Neil MacKinnon

Heteronuclear Micro-Helmholtz Coil Facilitates µm-Range Spatial and Sub-Hz Spectral Resolution NMR of nL-Volume Samples on Customisable Microfluidic Chips.

We present a completely revised generation of a modular micro-NMR detector, featuring an active sample volume of ∼ 100 nL, and an improvement of 87% in probe efficiency. The detector is capable of rapidly screening different samples using exchangeable, application-specific, MEMS-fabricated, microfluidic sample containers. In contrast to our previous design, the sample holder chips can be simply sealed with adhesive tape, with excellent adhesion due to the smooth surfaces surrounding the fluidic ports, and so withstand pressures of ∼2.5 bar, while simultaneously enabling high spectral resolution up to 0.62 Hz for H2O, due to its optimised geometry. We have additionally reworked the coil design and fabrication processes, replacing liquid photoresists by dry film stock, whose final thickness does not depend on accurate volume dispensing or precise levelling during curing. We further introduced mechanical alignment structures to avoid time-intensive optical alignment of the chip stacks during assembly, while we exchanged the laser-cut, PMMA spacers by diced glass spacers, which are not susceptible to melting during cutting. Doing so led to an overall simplification of the entire fabrication chain, while simultaneously increasing the yield, due to an improved uniformity of thickness of the individual layers, and in addition, due to more accurate vertical positioning of the wirebonded coils, now delimited by a post base plateau. We demonstrate the capability of the design by acquiring a 1H spectrum of ∼ 11 nmol sucrose dissolved in D2O, where we achieved a linewidth of 1.25 Hz for the TSP reference peak. Chemical shift imaging experiments were further recorded from voxel volumes of only ∼ 1.5nL, which corresponded to amounts of just 1.5 nmol per voxel for a 1 M concentration. To extend the micro-detector to other nuclei of interest, we have implemented a trap circuit, enabling heteronuclear spectroscopy, demonstrated by two 1H/13C 2D HSQC experiments.

3D Carbon Scaffolds for Neural Stem Cell Culture and Magnetic Resonance Imaging

3D glassy carbon structures with percolated macropores are obtained by pyrolysis of chemically synthesized cryogels featuring tunable porosity. These batch-fabricated structures are used as scaffolds for culturing neural stem cells (NSCs) and are characterized by magnetic resonance imaging (MRI). With the aid of MRI, the successful cultivation of NSCs on a glassy carbon surface and the precise 3D locations of these cell clusters within the opaque scaffold are demonstrated. MRI also yields pore morphology and porosity analyses, pre- and post-pyrolysis. This integrated approach yields a complete 3D dataset of the NSC network, which enables the visual inspection of the morphological details of individual cell clusters without disturbing them or destroying the scaffold. Reported experimental methodology is expected to have an impact on studies designed to understand the mechanism of neurodegenerative disease (ND) development, and can serve as a protocol for the culture of various other types of cells that display compatibility with glassy carbon surfaces.

Micro and nano patternable magnetic carbon

Carbon is conventionally not associated with magnetism, causing much of the discussion of its perspectives in nanotechnology to be centred on its electron-transport properties. Among the few existing examples of magnetic carbon production, none has found a direct route into scalable micro- and nanofabrication. Here we introduce a magnetic form of carbon whose precursor polymers can be lithographically patterned into micro- and nano-structures prior to pyrolysis. This unreactive and thermally robust material features a strong, room-temperature magnetism owing to a large number of unpaired electron spins with restricted mobility, which is achieved by controlling the progression of bond dissociation and formation during pyrolysis. The micro-manufacture of pyrolytic magnetic carbon, having (3.5±0.7)×1015 spins/mg, can immediately benefit a number of spintronic and magnetic-microelectromechanical system applications, and the fabrication of composite magnetic materials. The material could also complement the ma...

Advanced Microfluidic Assays for Caenorhabditis elegans

The in vivo analysis of a model organism, such as the nematode Caenorhabditis elegans, en‐ ables fundamental biomedical studies, including development, genetics, and neurobiolo‐ gy. In recent years, microfluidics technology has emerged as an attractive and enabling tool for the study of the multicellular organism. Advances in the application of microflui‐ dics to C. elegans assays facilitate the manipulation of nematodes in high-throughput for‐ mat and allow for the precise spatial and temporal control of their environment. In this chapter, we aim to illustrate the current microfluidic approaches for the investigation of behavior and neurobiology in C. elegans and discuss the trends of future development.

Novel selective TOCSY method enables NMR spectral elucidation of metabolomic mixtures

Complex mixture analysis is routinely encountered in NMR-based investigations. With the aim of component identification, spectral complexity may be addressed chromatographically or spectroscopically, the latter being favored to reduce sample handling requirements. An attractive experiment is selective total correlation spectroscopy (sel-TOCSY), which is capable of providing tremendous spectral simplification and thereby enhancing assignment capability. Unfortunately, isolating a well resolved resonance is increasingly difficult as the complexity of the mixture increases and the assumption of single spin system excitation is no longer robust. We present TOCSY optimized mixture elucidation (TOOMIXED), a technique capable of performing spectral assignment particularly in the case where the assumption of single spin system excitation is relaxed. Key to the technique is the collection of a series of 1D sel-TOCSY experiments as a function of the isotropic mixing time (τm), resulting in a series of resonance intensities indicative of the underlying molecular structure. By comparing these τm-dependent intensity patterns with a library of pre-determined component spectra, one is able to regain assignment capability. After consideration of the techniques robustness, we tested TOOMIXED firstly on a model mixture. As a benchmark we were able to assign a molecule with high confidence in the case of selectively exciting an isolated resonance. Assignment confidence was not compromised when performing TOOMIXED on a resonance known to contain multiple overlapping signals, and in the worst case the method suggested a follow-up sel-TOCSY experiment to confirm an ambiguous assignment. TOOMIXED was then demonstrated on two realistic samples (whisky and urine), where under our conditions an approximate limit of detection of 0.6mM was determined. Taking into account literature reports for the sel-TOCSY limit of detection, the technique should reach on the order of 10μM sensitivity. We anticipate this technique will be highly attractive to various analytical fields facing mixture analysis, including metabolomics, foodstuff analysis, pharmaceutical analysis, and forensics.

Novel ionic liquid - polymer composite and an approach for its patterning by conventional photolithography

A novel crosslinkable, conductive, highly transparent composite material based on a photoresist and an ionic liquid (the names of the composites are not announced here due to the current procedure of patenting) is presented. The composite possesses a good and stable ionic conductivity (up to 10 mS cm-1 at room temperature) over a wide frequency bandwidth (1 kHz - 1 MHz) and is optically transparent (transmission value of 90 % for a 170 μm thick film). In addition, an approach for the patterning of the composite material by conventional photolithography with a good spatial resolution (line width of 20 - 30 μm) is introduced. The unique properties of the material are utilized for time- and cost-saving direct manufacturing of electrically conductive, highly transparent microcomponents.

Micro-NMR elucidates altered metabolites in the Parkinson’s disease-related catp-6 genotype of Caenorhabditis elegans

AbstractIntroductionA severe form of Parkinson’s disease (PD) is the Kufor-Rakeb syndrome. Here mutations in the ATP13A2 (PARK9) gene lead to an early juvenile-onset Parkinsonism often accompanied by dementia. ATP13A2 encodes a lysosomal P-type ATPase. Its ortholog in Caenorhabditis elegans is the catp-6 gene where phenotypes with mutations in the alleles ok3473 and tm3190 show high mortality and low reproduction.ObjectivesSince PD is difficult to study in humans we wanted to investigate the potential to use C. elegans as model for the Kufor-Rakeb syndrome. As it is difficult to obtain enough catp-6 mutant worms for standard NMR metabolic profiling, we explored focused ultrasonication extraction and miniaturized NMR as techniques to overcome this limitation.Methods One- and two-dimensional NMR experiments (1H, JRES, TOCSY) were performed with a commercial high-resolution magic angle spinning (HR-MAS) probe (25 µL sample volume). Significant features were identified through analysis of variance (ANOVA, p < 0.05), volcano plots (p < 0.05, fold change >1.5), PCA, and PLS-DA.ResultsAssignment of statistically relevant peaks resulted in the identification of twenty altered metabolites. Previous studies on catp-6 mutants identified strong morphological and functional changes in their mitochondria. Our findings of altered TCA metabolites (fumarate, succinate), branched-chain amino acids (leucine, isoleucine and valine) and nucleotides (AMP, ATP and GTP), formate and hypoxanthine appear to support these findings. Highest fold changes (< −5) in wildtype relative to both catp-6 strains were found for GTP. Formic acid is known to inhibit the mitochondrial respiratory chain complex IV and high hypoxanthine in catp-6 indicates an increased nucleotide salvage pathway. Alterations in most of the remaining metabolites may be the result of the recently discovered activation of AMPK (AMP-activated protein kinase) and inhibition of mTOR (mechanistic target of rapamycin) pathways together with a catabolic response to recover energy production.ConclusionsIf the effect of the catp-6 mutation in C. elegans at the level of metabolites is correlated to the metabolic dysfunction in the human PARK9 ortholog, then it may be possible to uncover the molecular mechanism behind Parkinsonism and the Kufor-Rakeb syndrome.

Two-photon nanolithography for patterning ionic liquid-polymer composites

An important challenge in microand nanotechnology is direct patterning of functional structures. Moreover, the manufacture of 3D structures that have additional properties (e.g., conductivity and optical transparency) already in the directly patterned materials is highly desirable in a number of modern applications.1, 2 Such applications include sensors, solar cells, and electro-optical display devices (e.g., LCDs and electroluminescent display devices). Transparent conductive oxides (TCOs), such as indium tin oxide (ITO), antimony-doped tin oxide, and cadmium stannate (cadmium tin oxide), are commonly used materials for the functional 3D structures (e.g., as transparent electrodes). Of these, ITO is the most prominent on a commercial scale, and sputtered ITO films have conductivities of up to 104Scm 1. However, high substrate temperature conditions during coating of ITO, the complexity of fabrication methods, and the high cost of indium limit the range of potential ITO applications. Intrinsically conductive polymers, such as poly(3,4-ethylenedioxythiophene), also known as PEDOT, as well as electrically conductive polymer composites (ECPCs), have emerged over the past few years as alternatives to TCOs. In several ECPCs, a photoresist (an electronic insulator in its pure state, typically used for the direct manufacture of permanent patterns) is mixed with various conductive filler particles to significantly increase the conductivity of the polymerized material.3–8 Such composites allow the fabrication of low-cost devices with new properties. For instance, flexible plastic substrates with an electroconductive polymer layer can be produced through the use of continuous hopper or roller coating methods (rather than a batch process such as sputtering). The resulting organic electrodes Figure 1. High-resolution scanning electron microscope images of fabricated transparent and conductive 3D structures with (A) an aspect ratio of up to 1:10 and a resolution down to 150nm. The image in (B) shows that the closely spaced high-aspect-ratio nanostructures collapsed after they had been produced.

Advanced two-photon photolithography for patterning of transparent, electrically conductive ionic liquid-polymer nanostructures

A key challenge in micro- and nanotechnology is the direct patterning of functional structures. For example, it is highly desirable to possess the ability to create three-dimensional (3D), conductive, and optically transparent structures. Efforts in this direction have, to date, yielded less than optimal results since the polymer composites had low optical transparency over the visible range, were only slightly conductive, or incompatible with high resolution structuring. We have previously presented the novel cross-linkable, conductive, highly transparent composite material based on a photoresist (IP-L 780, OrmoComp, or SU-8) and the ionic liquid 1-butyl-3-methylimidazolium dicyanamide. Material patterning by conventional and two-photon photolithography has been demonstrated as proof-of-concept. Aiming to increase the resolution and to extend the spectrum of exciting applications we continued our research into identifying new ionic liquid - polymer composites. In this paper, we report the precise 3D single-step structuring of optically transparent and electrically conductive ionic liquid - polymer nanostructures with the highest spatial resolution (down to 150 nm) achieved to date. This was achieved via the development of novel cross-linkable composite based on the photoresist IP-G 780 and the ionic liquid 1-butyl-3-methylimidazolium dicyanamide. The successful combination of the developed material with the advanced direct laser writing technique enabled the time- and cost-saving direct manufacturing of transparent, electrically conductive components. We believe that the excellent characteristics of the structured material will open a wider range of exciting applications.

A microwave resonator integrated on a polymer microfluidic chip

We describe a novel stacked split-ring type microwave (MW) resonator that is integrated into a 10mm by 10mm sized microfluidic chip. A straightforward and scalable batch fabrication process renders the chip suitable for single-use applications. The resonator volume can be conveniently loaded with liquid sample via microfluidic channels patterned into the mid layer of the chip. The proposed MW resonator offers an alternative solution for compact in-field measurements, such as low-field magnetic resonance (MR) experiments requiring convenient sample exchange. A microstrip line was used to inductively couple MWs into the resonator. We characterised the proposed resonator topology by electromagnetic (EM) field simulations, a field perturbation method, as well as by return loss measurements. Electron paramagnetic resonance (EPR) spectra at X-band frequencies were recorded, revealing an electron-spin sensitivity of 3.7·10(11)spins·Hz(-1/2)G(-1) for a single EPR transition. Preliminary time-resolved EPR experiments on light-induced triplet states in pentacene were performed to estimate the MW conversion efficiency of the resonator.