In Situ Imaging of Live-Cell Extracellular pH during Cell Apoptosis with Surface-Enhanced Raman Spectroscopy

Abstract

Extracellular pH (pHe) is an important regulating factor that determines many cellular processes, including proliferation, differentiation, and apoptosis. In our previous work, we developed 4-MPy (4-mercaptopyridine) modified Au nanoparticles as intracellular pH sensors based on surface-enhanced Raman spectroscopy (SERS). We herein modified a Au-nanoparticle-assembled solid SERS substrate with 4-MPy molecules for in situ pHe sensing during apoptosis. We found a more acidic extracellular environment of cancer cells than that of normal cells from the pH imaging. We then in situ investigated the temporal and spatial evolution of pHe of cancer cells after addition of transforming growth factor-β (TGF-β). The pHe showed a fast decrease at the beginning, followed by a slow decrease until the complete loss of cellular functions, and the pH values in and out of the cells became similar. This work shows that our SERS substrate combined with an in situ cell culture system is well suitable for in situ pHe sensing during cell processes and will be a promising technique for understanding more pHe-related biological and pathological issues.

Mechanistic insights into the pH-dependent membrane peptide ATRAM

“…The local extracellular pH of cancer cells can be 0.5-1 pH units lower than their healthy counterparts due to altered glucose metabolism, vascularization, and induction of carbonic anhydrase IX, among other factors [23][24][25] . This process is accentuated by the local concentration of protons on the surface of cells, which results in a significantly more acidic pH than in the bulk microenvionment 26,27 .Using pH-sensitive peptides as a tool to target the plasma membrane of diseased cells can allow for higher targeting specificity of acidic diseased tissues compared to healthy ones 28,29 .…”

Live-Cell Surface-Enhanced Raman Spectroscopy Imaging of Intracellular pH: From Two Dimensions to Three Dimensions

“…imaging. Commonly used pH sensitive Raman reporters (RaRs), including 4MBA, 12,13,[21][22][23][24] 4-mercaptopyridine, 6,14,15 and para-aminothiophenol, 26 among others, are often hydrophobic. The combination of such molecules with a SERS-active AuNP would thus inevitably lead to colloidal instability and aggregation, especially in complex solutions with high ion and protein concentration such as cell culture media.…”

“…8,10,11 Many advances have been reported on SERS imaging of i-pH in living cells. 6,[12][13][14][15] However, they are consistently limited to analysis in two dimensions (2D). Despite the relatively rapid image acquisition in 2D, information in the vertical z axis has been sacrificed so far, meaning that a detailed i-pH profile in 3D has not been reported.…”

Application of molecular SERS nanosensors: where we stand and where we are headed towards?

“…The sensitive and reliable determination of these values could be important for the early diagnosis of many medical conditions. 4-Mercaptopyridine (4MPy) and 4mercaptobenzoic acid (4MBA) are the two pH sensing molecules mostly used for SERS-based detection [11][12][13][14][15]. They offer the possibility to monitor the pH variations over a large window (pH 4 to 9) and they also show good chemical affinity towards the surface of gold NPs.…”

“…Two recent studies report on the successful determination of pH e of living cells. One approach uses NPs [ 14 ], whereas the second one uses planar SERS substrates [ 15 ]. For the first approach, the membrane proteins of cells were biotinylated.…”