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Monitoring the Changes of pH in Lysosomes during Autophagy and Apoptosis by Plasmon Enhanced Raman Imaging Shan-Shan Li, Miao Zhang, Jian-Hua Wang, Fan Yang, Bin Kang, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01250 • Publication Date (Web): 30 May 2019 Downloaded from http://pubs.acs.org on June 1, 2019
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Analytical Chemistry
Monitoring the Changes of pH in Lysosomes during Autophagy and Apoptosis by Plasmon Enhanced Raman Imaging Shan-Shan Lia,b, Miao Zhanga, Jian-Hua Wanga, Fan Yanga, Bin Kanga,*, Jing-Juan Xua, and HongYuan Chena State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, 210023, China b Institute for Biosensing, and Collenge of Chemistry and Chemical Engineering, Qingdao University, 308 Ningxia Road, Qingdao 266071, China a
ABSTRACT: Lysosomes are acidic organelles that not only participate in intracellular degradation but also relate to various cellular functions. Abnormal pH in lysosomes would lead to lysosomal dysfunction, which may further result in many diseases. In this work, we statistically analyze the pH change in the lysosomes of Hela cells model by using surface enhanced Raman Scattering (SERS) imaging technique. We prepared a plasmon Raman pH probe and localized the pH probe to lysosomes via an incubation-depletion method. The pH profiles within lysosomes during the process of cellular autophagy and apoptosis were monitored in situ by SERS imaging. The pH in lysosomes decreased slightly during the process of autophagy, while the pH in lysosomes increased during apoptosis. The phenomenon, in general, is consistent with our current biological knowledge. However, we did not observe significant variation of pH between different individual cells. This information might provide an in depth understanding about the relationship of lysosomal pH with fundamental cellular functions and mechanism of diseases.
Intracellular pH plays many key roles in proliferation and apoptosis,1,2 ion transport,3,4 multidrug resistance,5 and muscle contraction.6 Changes of intracellular pH affect synaptic transmission and neuronal excitability.7,8 Intracellular abnormal pH is often associated with inappropriate growth and division, which are observed in some diseases.9,10 For some subcellular compartments, low pH in organelles contributes to protein degradation or activates enzyme and protein functions. Particularly, there are more than 50 different hydrolases involved in substrates degradation in lysosomes, and most of them exhibit pHdependent activities, which may be a functional protection against lysosomal rupture. Importantly, the acidic environment in the lysosomes (pH 4.5-5.5) is benefit for the degradation of proteins in cellular metabolism, nevertheless the activity of these enzymes is greatly reduced under the pH value of cytoplasm or extracellular environment.11,12 In other words, abnormal pH in the lysosomes leads to lysosomal dysfunction, which might result in many diseases in humans, including bone and immune system diseases, lipid and glucose metabolism diseases, neurodegenerative diseases, infectious diseases, etc.13 Therefore, monitoring the pH changes in the lysosomes can provide important information for studying physiological and pathological processes, but it is also a major challenge. Up to now, the reported methods for pH determination include nuclear magnetic resonance (NMR), microelectrode, absorption spectroscopy, fluorescence spectroscopy, fluorescence imaging, surface enhance Raman Scattering (SERS) spectroscopy and SERS imaging.14 Among them, fluorescence and SERS imaging could be used for in situ intracellular imaging analysis, and fluorescence imaging is quite mature and act as
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the most common way to detect intracellular pH.15,16 Although ratiometric fluorescence imaging could provide the average pH of cells in the imaging area, in biological systems, the organic fluorescent dye bears disadvantages of quenching, photobleaching, blinking, and the interference from background fluorescence, especially for single cell analysis, the interference from cell autofluorescence is extremely serious. As a fingerprint vibrational method, SERS enables in situ imaging analysis in cells with several advantages: no quenching or photobleaching, low damage to cells, high sensitivity, flexible excitation wavelength, and hyperspectral resolution, etc.17,18 To achieve SERS based pH sensing, reporter molecules with sensitive pH responses and strong Raman signals are modified on plasmonic nanoparticles. When the reporter molecules are in different pH environments, they undergo protonation and deprotonation processes, resulting in significant changes in the SERS spectra. Due to the simple molecular structure and strong combination with plasmonic nanoparticles, 4-mercaptobenzoic acid (4-MBA) and 4-mercaptopyridine (4-MPy) are the most commonly used reporter molecules.12,19-21 In addition, for the determination of acidic environment (3
