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.
Antonia Lai, Salvatore Almaviva, Valeria Spizzichino, Lorella Addari, Antonio Palucci, Domenico Luciani, Roberto Viola, Sandro Mengali, Christophe A. Marquette, Bartlomiej Jankiewicz, and Luigi Pierno, "Selectivity evaluation of label-free detection of Bacillus spp spores using functionalized SERS substrates (Conference Presentation)," Proc. SPIE 9994, Optical Materials and Biomaterials in Security and Defence Systems Technology XIII, 999408 (Presented at SPIE Security + Defence: September 28, 2016; Published: 5 December 2016); https://doi.org/10.1117/12.2240925.5229530549001.
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