Accurate Quantitative Sensing of Intracellular pH based on Self-ratiometric Upconversion Luminescent Nanoprobe

Cuixia Li,Hong Zhang,Qiqing Li,Youlin Zhang,Li Zhang,Xiaomin Liu,Langping Tu,Bin Xue,Jing Zuo,Huiying Zhao,Yulei Chang,Xianggui Kong

doi:10.1038/srep38617

Abstract

Accurate quantitation of intracellular pH (pHi) is of great importance in revealing the cellular activities and early warning of diseases. A series of fluorescence-based nano-bioprobes composed of different nanoparticles or/and dye pairs have already been developed for pHi sensing. Till now, biological auto-fluorescence background upon UV-Vis excitation and severe photo-bleaching of dyes are the two main factors impeding the accurate quantitative detection of pHi. Herein, we have developed a self-ratiometric luminescence nanoprobe based on förster resonant energy transfer (FRET) for probing pHi, in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Under 980 nm excitation, upconversion emission bands at 475 nm and 645 nm of NaYF4:Yb3+, Tm3+ UCNPs were used as pHi response and self-ratiometric reference signal, respectively. This direct quantitative sensing approach has circumvented the traditional software-based subsequent processing of images which may lead to relatively large uncertainty of the results. Due to efficient FRET and fluorescence background free, a highly-sensitive and accurate sensing has been achieved, featured by 3.56 per unit change in pHi value 3.0–7.0 with deviation less than 0.43. This approach shall facilitate the researches in pHi related areas and development of the intracellular drug delivery systems.

Highlights

PHi, in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively
FITC as pH indicator was conjugated on the surface of PEI-UCNPs by the PEI ligand
The intensity of the one at 475 nm, which was subject to the energy transfer between UCNPs and FITC, was used as response signal

Summary

Introduction

PHi, in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Under 980 nm excitation, upconversion emission bands at 475 nm and 645 nm of NaYF4:Yb3+, Tm3+ UCNPs were used as pHi response and selfratiometric reference signal, respectively This direct quantitative sensing approach has circumvented the traditional software-based subsequent processing of images which may lead to relatively large uncertainty of the results. The software-based subsequent processing (such as MATLAB, Olympus software etc.) is essential in most current quantification approaches, where the possible presence of large deviation is a critical concern[3,5,9] It is an exceeding desire of developing a novel nanoprobe which can circumvent these problems and realize a reliable, sensitive and accurate pHi measurement. Under NIR-excitation without the undesired fluorescence background and photo damage, an accurate quantitative measurement has been realized using the self-ratiometric nanoprobe, avoiding the popularly used imaging-based subsequent software processing which may bring in large deviation

Methods

Results

Conclusion