Abstract
The merging of the molecular specificity of Raman spectroscopy with the extraordinary optical properties of metallic nanoarchitectures is at the heart of Surface Enhanced Raman Spectroscopy (SERS), which in the last few decades proved its worth as powerful analytical tool with detection limits pushed to the single molecule recognition. Within this frame, SERS-based nanosensors for localized pH measurements have been developed and employed for a wide range of applications. Nevertheless, to improve the performances of such nanosensors, many key issues concerning their assembling, calibration and stability, that could significantly impact on the outcome of the pH measurements, need to be clarified. Here, we report on the detailed characterization of a case study SERS-active pH nanosensor, based on the conjugation of gold nanoparticles with the pH-sensitive molecular probe 4-mercaptobenzoic acid (4MBA). We analyzed and optimized all the aspects of the synthesis procedure and of the operating conditions to preserve the sensor stability and provide the highest responsiveness to pH. Exploiting the dependence of the SERS spectrum on the protonation degree of the carboxylic group at the edge of the 4MBA molecules, we derived a calibration curve for the nanosensor. The extrapolated working point, i.e., the pH value corresponding to the highest sensitivity, falls at pH 5.6, which corresponds to the pKa value of the molecule confined at the nanoparticle surface. A shift of the pKa of 4MBA, observed on the molecules confined at the nanostructured interface respect to the bulk counterpart, unveils the opportunity to assembly a SERS-based pH nanosensor with the ability to select its working point in the sensitivity region of interest, by acting on the nanostructured surface on which the molecular probe is confined. As a proof-of-concept, the nanosensor was successfully employed to measure the extracellular pH of normal and cancer cells, demonstrating the capability to discriminate between them.
Highlights
In the last decades, the advances in the field of plasmonics enabled to tailor and enhance electromagnetic fields at the subwavelength scale, with a wide range of applications spreading from electronics to biomedicine (Stockman et al, 2018)
In this paragraph we report on all the aspects concerning the fabrication and characterization of the nanosensor with special focus on the colloidal stability, pivotal to refine the performances in liquid environment, and on the chemical alterations that might occur in the molecular probe due to the laser illumination
The employed nanosensor consists of a plasmonic core made of 60 nm AuNPs conjugated with 4-mercaptobenzoic acid (4MBA), according to the protocol described in section Nanosensor Assembling
Summary
The advances in the field of plasmonics enabled to tailor and enhance electromagnetic fields at the subwavelength scale, with a wide range of applications spreading from electronics to biomedicine (Stockman et al, 2018). Beyond the application as ultrasensitive spectroscopy, SERS turned out to be an invaluable technique capable to actively and selectively interact with complex biological systems, including cells and tissues (Fasolato et al, 2016; Cialla-May et al, 2017); (Kneipp, 2017). In this context, colloidal noble metal nanoparticles play a central role thanks to their high surface-to-volume ratio and to their ease of synthesis and functionalization in different environmental conditions, which make them a versatile platform to develop SERS-active chemical sensors (Yeh et al, 2012). The extracellular pH of cancer cells is expected to be more acidic with respect to that of healthy ones (Webb et al, 2011)
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