New post-processing method of preparing nanofibrous SERS substrates with a high density of silver nanoparticles E. S. Prikhozhdenko, a V. S. Atkin, b B. V. Parakhonskiy, acd I. A. Rybkin, a A. Lapanje, ae G. B. Sukhorukov, fg D. A. Gorin ag and A. M. Yashchenok * ah SERS enabling substrates with increased sensitivity, their manufacturing reproducibility and enhanced structural stability are highly demanded in SERS applications. Therefore our main aim was to elaborate a simple and inexpensive method of functionalization of electrospun chitosan nanofibers by using silver nanoparticles (AgNP). For that purpose we established a protocol where we were able to control the density of AgNP on the surfaces of nanofibers, and thus electromagnetic hotspots, simply by variation of Tollens' reagent. By webbing of such fibers we were able to prepare films that were enabling SERS either of solutes or macromolecular structures such as bacterial cells. Especially, to detect bacterial cells we established two approaches of immobilisation of cells within the films, which enabled their detection with enhancement of sensitivity by a factor of 10 5 and an average of 25% spot-to-spot variation. 1 Introduction Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical method, since it enables several orders of magnitude enhancement of Raman signal. Enhancement of the signal is generated by plasmonic nanoparticles that are in most cases made up of gold or silver. On the surface of such metal moieties within plasmonic nanoparticles the localized surface plasmon resonance of electrons is occurring upon light illumination. 1 Theoretically, since the enhancement factor can be as large as 10 12 , it gives possibilities to detect single molecules. 2 Because of these features SERS can be a very appropriate analytical method for use in biomedical applications, 3 such as measuring pH either in living cells or in surrounding milieu, 4 cancer target- ing, 5 glucose, 5 protein 6 as well as nucleic acids. 7 In such appli- cations robust and uniform SERS substrates are more convenient than aggregated nanoparticles although currently the latter are more frequently used. 8,9 So far, most studies have been focused on the creation of 1D and 2D SERS substrates. 10–12 For example, single-walled carbon nanotubes decorated either with silver or gold nanoparticles are an interesting 1D SERS multifunctional nanoprobe for detection and imaging of bio- logical samples. 13,14 Single polyaniline@Ag nanobers, silver nanowires decorated with gold nanoparticles and multilayered rod-like nanocapsules oriented by electric tweezers have been designed for sensitive analyte detection. 15–17 Although the effi- cacy of 1D SERS nanoprobes their practical use is restricted due to limited concentration of hot spots in the irradiated area. In this regard, much attention has been paid to three-dimensional (3D) organization of SERS substrate. 18 The large surface area together with porous structure of gold nanosponges, aluminum membrane, nano-dendritic crystal lm, paper and cellulose lter, vertically aligned nanotubes have been suggested to concentrate the number of hot spots and sites for adsorption of analyte molecules and therefore improve the sensitivity of SERS substrate. 19–22 In this respect, nanobrous-based SERS substrates obtained by electrospinning method can be easily impregnated with plasmonic nanoparticles providing SERS activity, reveal excellent mechanical strength and exibility, cost-effective, and can be integrated within microuidic based systems and ultra-thin layer chromatography to automatize measurements. 23–26 These so lms can be also suitable for stand-off measurements, by means to provide hands off SERS based diagnostics. 27 Additionally, intrinsic porosity of such material made out of nanober mesh can be effectively used for capturing and concentrating of analytes from uids such as small molecules 28 and whole microbial cells. 29 a Remote Controlled Theranostic Systems Lab, Educational Research Institute of Nanostructures and Biosystem, Saratov State University, Astrakhanskaya 83, Saratov, 410026, Russia. E-mail: yashenokam@gmail.com b Educational Research Institute of Nanostructures and Biosystem, Saratov State University, Saratov, Russia c A. V. Shubnikov Institute of Crystallography Russian Academy of Science, Moscow, Russia d Department of Molecular Biotechnology, Ghent University, Ghent, Belgium e Josef Stefan Institute, Ljubljana, Slovenia f School of Engineering and Materials Science, Queen Mary University of London, London, UK g RASA Center in St. Petersburg, Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia h RASA Center in Tomsk, Tomsk Polytechnic University, Tomsk, Russia Cite this: RSC Adv. , 2016, 6, 84505 Received 22nd July 2016 Accepted 29th August 2016 DOI: 10.1039/c6ra18636j www.rsc.org/advances This journal is © The Royal Society of Chemistry 2016 RSC Adv. , 2016, 6, 84505–84511 | 84505 RSC Advances PAPER