The real-time monitoring of nitric oxide (NO) at the subcellular level is still a great challenge. To attain this goal, we developed a fast and selective near-infrared (NIR) fluorescent probe for the targeted tracing of endogenous NO. This probe possesses vital features for the real-time detection of intracellular NO including a significant turn-on NIR response, high specificity, and a fast response by a controlled photoinduced electron transfer (PET) process, which is applicable to the real-time monitoring of endogenous NO in mitochondria.

Curcuminoid difluoroboron has attractive performance as a promising near-infrared (NIR) fluorescent dye. In this contribution, we designed and synthesized a mitochondria-targeted NIR fluorescent probe DFB1 based on difluoroboron curcuminoid scaffold for the detection of Cys (cysteine). DFB1 employs a curcumin analog as the NIR fluorophore, an acrylate group containing α, β-unsaturated ketone as a functional trigger moiety for Cys, and a triphenylphosphonium (TPP) cation moiety for specifically targeting mitochondria. The remarkable shift of DFB1 with Cys was observed from 470 nm to 590 nm in absorption spectra and from 560 nm to 680 nm in emission spectra. Notably, DFB1 manifests significantly dual-channel and turn-on NIR fluorescent signals simultaneously in response to Cys concentration, which make it favorable for monitoring endogenous Cys activity in vivo. This probe has high sensitivity and selectivity for the detection of Cys over homocysteine (Hcy) and glutathione (GSH). This specific response for Cys was based on differences kinetics of intramolecular adduct/cyclizations. More importantly, biological experiments indicated that this probe could be utilized for the detection of endogenous mitochondrial Cys in living cells.A highly selective dual-channel NIR fluorescent probe (DFB1) based on curcuminoid difluoroboron is developed for discrimination Cys over GSH, Hcy and other amino acids in mitochondria of living cells.Download high-res image (162KB)Download full-size image

Development of “smart” noninvasive bioimaging probes for trapping specific enzyme activities is highly desirable for cancer therapy in vivo. Given that β-galactosidase (β-gal) is an important biomarker for cell senescence and primary ovarian cancers, we design an enzyme-activatable ratiometric near-infrared (NIR) probe (DCM-βgal) for the real-time fluorescent quantification and trapping of β-gal activity in vivo and in situ. DCM-βgal manifests significantly ratiometric and turn-on NIR fluorescent signals simultaneously in response to β-gal concentration, which makes it favorable for monitoring dynamic β-gal activity in vivo with self-calibration in fluorescent mode. We exemplify DCM-βgal for the ratiometric tracking of endogenously overexpressed β-gal distribution in living 293T cells via the lacZ gene transfection method and OVCAR-3 cells, and further realize real-time in vivo bioimaging of β-gal activity in colorectal tumor-bearing nude mice. Advantages of our system include light-up ratiometric NIR fluorescence with large Stokes shift, high photostability, and pH independency under the physiological range, allowing for the in vivo real-time evaluation of β-gal activity at the tumor site with high-resolution three-dimensional bioimaging for the first time. Our work provides a potential tool for in vivo real-time tracking enzyme activity in preclinical applications.

Real-time tracking for where (W), when (W), and how (H) prodrugs are delivered and activated in vivo is a great challenge for prodrug development. Disulfide linkage-based prodrugs as well as their delivery systems have been studied extensively, but the WWH question in spatial and temporal (spatiotemporal) precision remains unanswered. Herein, we present a novel prodrug of camptothecin (CPT) linked to a near-infrared (NIR) cyanine dye via a disulfide linkage (Cy-S-CPT). The cleavage of the disulfide bond in Cy-S-CPT by endogenous glutathione (GSH) can activate the anti-cancer drug CPT and induce a remarkable fluorescence shift from 825 to 650 nm, thereby providing dual fluorescent channels to real-time track the prodrug biodistribution and activation in vivo. Impressively, the dual-channel NIR fluorescence bioimaging exhibits the pervasive drug distribution, i.e., the biodistribution of the intact prodrug was traced at the 825 nm-NIR fluorescence channel, whereas the activated drug was tracked at the 650 nm red fluorescence channel. In this way, we can overcome the blind spot in the metabolism kinetics of prodrugs in a certain organ or tissue. As demonstrated, the prodrug prompts activation in all the organs, particularly in the liver after an intravenous injection, and achieves predominant accumulation and activation in tumors at 24 h post injection. Cy-S-CPT loaded in PEG–PLA nanoparticles display significantly improved therapeutic efficacy and low side effects with respect to the clinical used drug CPT-11. As a consequence, the NIR spatiotemporal bioimaging in vivo with dual fluorescence channels allows the prodrug release profile to be extracted precisely, particularly in visualizing drug-released information from complex biological systems such as mice, thereby providing a unique opportunity to take insight into the relationship between theranosis and pharmacokinetics.

The development of innovative strategies for high-performance near-infrared (NIR) fluorescent materials is in urgent demand for bioimaging. By replacing the stronger electron-withdrawing groups or extending the π-conjugated system, novel NIR fluorescent materials of DCM analogues have been developed, along with several striking characteristics: bright NIR fluorescence over 700 nm, large Stokes shift and good photo-stability. It is demonstrated that introducing a stronger electron-withdrawing unit to the acceptor moiety of DCM analogues is a favourably efficient strategy to tune and prolong the emission wavelength into the NIR region with a large Stokes shift. In comparison with the commercial NIR dye ICG, S-DCM-N and S-DCM-P display excellent photostability and low photobleaching. The large Stokes Shift and NIR fluorescence channel of S-DCM-N and S-DCM-P are very favourable for fluorescence labelling with a high signal-to-noise ratio in living species.

Leucine aminopeptidase (LAP), one of the important proteolytic enzymes, is intertwined with the progress of many pathological disorders as a well-defined biomarker. To explore fluorescent aminopeptidase probe for quantitative detection of LAP distribution and dynamic changes, herein we report a LAP-targeting near-infrared (NIR) fluorescent probe (DCM–Leu) for ratiometric quantitative trapping of LAP activity in different kinds of living cells. DCM–Leu is composed of a NIR-emitting fluorophore (DCM) as a reporter and l-leucine as a triggered moiety, which are linked together by an amide bond specific for LAP cleavage. High contrast on the ratiometric NIR fluorescence signal can be achieved in response to LAP activity, thus enabling quantification of endogenous LAP with “build-in calibration” as well as minimal background interference. Its ratiometric NIR signal can be blocked in a dose-dependent manner by bestatin, an LAP inhibitor, indicating that the alteration of endogenous LAP activity results in these obviously fluorescent signal responses. It is worth noting that DCM–Leu features striking characteristics such as a large Stokes shift (∼205 nm), superior selectivity, and strong photostability responding to LAP. Impressively, not only did we successfully exemplify DCM–Leu in situ ratiometric trapping and quantification of endogenous LAP activity in various types of living cells, but also, with the aid of three-dimensional confocal imaging, the intracellular LAP distribution is clearly observed from different perspectives for the first time, owing to the high signal-to-noise of ratiometric NIR fluorescent response. Collectively, these results demonstrate preclinical potential value of DCM–Leu serving as a useful NIR fluorescent probe for early detection of LAP-associated disease and screening inhibitor.Keywords: fluorescent probe; in situ; leucine aminopeptidase; near-infrared; ratiometric

Flash nanoprecipitation (FNP) is an easily scalable and fast processing method for the preparation of nanoparticles (NPs) with simple vortex equipment. By using the FNP method, fluorescent NPs are prepared in less than 1 s in a multi-inlet vortex mixer, in which hydrophobic aggregation-induced emission (AIE)-active dye of EDP is incorporated within the biocompatible block copolymer poly(ethylene glycol)-b-poly(ε-caprolactone) for EDP NP assembly. The formulation parameters of stream velocity, dyes, and loading and concentration in FNP are optimized. The sizes of the NPs ranged from 20 to 60 nm with a ratio change of mixed solvents. As a control, an aggregation-caused quenching (ACQ) molecule of BDP was also synthesized for BDP NPs. To gain insight into the effect of the polymer on the aggregation state of hydrophobic dyes, the preparation of EDP and BDP NPs without block copolymer was also investigated. Apparently, the sizes of the NPs display large distributions without an amphiphilic block copolymer as the engineering template, suggesting that the block of polymers plays a key role in tuning the aggregation state of encapsulated dyes in FNP processes. Moreover, the peak shifts of dye with different microenvironments also confirmed the successful encapsulation of fluorescent dye in the NP cores. Finally, by externally applied forces in the FNP method, the engineered assembly of AIE-active fluorescent NPs possessing a narrow size distribution with desirable fluorescence properties was obtained. These features provide the possibility of rapidly constructing controllable AIE-active fluorescent NPs as biomedical tracers.

α-Ketoglutarate (α-KA) can convert to 2-hydroxyglutarate (2-HG), which is confirmed to be associated with many diseases, especially with acute myeloid leukemia (AML). In this paper, a novel reaction-based chemosensor DT based on the typical Schiff-base reaction was designed for sensing the biomarker of α-KA, in which a diazanyl group as the recognition group was linked with a benzothiadiazole unit as the fluorophore moiety. Considering the typical Schiff-base reaction to generate hydrazones suffering from slow kinetics, particularly under neutral conditions, a series of parallel experiments was conducted for optimizing the chemical recognition process, including varying the solvent, reaction temperature, reactant concentration, and reaction rate. The optimum condition was established as a pH value, temperature, α-KA concentration, and response time of 5.7, 30 °C, 100 μM, and 20 min, respectively. Notably, in contrast with the initial 6.3-fold fluorescence enhancement, the remarkable 75-fold fluorescence enhancement ((I – I0)/I0 at 560 nm) was observed by optimizing the chemical recognition process of DT and α-KA. Finally, DT was carried out for the chemical recognition processing of α-KA in serum. We demonstrated that DT is selective for α-KA over other potential biologically interferences with similar structures and thus is suitable for detecting α-KA in serum. On the basis of the optimized chemical recognition process, DT shows high potential application for sensing α-KA with remarkable fluorescence enhancement. This work provided a potential method that is quick and convenient for sensing biomarker α-KA in serum. It is worth noting that without complicated pretreatment, utilizing a novel reaction-based fluorescent chemosensor may establish a new promising platform for clinical diagnosis biomarker.

In vivo monitoring of the biodistribution and activation of prodrugs is urgently required. Near infrared (NIR) fluorescence-active fluorophores with excellent photostability are preferable for tracking drug release in vivo. Herein, we describe a NIR prodrug DCM-S-CPT and its polyethylene glycol–polylactic acid (PEG-PLA) loaded nanoparticles as a potent cancer therapy. We have conjugated a dicyanomethylene-4H-pyran derivative as the NIR fluorophore with camptothecin (CPT) as the anticancer drug using a disulfide linker. In vitro experiments verify that the high intracellular glutathione (GSH) concentrations in tumor cells cause cleavage of the disulfide linker, resulting in concomitantly the active drug CPT release and significant NIR fluorescence turn-on with large Stokes shift (200 nm). The NIR fluorescence of DCM-S-CPT at 665 nm with fast response to GSH can act as a direct off–on signal reporter for the GSH-activatable prodrug. Particularly, DCM-S-CPT possesses much better photostability than ICG, which is highly desirable for in situ fluorescence-tracking of cancer chemotherapy. DCM-S-CPT has been successfully utilized for in vivo and in situ tracking of drug release and cancer therapeutic efficacy in living animals by NIR fluorescence. DCM-S-CPT exhibits excellent tumor-activatable performance when intravenously injected into tumor-bearing nude mice, as well as specific cancer therapy with few side effects. DCM-S-CPT loaded in PEG-PLA nanoparticles shows even higher antitumor activity than free CPT, and is also retained longer in the plasma. The tumor-targeting ability and the specific drug release in tumors make DCM-S-CPT as a promising prodrug, providing significant advances toward deeper understanding and exploration of theranostic drug-delivery systems.

Water-soluble, long wavelength fluorescent aggregation-induced emission (AIE)-active materials are in great demand for high contrast biosensing and bioimaging. The substitution position effects of the sulfonate group on the basis of two quinoline-malononitrile (QM) derivatives (EDS and EDPS) provide insight into efficient modulation in the hydrophilicity, emitting color, and specific AIE characteristics. EDS shows a unique AIE behaviour in aqueous solution, but EDPS does not. The abnormal non-fluorescence aggregation for EDS in pure water is capsule-like with loose packing characteristics, but still has enough cavities or free volume to consume the radiative energy, resulting in nearly no fluorescence. When binding with the protein BSA, the sulfonate unit as a conformation function group (CFG) plays a vital role in altering its initial loose ensemble into tightly compact aggregation with light-up AIE characteristics. By cell tracking, dynamic light scattering (DLS) and transmission electron microscopy (TEM), the key role of sulfonate groups in the conformation alteration has been well demonstrated for the first time. Moreover, EDS is successfully exploited in a label-free real time AIE fluorescent assay for trypsin detection and inhibitor screening. The hydrophilic sulfonate group from the different substitution position in the AIE-active QM building blocks provides an effective way to tailor the intermolecular aggregation associated with molecular stacking, especially for in situ cell tracking and real-time trypsin detection.

Detection of biomarkers via optical microfluidic chips is in great demand for high contrast biosensing and bioimaging. In distinct contrast with traditional chromatographic methods, which require tedious pretreatment and are not directly applicable in blood serum, a “turn-on” fluorescent sensor for the cancer cell damaging agent α-ketoglutaric acid (α-KA) has been established. A hydrazino group is introduced into the naphthalimide moiety as the reaction trigger for the specific fluorescence turn-on response. Under the rational design, probe 3 can successfully detect α-KA in a purely aqueous system, along with approximately 7-fold fluorescence enhancement and a rapid response with the aid of micelles. The sensor exhibits good selectivity among 20 common amino acids, in particular showing little interference with various dicarbonyl derivatives and reactive oxygen species. Finally, the detection of α-KA in human serum is demonstrated in a microfluidic chip, indicative of a potential platform for high-throughput screening and monitoring of kinetics, especially in biological fields.

A ratiometric and colorimetric cyanine-based palladium sensor with an excellent selectivity and sensitivity has been designed. Notably, the modulation of π-conjugated electrons in cyanine dyes can result in a ratiometric fluorescence change with a large Stokes shift (270 nm), especially for realizing palladium detection in aqueous samples using indicator paper and in living cells by ratiometric mode. The limit of detection is as low as 0.3 ppb.

The donor–acceptor system of indoline–benzothiadiazole is established as the novel and reactive platform for generating amine radical cations with the interaction of Cu2+, which has been successfully exploited as the building block to be highly sensitive and selective near infrared (NIR) colorimetric and fluorescent Cu2+ sensors. Upon the addition of Cu2+, an instantaneous red shift of absorption spectra as well as the quenched NIR fluorescence of the substrates is observed. The feasibility and validity of the radical cation generation are confirmed by cyclic voltammetry and electron paramagnetic resonance spectra. Moreover, the introduction of an aldehyde group extends the electron spin density and changes the charge distribution. Our system demonstrates the large scope and diversity in terms of activation mechanism, response time, and property control in the design of Cu2+ sensors.Keywords: copper sensors; donor−acceptor; indoline; near-infrared; radical cations;

Detection of biomarkers via optical microfluidic chips is in great demand for high contrast biosensing and bioimaging. In distinct contrast with traditional chromatographic methods, which require tedious pretreatment and are not directly applicable in blood serum, a “turn-on” fluorescent sensor for the cancer cell damaging agent α-ketoglutaric acid (α-KA) has been established. A hydrazino group is introduced into the naphthalimide moiety as the reaction trigger for the specific fluorescence turn-on response. Under the rational design, probe 3 can successfully detect α-KA in a purely aqueous system, along with approximately 7-fold fluorescence enhancement and a rapid response with the aid of micelles. The sensor exhibits good selectivity among 20 common amino acids, in particular showing little interference with various dicarbonyl derivatives and reactive oxygen species. Finally, the detection of α-KA in human serum is demonstrated in a microfluidic chip, indicative of a potential platform for high-throughput screening and monitoring of kinetics, especially in biological fields.

A ratiometric and colorimetric cyanine-based palladium sensor with an excellent selectivity and sensitivity has been designed. Notably, the modulation of π-conjugated electrons in cyanine dyes can result in a ratiometric fluorescence change with a large Stokes shift (270 nm), especially for realizing palladium detection in aqueous samples using indicator paper and in living cells by ratiometric mode. The limit of detection is as low as 0.3 ppb.

Real-time tracking for where (W), when (W), and how (H) prodrugs are delivered and activated in vivo is a great challenge for prodrug development. Disulfide linkage-based prodrugs as well as their delivery systems have been studied extensively, but the WWH question in spatial and temporal (spatiotemporal) precision remains unanswered. Herein, we present a novel prodrug of camptothecin (CPT) linked to a near-infrared (NIR) cyanine dye via a disulfide linkage (Cy-S-CPT). The cleavage of the disulfide bond in Cy-S-CPT by endogenous glutathione (GSH) can activate the anti-cancer drug CPT and induce a remarkable fluorescence shift from 825 to 650 nm, thereby providing dual fluorescent channels to real-time track the prodrug biodistribution and activation in vivo. Impressively, the dual-channel NIR fluorescence bioimaging exhibits the pervasive drug distribution, i.e., the biodistribution of the intact prodrug was traced at the 825 nm-NIR fluorescence channel, whereas the activated drug was tracked at the 650 nm red fluorescence channel. In this way, we can overcome the blind spot in the metabolism kinetics of prodrugs in a certain organ or tissue. As demonstrated, the prodrug prompts activation in all the organs, particularly in the liver after an intravenous injection, and achieves predominant accumulation and activation in tumors at 24 h post injection. Cy-S-CPT loaded in PEG–PLA nanoparticles display significantly improved therapeutic efficacy and low side effects with respect to the clinical used drug CPT-11. As a consequence, the NIR spatiotemporal bioimaging in vivo with dual fluorescence channels allows the prodrug release profile to be extracted precisely, particularly in visualizing drug-released information from complex biological systems such as mice, thereby providing a unique opportunity to take insight into the relationship between theranosis and pharmacokinetics.

Water-soluble, long wavelength fluorescent aggregation-induced emission (AIE)-active materials are in great demand for high contrast biosensing and bioimaging. The substitution position effects of the sulfonate group on the basis of two quinoline-malononitrile (QM) derivatives (EDS and EDPS) provide insight into efficient modulation in the hydrophilicity, emitting color, and specific AIE characteristics. EDS shows a unique AIE behaviour in aqueous solution, but EDPS does not. The abnormal non-fluorescence aggregation for EDS in pure water is capsule-like with loose packing characteristics, but still has enough cavities or free volume to consume the radiative energy, resulting in nearly no fluorescence. When binding with the protein BSA, the sulfonate unit as a conformation function group (CFG) plays a vital role in altering its initial loose ensemble into tightly compact aggregation with light-up AIE characteristics. By cell tracking, dynamic light scattering (DLS) and transmission electron microscopy (TEM), the key role of sulfonate groups in the conformation alteration has been well demonstrated for the first time. Moreover, EDS is successfully exploited in a label-free real time AIE fluorescent assay for trypsin detection and inhibitor screening. The hydrophilic sulfonate group from the different substitution position in the AIE-active QM building blocks provides an effective way to tailor the intermolecular aggregation associated with molecular stacking, especially for in situ cell tracking and real-time trypsin detection.

The development of innovative strategies for high-performance near-infrared (NIR) fluorescent materials is in urgent demand for bioimaging. By replacing the stronger electron-withdrawing groups or extending the π-conjugated system, novel NIR fluorescent materials of DCM analogues have been developed, along with several striking characteristics: bright NIR fluorescence over 700 nm, large Stokes shift and good photo-stability. It is demonstrated that introducing a stronger electron-withdrawing unit to the acceptor moiety of DCM analogues is a favourably efficient strategy to tune and prolong the emission wavelength into the NIR region with a large Stokes shift. In comparison with the commercial NIR dye ICG, S-DCM-N and S-DCM-P display excellent photostability and low photobleaching. The large Stokes Shift and NIR fluorescence channel of S-DCM-N and S-DCM-P are very favourable for fluorescence labelling with a high signal-to-noise ratio in living species.