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Desorption/ionization on porous silicon mass spectrometry (DIOS-MS) is shown to be a powerful technique for the sensing of low-molecular-weight compounds, including drugs and their metabolites. Surface modification of DIOS surfaces is required to increase analytical performance and ensure stability. However, common wet chemical modification techniques use fluorosilanes, which are less suitable for high-throughput manufacturing and analytical repeatability. Here, we report an alternative, rapid functionalization technique for DIOS surfaces using plasma polymerization (ppDIOS). We demonstrate the detection of drugs, metabolites, pesticides, and doping agents, directly from biological matrices, with molecular confirmation performed using the fragmentation capabilities of a tandem MS instrument. Furthermore, the ppDIOS surfaces were found to be stable over a 162 day period with no loss of reproducibility and sensitivity. This alternative functionalization technique is cost-effective and amenable to upscaling, ensuring avenues for the high-throughput manufacture and detection of hundreds of analytes across various applications while still maintaining the gold-standard clinical technique using mass spectrometry.

Novel doping agents and doping strategies are continually entering the market, placing a burden on analytical methods to detect, adapt, and respond to subtle changes in the composition of biological samples. Therefore, there is a growing interest in rapid, adaptable, and ideally confirmatory analytical methods for the fight against doping. Nanostructured silicon (nano-Si)-based surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) can effectively address this need, allowing fast and sensitive detection of prohibited compounds used in sport doping. Here, we demonstrate the detection of growth hormone peptides, anabolic-androgenic steroids, and narcotics at low concentrations directly from biological matrices. Molecular confirmation was performed using the fragmentation data of the structures, obtained with the tandem mass spectrometry capabilities of the SALDI instrument. The obtained data were in excellent agreement with those obtained using leading triple quadrupole liquid chromatography-mass spectrometry instruments. Furthermore, nano-Si SALDI-MS has the capacity for high-throughput analysis of hundreds of biological samples, providing opportunities for real-time MS analysis at sporting events.

Indole derivatives are a structurally diverse group of compounds found in food, toxins, medicines, and produced by commensal microbiota. On contact with acidic stomach conditions, indoles undergo condensation to generate metabolites that vary in solubility, activity and toxicity as they move through the gut. Here, using halogenated ions, we map promising chemo-preventative indoles, i) 6-bromoisatin (6Br), ii) the mixed indole natural extract (NE) 6Br is found in, and iii) the highly insoluble metabolites formed in vivo using desorption/ionisation on porous silicon-mass spectrometry imaging (DIOS-MSI). The functionalised porous silicon architecture allowed insoluble metabolites to be detected that would otherwise evade most analytical platforms, providing direct evidence for identifying the therapeutic component, 6Br, from the mixed indole NE. As a therapeutic lead, 0.025 mg/g 6Br acts as a chemo-preventative compound in a 12 week genotoxic mouse model; at this dose 6Br significantly reduces epithelial cell proliferation, tumour precursors (aberrant crypt foci; ACF); and tumour numbers while having minimal effects on liver, blood biochemistry and weight parameters compared to controls. The same could not be said for the NE where 6Br originates, which significantly increased liver damage markers. DIOS-MSI revealed a large range of previously unknown insoluble metabolites that could contribute to reduced efficacy and increased toxicity.

Porous silicon microparticles (pSi MPs) functionalized with fluorescent dyes (lissamine and carboxy-5-fluorescein) and intrinsically luminescent pSi MPs were explored as novel fingerprint dusting powders. The versatility of luminescent pSi MPs is demonstrated through time-gated imaging of their long-lived (lifetime>28μs) near-IR emission, and mass spectrometry analysis of fingerprints dusted with luminescent pSi MPs to provide further information on exogenous small molecules present in latent fingerprints. Refereed/Peer-reviewed

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 micro-patterning.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. Refereed/Peer-reviewed

This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 24 month embargo from date of publication (Aug 2017) in accordance with the publisher’s archiving policy, In order to deal with the range of surfaces encountered in crime scenes and items associated with crimes, forensic fingermark examiners must have access to a range of latent mark enhancement techniques, each compatible with a particular type of surface. Consequently, the development of techniques with universal or even wide-ranging surface compatibility would be valuable to law enforcement. Herein, we describe a one-step silver sputtering method for the enhancement of latent fingermarks on plastic, glass, paper and metal substrates. This method allows for the ridge pattern to be captured for human identification purposes and, more importantly, for the downstream mass spectrometric imaging of the fingermark in order to display the spatial distribution of common endogenous and exogenous substances such as illicit drugs

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. Refereed/Peer-reviewed

Desorption ionisation on porous silicon (DIOS) was used for the detection of nicotine from exhaled breath. This result represents proof-of-principle of the ability of DIOS to detect small molecular analytes in breath including biomarkers and illicit drugs. Refereed/Peer-reviewed

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. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 34: 627–640, 2015.

Rationale Nanostructure-based mass spectrometry imaging (MSI) is a promising technology for molecular imaging of small molecules, without the complex chemical background typically encountered in matrix-assisted molecular imaging approaches. Here, we have enhanced these surfaces with silver (Ag) to provide a second tier of MSI data from a single sample. Methods MSI data was acquired through the application of laser desorption/ionization mass spectrometry to biological samples imprinted onto desorption/ionization on silicon (DIOS) substrates. Following initial analysis, ultra-thin Ag layers were overlaid onto the followed by MSI analysis (Ag-DIOS MSI). This approach was first demonstrated for fingermark small molecules including environmental contaminants and sebum components. Subsequently, this bimodal method was translated to lipids and metabolites in fore-stomach sections from a 6-bromoisatin chemopreventative murine mouse model. Results DIOS MSI allowed mapping of common ions in fingermarks as well as 6-bromoisatin metabolites and lipids in murine fore-stomach. Furthermore, DIOS MSI was complemented by the Ag-DIOS MSI of Ag-adductable lipids such as wax esters in fingermarks and cholesterol in murine fore-stomach. Gastrointestinal acid condensation products of 6-bromoisatin, such as the 6,6'-dibromoindirubin mapped herein, are very challenging to isolate and characterize. By re-analyzing the same tissue imprints, this metabolite was readily detected by DIOS, placed in a tissue-specific spatial context, and subsequently overlaid with additional lipid distributions acquired using Ag-DIOS MSI. Conclusions The ability to place metabolite and lipid classes in a tissue-specific context makes this novel method suited to MSI analyses where the collection of additional information from the same sample maximises resource use, and also maximises the number of annotated small molecules, in particular for metabolites that are typically undetectable with traditional platforms. Copyright © 2017 John Wiley & Sons, Ltd.

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. Refereed/Peer-reviewed

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.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is a high throughput analytical technique capable of detecting low molecular weight analytes, including illicit drugs, and with potential applications in forensic toxicology as well as athlete and workplace testing, particularly for biological fluids (oral fluids, urine and blood). However, successful detection of illicit drugs using SALDI-MS often requires extraction steps to reduce the inherent complexity of biological fluids. Here, we demonstrate an all-in-one extraction and analytical system consisting of hydrophobically functionalized porous silicon microparticles (pSi-MPs) for affinity SALDI-MS of prescription and illicit drugs. This novel approach allows for the analysis of drugs from multiple biological fluids without sample preparation protocols. The effect of pSi-MP size, pore diameter, pore depth and functionalization on analytical performance is investigated. pSi-MPs were optimized for the rapid and high sensitivity detection of methadone, cocaine and 3,4-methylenedioxymethamphetamine (MDMA). This optimized system allowed extraction and detection of methadone from spiked saliva and clinical urine samples. Furthermore, by detecting oxycodone in additional clinical saliva and plasma samples, we were able to demonstrate the versatility of the pSi-MP SALDI-MS technique. Refereed/Peer-reviewed

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. Refereed/Peer-reviewed

Here, we report a novel surface modification for porous silicon (pSi). Hydroxyl-terminated pSi surfaces are modified with a hydrosilane via Si–H activation using the Lewis acid catalyst tris(pentafluorophenyl) borane. This surface reaction is fast and efficient at room temperature, and leads to a surface stabilised against hydrolytic attack in aqueous media. The resulting surface shows promise as a substrate for surface-assisted laser desorption/ionisation mass spectrometry Refereed/Peer-reviewed

Desorption ionisation on porous silicon mass spectrometry imaging (DIOS-MSI) was used on fingerprints to map the distribution of exogenous and endogenous molecules present in sweat. Our attention was focused on the proof-of-principle to detect illicit drugs and their metabolites to exemplify the technique's potential in the area of forensic and workplace testing. Refereed/Peer-reviewed

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence (http://creativecommons.org/licenses/by/3.0/), Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is ideally suited for the highthroughput analysis of small molecules in bodily fluids (e.g. saliva, urine, and blood plasma). A key application for this technique is the testing of drug consumption in the context of workplace, roadside, athlete sports and anti-addictive drug compliance. Here, we show that vertically-aligned ordered silicon nanopillar (SiNP) arrays fabricated using nanosphere lithography followed by metal-assisted chemical etching (MACE) are suitable substrates for the SALDI-MS detection of methadone and small peptides. Porosity, length and diameter are fabrication parameters that we have explored here in order to optimize analytical performance. We demonstrate the quantitative analysis of methadone in MilliQ water down to 32 ng mL 1. Finally, the capability of SiNP arrays to facilitate the detection of methadone in clinical samples is also demonstrated.

Surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) is an emerging technique used for the detection of small molecules (o700 Da) such as illicit drugs. In recent times, this techniquehas 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 massspectrometry (NIMS) and nanostructured laser desorption ionization (NALDI™) are compared for the detection of four drug classes, amphetamines, benzodiazepines, opiates and tropane alkaloids. We focusin our comparison on structural and chemical characteristics, as well as analytical performance and longevity. Refereed/Peer-reviewed

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.

International audience; 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.

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. Refereed/Peer-reviewed

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