Development of nanostructured substrates for quantification of anticancer drugs in biofluids with Surface Enhanced Raman Scattering (SERS)

To date, therapeutic drug monitoring (TDM), that is the quantification of anticancer drugs in biofluids of oncological patients, is expensive and time consuming. Such a quantitative determination is crucial for a successful chemotherapic treatment due to the high interpatient variability of the anticancer drugs levels in biofluids. A frequent TDM turns out to be necessary to limit as far as possible the heavy side-effects of the treatment. Thus, the development of a new Point Of Care (POC) tool for a faster and less expensive TDM is highly desirable. Surface Enhanced Raman Scattering (SERS) is a surface-sensitive technique able to detect low concentrations of specific analytes in liquid environments. The high sensitivity given by metal nanostructured materials, the specificity given by Raman spectroscopy, the rapidity of the measurements and the possibility to develop inexpensive solid SERS substrates point to SERS as an appropriate platform for TDM. Moreover, solid SERS substrates together with the availability of portable instruments make this technique a possible new POC tool to quantify the drugs were a quick response is needed. The aim of this doctoral project is the development and the optimization of cost-efficient, repeatable and sensitive solid SERS substrates for quantitative analysis of antitumoral drugs in biofluids such as human serum. In the first part of this doctoral thesis, a general introduction about Raman spectroscopy, SERS and SERS as quantitative technique will be reported. In the second chapter, the development and the optimization of repeatable solid SERS substrates constituted by metal nanoparticles deposited on filter paper with a simple dipping method, it will be presented. Specifically, four kinds of substrates developed with dip-coating method will be presented, differing depending on the type of nanoparticles used. As the first step, I performed a systematic study on c-Au and c-Ag paper made substrates using two different non resonant analytes as probe molecules (i.e. Adenine and 4-Mercaptobenzoic acid). The aim was to optimize the parameters involved in the development procedure (i.e. the kind of filter paper, the NPs concentration and the presence of an aggregating agent) exploring how they affect the repeatability of the substrates. Optimized paper-based SERS substrates aiming at building a platform for quantitative analysis will be presented in terms of inter- and intra- sample repeatability, sensitivity and long term stability. Then, solid substrates based on different nanoparticles, namely h-Ag and NSs, will be presented and discussed from a repeatability point of view. The SERS signal variation, hence the repeatability, will be quantified calculating the relative standard deviation (RSD%). In the third part of the work, I will present some applications of the substrates developed in the first part. In particular, preliminary results will be reported, which are related to Methotrexate (MTX) and Imatinib (IMT) rapid quantification, in model solutions mimicking biofluids as well as in real biofluids such as human serum. Optimized c-Au and h-Ag paper-made substrates were used for the analysis of MTX and IMT, respectively. The direct quantification of MTX and IMT spiked in human serum will be presented by means of combined SERS with paper-made solid substrates and a multivariate Partial Least Squares Regression (PLSR) model. The model performance will be judged on the basis of the Root Mean Square Error of Prediction (RMSEP), which quantifies the quantification accuracy and precision.