A simple lysosome-targeted probe for detection of hypochlorous acid in living cells

Highlights

• A turn-on NDS probe has been synthesized and characterized.

• The NDS probe can detect endogenous HClO in living cells.

• The NDS probe could target lysosomes.

Abstract

Since HOCl plays an indispensable role in a variety of physiological and pathological processes, monitoring the level of hypochlorous acid (HOCl) in living cells is critical. Based on this, it is necessary to develop a convenient and simple technique to detect HOCl in living cells. In this contribution, a simple 2-hydroxy-6-morpholino-1H-benzo[de]isoquinoline-1,3(2 H)-dione (NDS) was developed as fluorescent probe for monitoring HOCl in live cells. Probe NDS exhibited preeminent specificity towards HOCl over other various bioactive substances. In addition, the probe could respond to HOCl in solutions within 16 s and the limit of detection was as low as 105.24 nM. NDS has good lysosomal targeting and has been successfully applied to the expression of exogenous and endogenous lysosomal HOCl in HeLa cells. This study provides a promising tool for studying physiological or pathological related processes.

Introduction

Hypochlorous acid/hypochlorite (HOCl/ClO⁻) is widely used in daily life for bleaching, drinking water disinfection and cooling water treatment. Hypochlorous acid, as one of the important species in reactive oxygen species, plays an important role in many biological processes [[1], [2], [3], [4]]. In vivo, HOCl is a normal metabolic by-product of cells that dynamically distributed in cells and does not accumulate in a certain location in the cell and it can be generated endogenously through the reaction of chlorine ion (Cl⁻) and hydrogen peroxide (H₂O₂) catalyzed by myeloperoxidase (MPO) [[5], [6], [7], [8], [9]]. HOCl is a weak acid (pK_a = 7.6 for HOCl), mainly distributed in the acidic organelle lysosomes of macrophages. Despite the extremely low concentration of HOCl, it plays a vital role in the immune defense against invasive bacteria and other pathogens [[10], [11], [12], [13]]. Abnormal levels of HOCl can damage the immune system and cause multiple diseases such as cardiovascular diseases, cystic fibrosis cancers, atherosclerosis, arthritis and neuron degeneration [[14], [15], [16]]. Therefore, it is of great significance to develop convenient and selective HOCl detection methods.

So far, several methods for detecting HOCl have been reported, such as colorimetry, electrochemistry, chemiluminescence and fluorescence analysis [17,18]. Among them, due to the high selectivity and sensitivity of the fluorescence probe, simple instrument implementation and ease of use, it has become an efficient tool for detecting HOCl [[19], [20], [21], [22]]. Up till now, many fluorescent probes have been synthesized and applied to the detection of HOCl, most of which are designed by utilizing the strong oxidation properties of HOCl such as chalcogenide (S, Se, Te) atoms [[23], [24], [25]], borate ester group [26], oxime [27], unsaturated carbon-carbon double bond [28], dimethylthiocarbamate and so on. Due to the tendency of these groups to be easily oxidized by HOCl, have been employed as reporters in fluorescent probe design. The selection of fluorophore for probe molecules is also diverse, such as rhodamine dye [[29], [30], [31]], boron-dipyrromethene (BODIPY) [[32], [33], [34]], 1,8-naphthalimide-derived [35], coumarin [36], anthocyanins [37,38] and so on.

In this paper, a new compound with naphthalimide as fluorophore and dimethylthioamino as responsive group was designed and synthesized, and Intramolecular Charge Transfer (ICT) mechanism was used to detect HOCl. At the same time, morpholine group was introduced as the target group of lysosomes to detect hypochlorous acid in Hela cell. NDS showed good linear relativity, a low limit of detection (105.24 nM), fast response (within 16 s) and was successfully applied to monitor the fluctuation of basal HOCl levels and exogenous HOCl levels in HeLa cell lysosomes.