Taryn Guinan

Surface-assisted laser desorption ionization mass spectrometry techniques for application in forensics

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is an excellent analytical technique for the rapid and sensitive analysis of macromolecules (>700 Da), such as peptides, proteins, nucleic acids, and synthetic polymers. However, the detection of smaller organic molecules with masses below 700 Da using MALDI-MS is challenging due to the appearance of matrix adducts and matrix fragment peaks in the same spectral range. Recently, nanostructured substrates have been developed that facilitate matrix-free laser desorption ionization (LDI), contributing to an emerging analytical paradigm referred to as surface-assisted laser desorption ionization (SALDI) MS. Since SALDI enables the detection of small organic molecules, it is rapidly growing in popularity, including in the field of forensics. At the same time, SALDI also holds significant potential as a high throughput analytical tool in roadside, work place and athlete drug testing. In this review, we discuss recent advances in SALDI techniques such as desorption ionization on porous silicon (DIOS), nano-initiator mass spectrometry (NIMS) and nano assisted laser desorption ionization (NALDI™) and compare their strengths and weaknesses with particular focus on forensic applications. These include the detection of illicit drug molecules and their metabolites in biological matrices and small molecule detection from forensic samples including banknotes and fingerprints. Finally, the review highlights recent advances in mass spectrometry imaging (MSI) using SALDI techniques.

Rapid detection of illicit drugs in neat saliva using desorption/ionization on porous silicon

The ability to detect illicit drugs directly in oral fluids is of major interest for roadside, workplace and athlete drug testing. For example, roadside testing for popular drugs of abuse is being rolled out by law enforcement agencies following the introduction of legislation in several countries all over the world. This paper reports on the direct analysis of methamphetamine, cocaine and 3,4-methylenedioxymethamphetamine in oral fluids using a hydrophobic porous silicon array as a combined drug extraction and concentration medium. Analysis by laser desorption/ionization time-of-flight mass spectrometry (MS) identified these drugs with a sensitivity in line with the suggested confirmatory cut-off concentrations, and 300 times faster. In addition, MS imaging demonstrated good spot-to-spot reproducibility of the signal. Our analytical approach is compatible with multiplexing and is therefore suitable for high-throughput analysis of samples obtained from drug testing in the field. Furthermore, the application of this analytical technology is not limited to illicit drugs or oral fluids. Indeed, we believe that this platform technology could be applied to the high-throughput analysis of diverse metabolites in body fluids.

Mass spectrometry imaging on porous silicon: investigating the distribution of bioactives in marine mollusc tissues

Desorption/ionization on porous silicon-mass spectrometry (DIOS-MS) is an attractive alternative to conventional matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of low molecular weight compounds. Porous silicon (pSi) chips are also suitable as support for mass spectrometry imaging (MSI). Here, we report an implementation of DIOS-MSI using the biosynthetic organs of a marine mollusc for proof of principle. The tissue section is stamped onto a fluorocarbon-functionalized pSi chip, which extracts and traps small hydrophobic molecules from the tissue under retention of their relative spatial distribution. The section is subsequently removed and the chip is imaged without any remaining tissue. We apply this novel tissue contact printing approach to investigate the distribution of biologically active brominated precursors to Tyrian purple in the hypobranchial gland of the marine mollusc, Dicathais orbita, using DIOS-MSI. The tissue contact printing is also compatible with other types of desorption/ionization surfaces, such as nanoassisted laser desorption/ionization (NALDI) targets.

Dense arrays of uniform submicron pores in silicon and their applications.

We report a versatile particle-based route to dense arrays of parallel submicron pores with high aspect ratio in silicon and explore the application of these arrays in sensors, optics, and polymer micropatterning. Polystyrene (PS) spheres are convectively assembled on gold-coated silicon wafers and sputter-etched, resulting in well-defined gold disc arrays with excellent long-range order. The gold discs act as catalysts in metal-assisted chemical etching, yielding uniform pores with straight walls, flat bottoms, and high aspect ratio. The resulting pore arrays can be used as robust antireflective surfaces, in biosensing applications, and as templates for polymer replica molding.

Comparison of the performance of different silicon-based SALDI substrates for illicit drug detection

Surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) is an emerging technique used for the detection of small molecules (<700 Da) such as illicit drugs. In recent times, this technique has been employed for the detection of illicit drugs in various body fluids including saliva. Three common SALDI techniques, desorption ionization on porous silicon (DIOS), nanostructure-initiator mass spectrometry (NIMS) and nanostructured laser desorption ionization (NALDI(™)) are compared for the detection of four drug classes, amphetamines, benzodiazepines, opiates and tropane alkaloids. We focus in our comparison on structural and chemical characteristics, as well as analytical performance and longevity.

Silver Coating for High-Mass-Accuracy Imaging Mass Spectrometry of Fingerprints on Nanostructured Silicon.

Nanostructure imaging mass spectrometry (NIMS) using porous silicon (pSi) is a key technique for molecular imaging of exogenous and endogenous low molecular weight compounds from fingerprints. However, high-mass-accuracy NIMS can be difficult to achieve as time-of-flight (ToF) mass analyzers, which dominate the field, cannot sufficiently compensate for shifts in measured m/z values. Here, we show internal recalibration using a thin layer of silver (Ag) sputter-coated onto functionalized pSi substrates. NIMS peaks for several previously reported fingerprint components were selected and mass accuracy was compared to theoretical values. Mass accuracy was improved by more than an order of magnitude in several cases. This straightforward method should form part of the standard guidelines for NIMS studies for spatial characterization of small molecules.

Interaction of Antibiotics with Lipid Vesicles on Thin Film Porous Silicon Using Reflectance Interferometric Fourier Transform Spectroscopy

The ability to observe interactions of drugs with cell membranes is an important area in pharmaceutical research. However, these processes are often difficult to understand due to the dynamic nature of cell membranes. Therefore, artificial systems composed of lipids have been used to study membrane properties and their interaction with drugs. Here, lipid vesicle adsorption, rupture, and formation of planar lipid bilayers induced by various antibiotics (surfactin, azithromycin, gramicidin, melittin and ciprofloxacin) and the detergent dodecyl-b-D-thiomaltoside (DOTM) was studied using reflective interferometric Fourier transform spectroscopy (RIFTS) on an oxidized porous silicon (pSi) surface as a transducer. The pSi transducer surfaces are prepared as thin films of 3 μm thickness with pore dimensions of a few nanometers in diameter by electrochemical etching of crystalline silicon followed by passivation with a thermal oxide layer. Furthermore, the sensitivity of RIFTS was investigated using three different concentrations of surfactin. Complementary techniques including atomic force microscopy, fluorescence recovery after photobleaching, and fluorescence microscopy were used to validate the RIFTS-based method and confirm adsorption and consequent rupture of vesicles to form a phospholipid bilayer upon the addition of antibiotics. The method provides a sensitive and real-time approach to monitor the antibiotic-induced transition of lipid vesicles to phospholipid bilayers.

A molecular probe for the detection of polar lipids in live cells

Lipids have an important role in many aspects of cell biology, including membrane architecture/compartment formation, intracellular traffic, signalling, hormone regulation, inflammation, energy storage and metabolism. Lipid biology is therefore integrally involved in major human diseases, including metabolic disorders, neurodegenerative diseases, obesity, heart disease, immune disorders and cancers, which commonly display altered lipid transport and metabolism. However, the investigation of these important cellular processes has been limited by the availability of specific tools to visualise lipids in live cells. Here we describe the potential for ReZolve-L1™ to localise to intracellular compartments containing polar lipids, such as for example sphingomyelin and phosphatidylethanolamine. In live Drosophila fat body tissue from third instar larvae, ReZolve-L1™ interacted mainly with lipid droplets, including the core region of these organelles. The presence of polar lipids in the core of these lipid droplets was confirmed by Raman mapping and while this was consistent with the distribution of ReZolve-L1™ it did not exclude that the molecular probe might be detecting other lipid species. In response to complete starvation conditions, ReZolve-L1™ was detected mainly in Atg8-GFP autophagic compartments, and showed reduced staining in the lipid droplets of fat body cells. The induction of autophagy by Tor inhibition also increased ReZolve-L1™ detection in autophagic compartments, whereas Atg9 knock down impaired autophagosome formation and altered the distribution of ReZolve-L1™. Finally, during Drosophila metamorphosis fat body tissues showed increased ReZolve-L1™ staining in autophagic compartments at two hours post puparium formation, when compared to earlier developmental time points. We concluded that ReZolve-L1™ is a new live cell imaging tool, which can be used as an imaging reagent for the detection of polar lipids in different intracellular compartments.

Fabrication of Nanostructured Mesoporous Germanium for Application in Laser Desorption Ionization Mass Spectrometry

Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is a high-throughput analytical technique ideally suited for small-molecule detection from different bodily fluids (e.g., saliva, urine, and blood plasma). Many SALDI-MS substrates require complex fabrication processes and further surface modifications. Furthermore, some substrates show instability upon exposure to ambient conditions and need to be kept under special inert conditions. We have successfully optimized mesoporous germanium (meso-pGe) using bipolar electrochemical etching and efficiently applied meso-pGe as a SALDI-MS substrate for the detection of illicit drugs such as in the context of workplace, roadside, and antiaddictive drug compliance. Argon plasma treatment improved the meso-pGe efficiency as a SALDI-MS substrate and eliminated the need for surface functionalization. The resulting substrate showed a precise surface geometry tuning by altering the etching parameters, and an outstanding performance for illicit drug detection with a limit of detection in Milli-Q water of 1.7 ng/mL and in spiked saliva as low as 5.3 ng/mL for cocaine. The meso-pGe substrate had a demonstrated stability over 56 days stored in ambient conditions. This proof-of-principle study demonstrates that meso-pGe can be reproducibly fabricated and applied as an analytical SALDI-MS substrate which opens the door for further analytical and forensic high-throughput applications.

Mass spectrometry imaging reveals new biological roles for choline esters and Tyrian purple precursors in muricid molluscs

Despite significant advances in chemical ecology, the biodistribution, temporal changes and ecological function of most marine secondary metabolites remain unknown. One such example is the association between choline esters and Tyrian purple precursors in muricid molluscs. Mass spectrometry imaging (MSI) on nano-structured surfaces has emerged as a sophisticated platform for spatial analysis of low molecular mass metabolites in heterogeneous tissues, ideal for low abundant secondary metabolites. Here we applied desorption-ionisation on porous silicon (DIOS) to examine in situ changes in biodistribution over the reproductive cycle. DIOS-MSI showed muscle-relaxing choline ester murexine to co-localise with tyrindoxyl sulfate in the biosynthetic hypobranchial glands. But during egg-laying, murexine was transferred to the capsule gland, and then to the egg capsules, where chemical ripening resulted in Tyrian purple formation. Murexine was found to tranquilise the larvae and may relax the reproductive tract. This study shows that DIOS-MSI is a powerful tool that can provide new insights into marine chemo-ecology.