(78a) Functionalized Plasmonic Nanostructure Arrays for Mapping Extracellular pH of Single Living Cells

The extracellular pH (pHe) of living cells is one of the major factors that influence cell behaviors including cycle progression, migration, and proliferation. Tumor cells are believed to become more invasive due to the dysregulated pHe. Thus, accurate sensing and mapping of the pHe is still a critical yet challenging task in the study of pHe-dependent cell behaviors. However, the spatial resolutions of traditional methods such as microelectrodes and magnetic resonance spectroscopic imaging are not high enough to achieve single cell study. We present a method to map pHe of single living cells based on surface-enhanced Raman spectroscopy (SERS). The novelty of our work is on two aspects. First, we used a gold quasi three-dimensional plasmonic nanostructure array (Q3D-PNA), instead of metallic nanoparticles, as a SERS-active substrate. With the immobilization of a pH probe molecule, 4-mercaptobenzoic acid (4-MBA), such Q3D-PNA SERS substrates enable exceptionally sensitive and reproducible pH measurement and only the distributions of extracellular pH, where cells attach to Q3D-PNAs, are detected. Second, we prudentially investigated the influences of cations and complexity of detection solutions on the responses of 4-MBA SERS spectra to pH variations to ensure the pHe mapping accuracy. This is because the pH indicator, intensity of the symmetric carboxylate stretching mode (νCOO-), is not only a function of pH but also affected by the presence of cations in the solution. As a proof of concept, a normal cell line (NIH/3T3) and a tumor cell line (HepG2) were cultured on the 4-MBA modified Q3D-PNA SERS substrates. Local pHe of single living cells was detected and mapped with good spatial resolution and pH sensitivity. The domains of different pHe on both cells were clearly observed. Moreover, the averaged pHe of tumor cells was shown to be more acidic compared with that of normal cells. This technique can be used for the differentiation between tumor and normal cells. In addition, it will benefit the study of metabolisms and functions of single living cells as well as transmembrane drug delivery ruled by the extracellular acidity.