**1. Introduction**

Human tears represent an exceptionally rich source of information regarding the health status of eyes and, more generally, of whole body functionality. This is mainly due to the presence in tears of a large variety of salts and organic components (including proteins, lipids, metabolites, nucleic acids, and electrolytes) whose concentrations can be altered by pathologies, eye diseases and/or inflammatory processes [1,2]. An increasing attention is presently given to the analysis of this human body fluid. The small amounts of substance considered and the typical low concentration of organic compounds hamper the access to direct analysis by biochemical methods even if the improvement of technology is overcoming some of the main obstacles and the route to a wider and reliable use of tear diagnostics is in progress. In this framework, micro-Raman spectroscopy can have an important contribution. Vibrational spectroscopies have been shown to be extremely useful to

analyze biological samples providing valuable insight into the nature of samples and biofluids [3–6] and into the determination of the chemical structure of specific molecules [7]. Raman spectroscopy has been also used for investigating tears [8–10]. More recently, the use of Surface Enhanced Raman Spectroscopy (SERS) has been proposed with interesting perspectives in terms of sensitivity and specificity of the signal response [11–13]. In the work of P. Hu et al. [11] SERS has been implemented by using silver nanoparticles and applied to the analysis of dried human tears. The spectroscopy of dried fluids, the so-called drop-coating deposition Raman spectroscopy (DCDRS), has many advantages with respect to liquid sample analysis and has been applied fruitfully to tear investigation [8,9]. SERS mechanisms add new potentialities to DCDRS. In the case of tears, SERS response has some differences with respect to DCDRS signal and the observed modes are mainly assignable to the proteins [11]. In particular, P. Hu et al. [11] evidenced a correlation between the intensity of SERS modes at 757 and 1557 cm−<sup>1</sup> spectral positions and the lysozyme content, in agreement with a previous work of J. Filik and N. Stones [14]. Gold coated nanostructured substrates have been used by S. Choi et al. to perform SERS investigation of dried tears aimed to characterize the spectral changes occurring in adenoviral conjunctivitis-infected subjects [12]. In that work, the principal component analysis (PCA) allowed a global analysis of the spectrum configuration that was able to distinguish between normal and infected tear fluids confirming that SERS measurements can offer valuable diagnostic tools. For this reason, we investigate the possibility of performing SERS measurements on human tears using a cheap and easy method to prepare home-made substrates, differently from reported works that used more sophisticated and expensive substrates [11,12] not suitable for large screening purposes. In this present work, human tear samples have been examinated by SERS using home-made SERS subtrates based on gold nanoparticles using a procedure simpler and faster than the one proposed in [11], which requires the mixing of each tear sample with colloids. In order to test the efficiency and sensibility of the method, preliminary SERS measurements have been performed on Rhodamine 6G aqueous solutions at different concentrations.
