Rational Development of a New Reaction-Based Ratiometric Fluorescent Probe with a Large Stokes Shift for Selective Detection of Bisulfite in Tap Water, Real Food Samples, Onion Tissues, and Zebrafish.

Bisulfite (HSO3-) is usually widely added to tap water and food because it has antibacterial, bleaching, and antioxidant effects. However, its abnormal addition would cause a series of serious diseases related to it. Therefore, development of an effective method for HSO3- detection was of great significance to human health. In this work, a new reaction-based ratiometric fluorescent probe KQ-SO2 was rationally designed, which could be used for the highly selective detection of HSO3- in tap water, real food samples, onion tissues, and zebrafish. Specifically, a positively charged benzo[e]indolium moiety and a carbazole group through a condensation reaction resulted in KQ-SO2, which displayed two well-resolved emission bands separated by 225 nm, fast response (1 min), and high selectivity and sensitivity toward HSO3- upon undergoing the Michael addition reaction, as well as low cytotoxicity in vitro. In addition, KQ-SO2 has been successfully applied for the detection of HSO3- in tap water, real food samples, onion tissues, and zebrafish with satisfactory results. We predict that KQ-SO2 could be used as a powerful tool to reveal the relationship between HSO3- and the human health.

A novel fluorescent probe for ratiometric detection of formaldehyde in real food samples, living tissues and zebrafish.

Herein, a two-photon (TP) ratiometric fluorescent probe (NpFA) was developed for detecting formaldehyde (FA) in real food samples, living onion tissues and zebrafish by fluorescence resonance energy transfer (FRET) strategy. Specifically, a TP fluorophore as the donor and a FA turn-on naphthalimide fluorophore as the acceptor were connected by a non-conjugated linker to construct the TP-FRET-based NpFA, which exhibited a target-modulated ratiometric fluorescence response to FA rapidly with high selectivity and sensitivity during 65 s, and a large ratio ~5-fold enhancement at I550/I410 after addition of FA, displaying ~60-fold enhancement at 550 nm and a quite low DOC of 5.8 ± 0.2 nM. Moreover, NpFA has a good imaging resolution and depth of deep tissue penetration. Therefore, based on the above results, NpFA has the capability to be a useful tool for investigating FA in real samples application, and we also hope NpFA will further study of the physiological and pathological function of FA.

TP-FRET-Based Fluorescent Sensor for Ratiometric Detection of Formaldehyde in Real Food Samples, Living Cells, Tissues, and Zebrafish.

Formaldehyde (FA, HCHO) is a highly reactive carbonyl species, which is very harmful to humans and the environment as a tissue fixative and preservative. Therefore, developing some highly sensitive, selective, and rapid detection methods is significant for human health in food safety and environmental protection. Herein, a two-photon (TP) ratiometric sensor, CmNp-CHO, has been constructed by conjugating a TP donor (Π-push-pull-structure) with a FA off-on acceptor (functioned with hydrazide moiety) via a nonconjugated linker through the fluorescence resonance energy transfer mechanism. Such a scaffold affords CmNp-CHO a reliable and specific probe for detecting FA with two well-resolved emission peaks separated by 124 nm. Also, it responds to FA rapidly with high selectivity and sensitivity during 1.0 min and a large ratio enhancement at I550/I426 with addition of 0-20µM FA, exhibiting ∼4-fold ratio increase and a fairly low LOD of 8.3 ± 0.3 nM. Moreover, CmNp-CHO has been successfully employed for detecting FA in live cells, onion tissues, and zebrafish, exhibiting that CmNp-CHO can serve as a useful tool for investigating FA in real food application and offering strong theoretical support and technical means for investigation of physiological and pathological functions of FA.

An efficient TP-FRET-based lysosome-targetable fluorescent probe for imaging peroxynitrite with two well-resolved emission channels in living cells, tissues and zebrafish.

Peroxynitrite (ONOO-), an important role of reactive oxygen species (ROS) in vivo, and studies showed abnormal of ROS can induce lysosomal membrane permeabilization (LMP) and lead to the death of cells. Thus, it is of great significance for designing an effective method for investigating relationship between physiology and pathology between ONOO- and lysosome. Herein, for the first time, we adopted a Förster resonance energy transfer (FRET) strategy to construct a lysosome-targetable small molecular ratiometric two-photon (TP) fluorescent probe (NpRh-ONOO) for detecting ONOO- in living cells, tissues and zebrafish. Specifically, a TP fluorophore and a rhodamine B fluorophore are directly connected by a flexible piperidine linker to form the TP-FRET-scaffold, a hydrazide as ONOO- reactive set, and a dimethylamino as lysosome targeting-group, which shows a target-modulated ratiometric TP fluorescence response, two well-resolved emission peaks separated by 73 nm, rapid response (<10 s), high selectivity and sensitivity with the detection limit is as low as 3.3 nM for ONOO-. These prominent features of probe were then applied for ratiometric bioimaging both exogenous and endogenous peroxynitrite in living cells, tissues and zebrafish, demonstrating it can be used as a powerful tool for biological research of lysosomal peroxynitrite in biological systems.

An effective FRET-based two-photon ratiometric fluorescent probe with double well-resolved emission bands for lysosomal pH changes in living cells and zebrafish.

In cells, lysosome is an acidic organelle (approximately pH 4.5-5.5), whose pH changes plays a key role in mediating various biological processes. To address this issue, a lot of fluorescent probes have been developed and prepared for tracking lysosomal pH changes. However, few of these probes can realize the imaging of lysosomal pH changes in biosystems. Herein, a new two-photon (TP) ratiometric fluorescent probe (NpRhLys-pH) by adopting the fluorescence resonance energy transfer (FRET) strategy has been developed for imaging of lysosomal pH changes in living cells and zebrafish. In this probe NpRhLys-pH, constructed by conjugating a TP fluorophore (D-Π-A-structured naphthalimide derivative) with a rhodamine B fluorophore via a non-conjugated flexible linker, the morpholine moiety serves as a targeting unit for anchoring lysosomes, and the xanthane derivative shows a pH-modulated open/close form of the spirocycle. Such a scaffold affords the NpRhLys-pH is a reliable and specific probe for anchoring lysosomes in living cells and zebrafish with dual-channel emission peaks separated by 85 nm, and responds to lysosomal pH rapidly and reversibly with high selectivity and sensitivity, demonstrating it can be used as a powerful tool for the biological research of the relationship between physiology and pathology and lysosomal pH changes in biological systems.

A TP-FRET-based fluorescent sensor for ratiometric visualization of selenocysteine derivatives in living cells, tissues and zebrafish.

Selenium is a biologically essential micronutrient element serving as an essential building block for selenoproteins (SePs), which is playing a key role in various cellular functions. Hence, it is of great significance to developing a reliable and rapid method for detection of Sec in biosystems. Compared with the previously reported probes that have been developed for selective detection of Sec, two-photon (TP) ratiometric Sec-specific probes would be advantageous for the NIR excitation and built-in correction of the dual emission bands. To quantitatively and selectively detect Sec over biothiols with rapid and sensitive response, we for the first time report a new fluorescence resonance energy transfer (FRET)-based TP ratiometric fluorescence probe CmNp-Sec, which was constructed by conjugating a TP fluorophore 6 (coumarin derivative with a D-π-A-structure) with a naphthalimide fluorophore 9 via a non-conjugated linker, and employed a 4-dinitrobenzene-ether (DNB) with a strong ICT effect as Sec responsive moiety. It exhibits quantitatively detect Sec in a wide range (0-50 µM) with a limit of detection of 7.88 nM within 10 min. More impressively, this probe can be conveniently used to detect Sec in living cells, tissues and zebrafish, demonstrating it has the latent capability in further biological applications.