Abstract
The monitoring of intracellular pH is of great importance for understanding intracellular trafficking and functions. It has various limitations for biosensing based on the fluorescence intensity or spectra study. In this research, pH-sensitive carbon dots (CDs) were employed for intracellular pH sensing with fluorescence lifetime imaging microscopy (FLIM) for the first time. FLIM is a highly sensitive method that is used to detect a microenvironment and it can overcome the limitations of biosensing methods based on fluorescence intensity. The different groups on the CDs surfaces changing with pH environments led to different fluorescence lifetime values. The CDs aqueous solution had a gradual change from 1.6 ns to 3.7 ns in the fluorescence lifetime with a pH range of 2.6–8.6. Similar fluorescence lifetime changes were found in pH buffer-treated living cells. The detection of lysosomes, cytoplasm, and nuclei in living cells was achieved by measuring the fluorescence lifetime of CDs. In particular, a phasor FLIM analysis was used to improve the pH imaging. Moreover, the effects of the coenzymes, amino acids, and proteins on the fluorescence lifetime of CDs were examined in order to mimic the complex microenvironment inside the cells.
Highlights
Intracellular pH is of great importance for maintaining a normal cell activity [1], which plays a crucial role in biological functions such as cell proliferation [2], apoptosis [3], ion transport [4], endocytosis [5], and tumor growth [6]
The mechanism of carbon dots (CDs) fluorescence is still in debate, but there are a number of reports that agree that the protonation or deprotonation level of the surface functional groups of CDs may be different at variable pH environments, which could result in the change of fluorescence [31,53,54]
This work demonstrated the pH-sensitive CDs for intracellular pH sensing with fluorescence lifetime imaging microscopy (FLIM) for the first time
Summary
Intracellular pH is of great importance for maintaining a normal cell activity [1], which plays a crucial role in biological functions such as cell proliferation [2], apoptosis [3], ion transport [4], endocytosis [5], and tumor growth [6]. The real-time monitoring of the intracellular pH may aid understanding of the pathogenesis of these diseases and aid with treatment. Monitoring intracellular pH in real time is of great significance. Among the traditional approaches for measuring intracellular pH, fluorescence technologies show important advantages due to non-invasiveness, high sensitivity and low cost [9].Organic dyes [10,11], fluorescent proteins [12], and fluorescent nanomaterials [13]. Organic dyes usually a have high susceptibility to photobleaching and relatively narrow absorption [14]. QDs are the most familiar fluorescent nanomaterials with high brightness and good photostability, but most of the QDs containing heavy metal components such as cadmium, mercury or lead, are highly toxic [16]. Polyethylenimine-coated upconversion of nanoprobes was reported with large particle size and not conductive to cell imaging though its ratiometric sensing in solutions can be achieved [17]
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