The merging of the molecular specificity of Raman spectroscopy with the extraordinary optical properties of metallic nanoparticles settled the basis of the so-called Surface Enhanced Raman Spectroscopy (SERS), which in the last few decades proved its worth as powerful analytical tool with detection limits improved to the single molecule recognition.
The study here proposed is aimed to the development of a SERS-based pH nanosensor made up of gold nanoparticles (AuNPs) conjugated with the pH-sensitive molecular probe 4-mercaptobenzoic acid (4MBA), a benzene derived consisting of a thiol group, which covalently binds to the AuNPs surface, and a carboxylic acid. The SERS band of such probe show a strong sensitivity to the pH of the environment, depending on the protonation degree of the carboxyl.
Primarily, the functionalization procedure will be optimized in order to obtain a stable sensor with a high coverage of 4MBA on the AuNPs. The dynamic range of sensitivity of the nanosensor will be identified by SERS measurements in liquids for different pH and a calibration curve will be provided in terms of the relative intensity of selected pH-dependent SERS bands. The possibility to tune the working point of the nanosensor will be further explored by employing nanoparticles of different sizes and material.
Our approach will enable to obtain a versatile tool for the evaluation of the pH on the nanoscale, showing a great potential for biological measurements at the single cell level.
The final goal of our project is the development of a plasmonic pH nanosensor based on the conjugation of AuNPs with a pH sensitive molecular probe. The current methods for pH measurements include from one side the employment of electrodes or optical fibres which require huge sample volumes and lack in spatial resolution. On the other side, dyes and quantum dots allow better spatial resolution, but are prone to photobleaching. Our strategy will allow to overcome such drawbacks since the pH measurement is obtained by the variation of the SERS spectra related to the chemical modification of the 4MBA molecules resulting from the changing in their protonation degree. In this way it will be possible to evaluate the pH on the nanoscale with a non-invasive, label-free technique. In particular, this method is well suitable for monitoring pH dependent biological processes at the single cell level. Alterations of the local pH of cellular compartment can have dramatic effects on cells and organelles, encouraging the occurrence of diseases. Furthermore, the extracellular pH of cancer cells has been proved to be more acidic respect to that of healthy ones [13]. Therefore, the sensitive and reliable detection of the cellular pH microenvironment may facilitate the understanding of the changes during the occurrence and progression of diseases providing novel diagnostic strategies.
Moreover, since 4MBA is a bifunctional linker, the exposed carboxylic group enables the further functionalization with other molecules. In this context, the possibility of conjugation with drugs makes of 4MBA-AuNP also a suitable platform for therapeutic purposes, approaching the concept of theranostics, i.e. the combination of ultra-sensible diagnostics with advanced therapies on a single device.
As said, the sensitivity range of the nanosensor is located around the pKa value of the molecular probe. Preliminary measurements show that the pKa value of the molecule linked to the AuNPs surface is higher respect to that of the pure/free molecule. In this framework, clarify how the acidic properties of the molecule can be influenced by the confinement on the nanostructure will allow to gain versatility in the nanosensor design, enabling the modulation of the pKa in the pH range of interest acting on the properties of the NPs, such as size and material.
[13] B.A. Webb et al., Nat.Rev.Cancer 9, 671¿677, (2011).