Modulation of Near-Infrared Mitochondria-Targetable fluorescent probe for H2S bioimaging through the modification of heavy atom iodine.

H2S is correlated with mitochondrial dysfunction, which results in the death of cells. Two near-infrared fluorescent probes, Mito-HS-1 and Mito-HS-2, were designed for mitochondrial H2S imaging. Initially, the synthesis protocol of expensive IR-780-based hemicyanine (HXPI) was optimized with an appreciate yield of 80 % as compared with 14-56 % previously reported. Iodine atom was introduced to HXPI to obtain iodine-HXPI whose Stokes shift was increased to be 90 nm. On account of the rapid and fast nucleophilic attack of H2S, HXPI-based Mito-HS-1 could be applied for the real time imaging of mitochondrial H2S. Besides some similar optical properties with Mito-HS-1, iodine-HXPI-based Mito-HS-2 exhibited wider linear range (3-150 µM), more stable fluorescent imaging and more favorable specificity in vitro. Both Mito-HS-1 and Mito-HS-2 could be used to image exogenous H2S in cells, with Mito-HS-2 showing fairly better signal-to-noise. Additionally, the Pearson correlation coefficient of two probes demonstrated that they could successfully monitor mitochondrial H2S in A549 cells and Hela cells.

Cu-In-S/ZnS:Gd3+ quantum dots with isolated fluorescent and paramagnetic modules for dual-modality imaging in vivo.

Gd3+-doped quantum dots (QDs) have been widely used as small-sized bifunctional contrast agents for fluorescence/magnetic resonance (FL/MR) dual-modality imaging. However, Gd3+ doping will always compromise the FL of host QDs. Therefore, balancing the Gd3+ doping and the optical properties of QDs is crucial for constructing high-performance bifunctional nanoprobes. Additionally, most paramagnetic QDs are synthesized in the organic phase and need to be transferred to the aqueous phase for bioimaging. Herein, ingeniously designed shell-doped Cu-In-S/ZnS:Gd3+ QDs have been prepared in the aqueous phase. It has been demonstrated that isolating paramagnetic Gd3+ from fluorescent Cu-In-S core via doping Gd3+ into ZnS shell not only avoided the decrease of FL quantum yield (QY), but also ensured the water accessibility of paramagnetic Gd3+ ions, by which the FL QY and r1 relaxivity of Cu-In-S/ZnS:Gd3+ QDs achieved as much as 15.6% and 15.33 mM-1·s-1, respectively. These high-performance QDs with excellent stability, low biotoxicity, and good tumor permeability were successfully applied for in vivo tumor FL/MR dual-modality imaging, and have shown significant potential in the precision detection and diagnosis of diseases.

Radical nucleophilic substitution/cyclization: A novel strategy for selective and ultrafast fluorescence imaging of cysteine levels in ferroptosis process.

Diphenylisoindolo[2,1-a]quinoline can be used to detect cysteine among homocysteine, glutathione, and other 19 natural amino acids. Unlike other reported probes, the response mechanism involves sulfhydryl radical nucleophilic substitution and cyclization, and thus the differences in ring-formation kinetics enable high selectivity. After treated with Cys, the response process was completed rapidly and the maximum fluorescence intensity (at 496 nm) was reached extremely fast (<1 s) when excited at 380 nm in MeCN-PBS buffer (10.0 mM, pH = 7.4, 3:7 (v/v)). The quantum yield after the reaction was increased almost 7 times to be 0.02 from 0.003. Fluorescence intensity displayed a good quantitative linear relationship in the range 1-10 µM Cys with a detection limit of 270 nM. Furthermore, the probe was demonstrated for real-time monitoring of intracellular cysteine levels within HepG2 cells in ferroptosis process.

Base excision-initiated terminal deoxynucleotide transferase-assisted amplification for simultaneous detection of multiple DNA glycosylases.

Various DNA glycosylases involved in base excision repair may be associated with a wide disease spectrum that includes cancer, myocardial infarction, neurodegenerative disorders, etc. In this paper, we developed a sensitive method for simultaneous detection of multiple DNA glycosylases based on the target-initiated removal of damaged base and terminal deoxynucleotidyl transferase (TdT)-assisted labeling and signal amplification. We designed three specific stem-loop probes which contained specific targeting damaged bases in the stem for uracil DNA glycosylase (UDG), human alkyladenine DNA glycosylase (hAAG), and human 8-oxoguanine DNA glycosylase 1 (hOGG1), respectively. Target DNA glycosylase can initiate the recognition and clearance of damaged base on immobilized 3' blocked stem-loop probe, releasing apurine/apyrimidine (AP) site which can be hydrolyzed by AP endonuclease to produce 3'OH probe fragment for TdT extension. Numerous biotin-modified dUTPs were successively labeled on the 3' terminus of the probe fragments, and then reacted with streptavidin-phycoerythrin (SA-PE) for analysis by using the Luminex xMAP array platform. The amplification strategy based on TdT has been utilized to simultaneously and sensitively detect three different DNA glycosylases with detection limits of 10-3 U/ml. Moreover, it could be applied for analyzing DNA glycosylase activity in complex HeLa cell lysate samples. Therefore, this strategy possesses the advantages of high sensitivity, specificity, and multiplex, holding great potential for DNA glycosylase-related biomedical research.

PEGylated near-infrared fluorescence probe for mitochondria-targetable hydrogen peroxide detection.

Oxidative stress plays significant roles in the development of various diseases. H2O2 acts as a signaling molecule physiologically or harmful substance pathologically and the mitochondria are one of the most active places for the generation of H2O2. Thus, a new mitochondria-targeted probe 1 for H2O2 detection was synthesized herein, based on D-π-A structure with a large Stokes shift (150 nm) due to its ICT process. To improve its water solubility and sensitivity, probe 2 with PEG chain and probe 3 with two responsive boronated groups were then designed based on the structure of probe 1. As a result, the fluorescence intensity of probe 2 was far higher than that of probe 1 and probe 3 not only in vitro experiment but in cell imaging study with a larger linear range and signal-to-noise ratio, rendering it the best probe for further exogenous and endogenous H2O2 detection in Hela cells.

Sensitive detection of transcription factor by coupled fluorescence-encoded microsphere with exonuclease protection.

Aberrant transcription factors (TFs) activities are closely related to the occurrence and development of various diseases. Herein, we presented a fluorescence-encoded microsphere-based approach for TFs detection coupling with common DNA footprinting assay. Target TFs specifically bound the binding sites of double-stranded DNA (dsDNA) probes which were conjugated to microspheres. Thus, the probes were protected from being hydrolyzed by exonuclease III (Exo III). Afterwards, biotins labeled on the probes reacted with streptavidin-phycoerythrin (SA-PE) to produce fluorescent signal; however, in the absence of target TFs, the dsDNA probes would be hydrolyzed by Exo III resulting in biotins falling off and thus fluorescence signal was not generated. This strategy can be used to detect nuclear factor-kappa B p50 (NF-κB p50) with a detection limit of 0.2 nM. The steric hindrance of microspheres overcome the disadvantage of Exo III that can nibble into the protein-bound DNA region. Meanwhile, the fluorescent label of microsphere was specific to each TF, enabling multiplex detection could be achieved by changing specific protein binding site of corresponding dsDNA probe. This method has been successfully applied for simultaneous detection of NF-κB p50, AP-1 and CREB in nuclear extract isolated from HeLa cells stimulated or unstimulated by TNF-α, showing great potential for biomedical researches and precise disease diagnosis.

Small-sized copolymeric nanoparticles for tumor penetration and intracellular drug release.

Poor solid-tumor penetration of nanocarriers limits the drug efficacy. Herein, small-sized copolymeric nanoparticles are prepared for delivering the chemotherapeutic drug DOX into solid tumors deeply and releasing the drug effectively. These small-sized copolymeric nanoparticles represent substantial potential for clinical translation.