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Several bacteria evolve in spore if the environmental conditions get to adverse e.g. for nutrient deprivation. The bacteria of genus Bacillus are Gram-positive aerobic bacteria and they are able to produce endospores (physiological inactive and resistant form), usually dispersed as aerosols. Endospores can survive for long time, until the conditions get back favourable. The genera Bacillus include pathogens, as Bacillus anthracis, used in the past as biological weapon. Prompt, accurate and sensitive detection is crucial for its control as pathogens or bioterrorism attacks. In case of the contamination with spores of B. anthracis, the time is essential to assure success in rescue. So, in this context, the early and fast analytical techniques, that need no or negligible sample preparation, is strongly required. Raman spectroscopy, and in particular Surface Enhanced Raman Spectroscopy (SERS), that can amplify nonlinearly the inherently weak Raman signal by several orders of magnitude, have become recognized and versatile analytical techniques also in microorganisms detection. These techniques can be used as sensitive tools for the detection and classification of biological threats, they can provide the chemical fingerprint of samples without complex and time-consuming pre-treatment samples preparation. Furthermore the development of in-field portable compact Raman platforms allows for using SERS for routine analysis. In the framework of the RAMBO (Rapid Air particle Monitoring against BiOlogical threats) project the feasibility of the SERS technique for the rapid identification and classification of few units of Bacillus spp (B. atrophaeus and B. thuringiensis) spores was investigated. B. atrophaeus and B. thuringiensis are harmless but genetically similar to the deadly B. anthracis. The RAMBO project purpose is the development of an advanced sensor with high performances, capable of detecting few spores or bacilli, with high selectivity and reliability, by means of two sensing techniques: SERS for early warning of bioagents dispersed in air or in water, and Polymerase Chain Reaction (PCR) technique for final recognition and validation. SERS and PCR will work in a microfluidic chip. In order to bind selectively the endospores, specific peptide receptors for B. thuringiensis have been selected to functionalize SERS substrates. To characterize the substrates, with and without spores to assess the effective immobilization of target, microscopy inspections, by optical microscope and Scanning Electron Microscope (SEM), were also carried out. The results show up the poor selectively of these peptides for B. thuringiensis, used as target, compared with the non specific Bacillus control. The performance of the system seems to be quite similar for both of them: the data processing by Principal Component Analysis and the following clustering analysis suggest the presence of indistinct answers for any bound endospore on the surface, and this is confirmed by microscope inspection. It could decrease the discrimination power of the sensor. Despite of such poor receptor selectivity, the SERS spectra of B. thuringiensis endospores show characteristic signals that can be related to DNA fragments or, much more probably, to the peptidoglycan (component of the external coat). This spectral feature could be used to detect the presence of B. thuringiensis endospores.

In the framework of RAMBO (Rapid-Air Monitoring particle against biological threats) project of the European Defense Agency (EDA), the feasibility of an unattended Surface Enhanced Raman Spectroscopy (SERS) sensor for biological threats detection was investigated. Its main goal concern Bacillus anthrax detection, both as vegetative cells and endospores. However since such bacteria are classified in Risk Group 3 (very dangerous microorganism), Bacillus thuringiensis and Bacillus atrophaeus were used as simulants. In order to bind selectively the target bacilli, Phages properly selected were immobilized on an active commercially available SERS substrate (functionalization). The Phages are a type of virus that infect selectively, by means of receptors, specific bacteria. Moreover they can resist on water or air environments without losing their binding capabilities. The sensing surface was characterized by standard micro-Raman equipments to assess the background Raman features. The Raman measurements have been carried out from 10X to 100X of magnification to differentiate between average and local features. Moreover the fast response was acquired by limiting the measure time at less than 1 minute. Samples of vegetative cells and endospores of Bacilli were randomly dispersed on the functionalized SERS substrates. The results obtained are promising: samples with and without bacilli could be distinguished one from the other. This is a step toward the use of SERS as an effective and fast technique for early warning of biological threats.

In the last decades there have been several terroristic attacks with improvised explosive devices (IED) that have raised the need for new instrumentation, for homeland security applications, to obtain a reliable and effective fight against terrorism. Public transportation has been around for about 150 years, but terroristic attacks against buses, trains, subways, etc., is a relatively recent phenomenon [1]. Since 1970, transportation has been an increasingly attractive target for terrorists. Most of the attacks to transport infrastructures take place in countries where public transportation is the primary way to move. Terrorists prefer to execute a smaller-scale attack with certainty of success rather than a complex and demanding operation to cause massive death and destruction. [1]. Many commonly available materials, such as fertilizer, gunpowder, and hydrogen peroxide, can be used as explosives and other materials, such as nails, glass, or metal fragments, can be used to increase the amount of shrapnel propelled by the explosion. The majority of substances that are classified as chemical explosives generally contain oxygen, nitrogen and oxidable elements such as carbon and hydrogen [2]. The most common functional group in military explosives is NO2. That functionality can be attached to oxygen (ONO2) in the nitrate esters (PETN), to carbon (C-NO2) in the nitroarenes (TNT) and nitroalkanes (Nitromethane), and to nitrogen (N-NO2) as in the nitramines (RDX). Some organic peroxides, such as TATP and HMTD, are popular amongst terrorists because they are powerful initiators that can be easily prepared from easily available ingredients. Azides are also powerful primary explosives commonly used as initiators (commercial detonators) in civilian and military operations, therefore they could be potentially used by terrorists as initiators for IEDs. © 2014 SPIE.

In this work we report the results of RS measurements on some common military explosives and some of the most common explosives precursors deposited on clothing fabrics, both synthetic and natural, such as polyester, leather and denim cotton at concentration comparable to those obtained from a single fingerprint. RS Spectra were obtained using an integrated portable Raman system equipped with an optical microscope, focusing the light of a solid state GaAlAs laser emitting at 785 nm. A maximum exposure time of 10 s was used, focusing the beam in a 45 μm diameter spot on the sample. The substances were deposited starting from commercial solutions with a Micropipetting Nano-Plotter, ideal for generating high-quality spots by non-contact dispensing of sub-nanoliter volumes of liquids, in order to simulate a homogeneous stain on the fabric surface. Images acquired with a Confocal Laser Scanning Microscope provided further details of the deposition process showing single particles of micrometric volume trapped or deposited on the underlying tissues. The spectral features of each substance was clearly identified and discriminated from those belonging to the substrate fabric or from the surrounding fluorescence. Our results show that the application of RS using a microscope-based apparatus can provide interpretable Raman spectra in a fast, in-situ analysis, directly from explosive particles of some μm3 as the ones that it could be found in a single fingerprint, despite the contribution of the substrate, leaving the sample completely unaltered for further, more specific and propaedeutic laboratory analysis. The same approach can be envisaged for the detection of other illicit substances like drugs. © 2013 SPIE.

At the Diagnostic and Metrology Laboratory (ENEA) is under development a stand-off apparatus for trace detection of explosive compounds. The system is based on the Raman technique due to the high discrimination capabilities. All the optoelectronics components of the apparatus have been carefully designed as well as their optical matching. The main goal will be to detect low trace components within the respect of the eye safe regulation.

Surface Enhanced Raman Spectroscopy measurements on some common military explosives were performed with a table-top micro-Raman system integrated with a Serstech R785 minispectrometer. Serstech R785 is a miniaturised spectrometer suitable for Raman and NIR measurements. Integration of R785 in our table-top system aims to the realization of a portable SERS detector, able to perform in-situ measurements. SERS Spectra were obtained exciting the substance of interest with a 785 nm diode-laser, while these substances were deposited starting from commercial solutions on commercial SERS substrates, to improve the detection sensitivity. The amount of the sampled substance was determined through the analysis of images of the substrate covered with the residue of explosive. In fact, once the solvent is completely evaporated, the residue of explosive was observed to be uniformly distributed on the substrate surface. Images acquired with a Scanning Electron Microscope provided further details of the deposition process showing that a fraction of the active SERS sites are completely covered with the analyte while other sites appear to be empty; from the analysis of the images the sampled quantity was estimated to be about 200 pg. The main Raman features of each substance were clearly identified, the spectral resolution was sufficiently high to clearly distinguish spectra belonging to different substances.

Among insulating materials containing point defects, Lithium Fluoride, LiF, is a radiation sensitive material well known in dosimetry and as active medium in optically pumped optolectronic devices. Primary and aggregate electronic defects, known as colour centres (CCs), can be efficiently produced in LiF by low-penetrating radiation. A novel imaging detector for soft X-ray microscopy, based on photoluminescence from laser active CCs, is currently under development. The continuous shrinking dimensions of photonic devices has prompted us to use thin LiF films, directly grown by thermal evaporation on different substrates, as recording media in Extreme Ultra-Violet contact-lithography experiments for the fabrication of permanent, regular, light-emitting microstructures, produced with high spatial resolution on large areas in short exposure times. The experiments were performed by using geometrical masks in an excimer-pumped laser-plasma source and the samples analyzed by confocal laser scanning microscopy. Strong visible photoluminescence at room temperature was measured from very thin surface layers. A preliminary comparison between the optical response of CCs in thin LiF films grown on glass and silicon substrates with respect to LiF crystals was performed. The polycrystalline LiF films show a higher sensitivity, which is discussed taking into account light confinement effect in the investigated planar structures.