Rachel Davida Lowe

Calcium(II) selectively induces α-synuclein annular oligomers via interaction with the C-terminal domain

α‐Synuclein filaments are the major component of intracytoplasmic inclusion bodies characteristic of Parkinsons disease and related disorders. The process of α‐synuclein filament formation proceeds via intermediate or protofibrillar species, each of which may be cytotoxic. Because high levels of calcium(II) and other metal ions may play a role in disease pathogenesis, we investigated the influence of calcium and other metals on α‐synuclein speciation. Here we report that calcium(II) and cobalt(II) selectively induce the rapid formation of discrete annular α‐synuclein oligomeric species. We used atomic force microscopy to monitor the aggregation state of α‐synuclein after 1 d at 4°C in the presence of a range of metal ions compared with the filament formation pathway in the absence of metal ions. Three classes of effect were observed with different groups of metal ions: (1) Copper(II), iron(III), and nickel(II) yielded 0.8–4 nm spherical particles, similar to α‐synuclein incubated without metal ions; (2) magnesium(II), cadmium(II), and zinc(II) gave larger, 5–8 nm spherical oligomers; and, (3) cobalt(II) and calcium(II) gave frequent annular oligomers, 70–90 nm in diameter with calcium(II) and 22–30 nm in diameter with cobalt(II). In the absence of metal ions, annular oligomers ranging 45–90 nm in diameter were observed after 10 d incubation, short branched structures appeared after a further 3 wk and extended filaments after 2–3 mo. Previous studies have shown that α‐synuclein calcium binding is mediated by the acidic C terminus. We found that truncated α‐synuclein (1–125), lacking the C‐terminal 15 amino acids, did not form annular oligomers upon calcium addition, indicating the involvement of the calcium‐binding domain.

Annular α‐synuclein species from purified multiple system atrophy inclusions

Oligodendroglial cytoplasmic inclusions composed of α‐synuclein filamentous aggregates are the pathological hallmark of multiple system atrophy (MSA). We found that cortical tissue from MSA cases contains increased detergent‐resistant high‐molecular‐weight α‐synuclein species. To analyse these species, we immunopurified α‐synuclein aggregates from pathological samples and examined their ultrastructures using scanning electron and atomic force microscopies. Purified aggregates consisted of bundles of filaments. After treatment with 1% sarcosine or 2% 3‐[(3‐cholamidopropyl) dimethyl‐ammonio]‐1‐propanesulfonate (CHAPS) detergents, we observed frequent 30–50 nm annular particles, probably released from pathological aggregates due to the dissociation of filaments by the detergents. Antibody recognition imaging using a specific anti‐α‐synuclein antibody confirmed that the annular structures were positive for α‐synuclein. In contrast to pathological α‐synuclein, detergent treatment of recombinant α‐synuclein yielded only smaller, 10–18 nm spherical particles. Our results demonstrate that detergent treatment of pathological MSA α‐synuclein aggregates, but not recombinant α‐synuclein, yields discrete α‐synuclein‐positive species with annular morphologies. The ability of the pathological α‐synuclein to form annular aggregates may be an important factor contributing to the toxicity of the protein in disease that may have implications in designing therapeutic strategies aimed at detoxifying α‐synuclein aggregates.

Combined immunocapture and laser desorption/ionization mass spectrometry on porous silicon.

There is considerable interest in the highly parallelized mass spectrometry analysis of complex sample mixtures without any time-consuming prepurification. Porous silicon-based laser desorption/ionization mass spectrometry (pSi LDI-MS) is enabling technology for such analysis. Previous studies have focused on pSi surface functionalization to enhance sensitivity of detection and engineer surfaces for sample capture and enrichment in LDI-MS analysis. In this report, we build on this work by showing that surface functionalization of thin pSi films can be extended to the covalent immobilization of antibodies, producing a porous immunoaffinity surface. We demonstrate highly selective mass spectrometric detection of illicit drugs (benzodiazepines) on pSi films displaying antibenzodiazepine antibodies covalently immobilized via isocyanate chemistry. The effects of antibody immobilization conditions, antibody concentration, and surface blocking on LDI-MS performance and selectivity were studied. X-ray photoelectron spectroscopy (XPS) was instrumental in characterizing surface chemistry and optimizing LDI-MS performance. Overall, our approach is suitable for rapid and sensitive confirmatory analysis in forensic toxicology requiring only minimal sample volume and may be applied to other areas requiring small molecular analysis such as metabolomics and pharmacology.

Rapid drug detection in oral samples by porous silicon assisted laser desorption/ionization mass spectrometry

The demand for analysis of oral fluid for illicit drugs has arisen with the increased adoption of roadside testing, particularly in countries where changes in legislation allow random roadside testing of drivers for the presence of a palette of illicit drugs such as methamphetamine (MA), 3,4-methylenedioxymethamphetamine (MDMA) and Delta9-tetrahydrocannabinol (THC). Oral samples are currently tested for such drugs at the roadside using an immunoassay-based commercial test kit. Positive roadside tests are sent for confirmatory laboratory analysis, traditionally by means of gas chromatography/mass spectrometry (GC/MS). We present here an alternative rapid analysis technique, porous silicon assisted laser desorption/ionization time-of-flight mass spectrometry (pSi LDI-MS), for the high-throughput analysis of oral fluids. This technique alleviates the need for sample derivatization, requires only sub-microliter sample volumes and allows fast analysis (of the order of seconds). In this study, the application of the technique is demonstrated with real samples from actual roadside testing. The analysis of oral samples resulted in detection of MA and MDMA with no extraction and analysis of THC after ethyl acetate extraction. We propose that, subject to miniaturization of a suitable mass spectrometer, this technique is well suited to underpin the deployment of oral fluid testing in the clinic, workplace and on the roadside.

Lateral heterogeneities in supported bilayers from pure and mixed phosphatidylethanolamine demonstrating hydrogen bonding capacity.

The phase behavior and lateral organization of saturated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) bilayers were investigated using atomic force microscopy (AFM) and force-volume (FV) imaging for both pure and two component mixed layers. The results demonstrated the existence of unexpected segregated domains in pure PE membranes at temperatures well below the transition temperature (Tm) of the component phospholipid. These domains were of low mechanical stability and lacked the capacity for hydrogen bonding between lipid headgroups. Temperature dependent studies for different PC/PE ratios using AFM also demonstrated the mixing of these phospholipid bilayers to exhibit only a single gel to liquid transition temperature. Further work performed using FV imaging and chemically modified probes established that no lipid segregation exists at the PC/PE ratios investigated.

Monitoring EDTA and endogenous metabolite biomarkers from serum with mass spectrometry

We describe a quantitative method for the determination of ethylenediaminetetraacetic acid (EDTA) in human serum by gas chromatography mass spectrometry (GC-MS), liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS), and desorption ionisation on silicon mass spectrometry (DIOS-MS). In the initial stages of the analysis, endogenous metabolites (1-palmitoyl-sn-glycero-3-phosphocholine and 1-stearoyl-sn-glycero-3-phosphocholine) were readily observed in LC-ESI-MS and DIOS-MS however, direct analysis of the EDTA free acid had limited sensitivity. In order to improve EDTA detection we employed a straightforward esterification derivatization. The most successful derivatization procedure converted EDTA to its methyl ester and, since 13C isotopes of these reagents are readily available, internal standards could be easily generated for quantitative analysis. This approach provided a limit of detection of 0.5 and 0.1 μM for GC-MS and LC-ESI-MS, and offers a viable method for the EDTA detection.

Biomedical Uses of Porous Silicon

The versatility of porous silicon (pSi), due to the myriad of possible structures, ease of chemical modification and inherent biocompatibility, has resulted in it being readily tailored for numerous biomedical applications. Commonly prepared via the anodisation of crystalline silicon wafers in HF electrolyte, pSi can be produced as films, microparticles, nanoparticles and free-standing membranes. The combination of both its unique physical properties and the incorporation of stable surface functionalities have been fundamental to its performance. Through an immense number of modification techniques, numerous species from antibodies to polymers can be integrated into pSi structures. This adaptability has produced materials with an increased half-life both in vitro and in vivo and enabled the development of both targeted detection platforms and local delivery of therapeutic payloads. As a result, modified pSi has been readily applied to a range of biomedical applications including molecular detection, drug delivery, cancer therapy, imaging and tissue engineering.

A fluorescence based method for the quantification of surface functional groups in closed micro- and nanofluidic channels

Surface analysis is critical for the validation of microfluidic surface modifications for biology, chemistry, and physics applications. However, until now quantitative analytical methods have mostly been focused on open surfaces. Here, we present a new fluorescence imaging method to directly measure the surface coverage of functional groups inside assembled microchannels over a wide dynamic range. A key advance of our work is the elimination of self-quenching to obtain a linear signal even with a high density of functional groups. This method is applied to image the density and monitor the stability of vapor deposited silane layers in bonded silicon/glass micro- and nanochannels.

The influence of pore size and oxidizing agent on the energetic properties of porous silicon

The explosive abilities of porous silicon (pSi) provide an alternative to existing carbon based explosives, in addition to the possibilities of explosions on a nanoscale. Here, an investigation into these explosive properties is conducted, by introducing an oxidiser onto freshly etched pSi with varying pore sizes. Explosions are triggered via the application of an electric spark. Light output and spectral data are collected to characterize the exxplosion. The energy output is observed via Differential Scanning Calorimetry (DSC), and surface images collected using SEM and AFM.