**1. Introduction**

Surface-enhanced Raman spectroscopy (SERS) is nowadays one of the most powerful and reliable method in a wide range of applications including biochemistry [1], biomedical analysis [2], forensics, bio- and chemical hazards [3] and environmental monitoring [4,5].

In SERS effect, for molecules adsorbed onto metallic nanostructures, the Raman signal is amplified up to 14 orders of magnitude. Two mechanisms, an electromagnetic (EM) and a chemical (CT) are involved in SERS effect. An electromagnetic enhancement results from the amplification of incident light due to excitation of localized surface plasmon resonance (LSPR) of the metal surface. It was

found that the morphology and dielectric environment of the plasmonic nanostructures plays a crucial role in the EM mechanism. The chemical enhancement process involves the charge-transfer occurring between the metal surface and adsorbed molecules, which can enhance the transition polarizability of adsorbates [6]. Typically, the electromagnetic enhancement can reach factors 103–1011, whilst the chemical enhancement contributes additional factor up to 10<sup>3</sup> [7,8]. As a result of these mechanisms, SERS ensures the ultratrace detection of analytes down to single molecules [9]. Besides ultrasensitvity and fingerprint specificity, SERS technique offers nondestructive, label–free, and fast detection of analytes under a wide range of conditions (excitation wavelength and power of the laser, temperature, pressure, presence of water).

These features lead to an increase of the practical applications of SERS technique especially in biological materials studies, starting from single macromolecules and ending with whole cells (for example, microorganisms) [10–14]. The SERS spectra of biomolecules carries the information about their structure. This technique also offers the quantitative and qualitative studies with the possibility of multiplexed detection of analytes in complex fluids such as blood, cerebrospinal fluid or urine.

However, the practical implementation of SERS technique in a clinical setting is hindered by the difficulties in generation of a model SERS–active nanostructures. Such SERS substrate should reveal homogenous and high enhancement factor (EF) across the surface, as well as chemical and physical stability. Additionally, the fabrication method of SERS substrates should be as cheap as possible and provide large-scale production. The commonly used SERS substrates are based on the nanostructured noble metals e.g. Ag, Au, Cu, however the most frequently used metal is silver as it is quite cheap in comparison with gold and provides very high SERS enhancement (much higher than for copper). Interestingly, SPR (surface plasmon resonance) application of Ag is not only important from the point of view of SERS studies, but also covers other technologically important fields such as photocatalysis [15,16].

Today, a variety of techniques have been applied to fabricate SERS nanostructures, such as electrochemical methods [17], nanosphere lithography [18], electron-beam lithography [19], nanoimprinting lithography [20], vapour layer deposition [21], colloidal suspension [22], and many other strategies.

Lately, our research group has developed several types of novel nanomaterials, which can be used in a wide range of biomedical studies [23–28]. In recent research works we have investigated the membrane-based SERS-platforms [26,29], where polymer mats covered with gold nanostructures enable filtration of bacteria from fluids (for example, blood plasm), their immobilization on the filter surface, and enhancement of Raman signal. This solution overcomes the problem of transferring bacteria from filter to the SERS platform and potential contamination of the sample, and, at the same time, enables detection of very low concentration of bacteria. Nowadays, there are many different types of materials which are used to produce SERS-active substrates, e.g. ceramics [30,31], silver nanorods [32], mesoporous silica [33], ultrafiltration membrane [34], filter paper [35,36] or metal nanocrystals [37]. Particularly interesting are the polymer-based manufacturing techniques, due to the low cost and ease of production. However, polymer substrates cannot meet the demands of high stiffness of the substrate and high heat transfer (i.e. heat dissipation). Therefore, we have expanded our research by developing the thermally resistant SERS platforms. Continuous irradiation of the laser beam over the SERS substrate can be a source of significant increase in the local temperature of metallic nanostructures, what is a critical issue in SERS measurements of heat-sensitive materials [38,39]. Thermal degradation or fragmentation of the analyte is a vital problem during examination of heat-sensitive materials like DNA, proteins, polymers or lipid bilayers [40]. Moreover, molecules that are adsorbed to the metal surface (e.g. thiols at gold) can desorb at a temperature of 60–100 ◦C [40]. Decomposition or fragmentation of the analyte often leads to broadening and reduction of the intensity of observed spectral bands, and as a result, to misinterpretation of analytical data [41,42].

Another issue is connected with possible degradation of the polymer in contact with water (biodegradable polymers like poly(L-lactide acid) (PLLA) or polylactid acid (PLA)) or in contact with organic solvents. Biological samples involve water-based samples (blood, urine, cerebrospinal fluid), therefore used polymers should not be biodegradable in water environment, especially when the filtration process is long.

To conclude, there is a strong need for cheap, versatile, durable, stable SERS-platform which can easily dissipate heat thus can be used with high power lasers. Such platform should, preferably, be resistant to water and other organic and inorganic solvents. In this paper, we present a new type of SERS platforms based on woven wire mesh made of stainless steel (referred here as a stainless-steel wire mesh, SSWM). There are four types of woven meshes: plain weave, twilled weave, plain Dutch weave, and twilled Dutch weave, all having periodic structure which makes them excellent base for SERS platform. They are used as a filtration media in chemical industry [43,44] due to their ability to withstand large pressures, high heat resistance, small aperture and reusability. We used twilled Dutch SSWM as a base for our SERS-platform: the SSWM was cleaned and sputtered with 50 nm thick layer of silver which ensured the high amplification of the Raman signal. The dense mesh structure also provided good deposition of the analyte on the surface.

The developed SERS platforms, named Ag/SSWM, show very high surface-enhancement factor (1 × 106), high stability (up to one month under ambient conditions), high reproducibility, and high thermal resistance to laser irradiation. These Ag/SSWM substrates demonstrated a grea<sup>t</sup> potential in sensitive and reproducible SERS-based detection of typical analyte like *p*-MBA, as well as whole microorganisms, that is, *Escherichia coli* and *Bacillus subtilis.*

### **2. Materials and Methods**

### *2.1. Chemicals and Materials*

*<sup>P</sup>*-mercaptobenzoic acid (*p*-MBA) and phosphate-buffered saline (PBS) packs (10 mM, pH = 7.2) were obtained from Sigma-Aldrich (Dorset, UK) and used without further purification. Water (resistivity over 18 M Ω), purified using a Milli-Q plus 185 system was used throughout the process. Stainless steel wire mesh was obtained with Anping County Huijin Wire Mesh Co., Ltd., Anping, China. Each type of mesh wire was purchased in quantity of 1 m<sup>2</sup> and deposited rolled in room temperature prior to use.
