/DNA of co-cultured pellets but still exceeded the sum of the GAG/DNA from the proportion of MCs and BMSCs in the co-cultured pellets. After IL-1β treatment, the expression of sox9, collagen I and II in co-cultured pellets was higher compared to their expression in pure pellets. IL-1β induced MMP-1 expression in mono-cultures of MCs but not significantly in mono-cultures of BMSCs or in co-cultured pellets. IL-1β induced MMP-13 expression in mono-cultured pellets of BMSCs and in co-cultured pellets.ConclusionsCo-cultures of MCs and BMSCs resulted in a synergistic production of cartilaginous matrix compared to mono-cultures of MCs and BMSCs. IL-1β did not abrogate the accumulated GAG matrix in co-cultures but mediated a decreased mRNA expression of aggrecan, collagen II and Sox9. These results strengthen the combinatorial use of primary MCs and BMSCs as a cell source for meniscus tissue engineering by demonstrating retention of fibrochondrogenic phenotype after exposure to IL-1β.

Fabrication of Porous Nanostructured Thin Films For Microfluidic and Microarray Applications

The functionality of microfluidic and microassay devices could be enhanced through further development of porous engineered microstructures. New structural elements fabricated with porous nanostructured thin films deposited by the glancing angle deposition (GLAD) technique have been developed for these devices, and are reviewed here. With the GLAD technique, engineered structures such as vertical posts, slanted posts, helices, and square spirals can be directly grown inside microfluidic channels. A high surface area channel (517cm 2 /cm 2 ) was made by depositing a silicon oxide porous film in glass microchannels (up to 4.5μm deep and 50μm wide). Similar channels were also fabricated by patterning channels in a photoresist-coated porous film. Self-sealed microchambers and channels were made by growing a SiO 2 porous film on 14μm high silicon mesas (2.5×2.5μm, 7×7μm, and 25×25μm) and lines (2.5 to 50μm wide). Devices with channels containing periodic arrays of Si pillars with controllable porosity and architecture were fabricated with only one lithography and deposition cycle. The entire device was made by depositing a single GLAD porous film with areas of different porosity defining the channels. The channels, 200μm wide and 10.5μm deep, contained helical pillars with pore sizes ranging from 100nm-2μm, while a more dense nanofibrous helical film made up the remainder of the device. Fluid flow activated by negative pressure was demonstrated in this device, using both a dye solution and a 50nm microsphere solution. Material selection is not limited to silicon or silicon oxide, but may include a wide range of semiconductors, insulators and metals. A GLAD film was used to separate a test dye solution, demonstrating its potential for use in thin layer chromatography. The reported elements are suitable for a range of applications, including – but not limited to – chromatography, nano-assays and capillary electrophoresis.