Yunfei Long

Selective visualization of endogenous hypochlorous acid in zebrafish during lipopolysaccharide-induced acute liver injury using a polymer micelles-based ratiometric fluorescent probe

The development of effective method for monitoring of endogenous hypochlorous acid (HClO) in vivo is of great significance for early diagnosis of lipopolysaccharide (LPS) induced acute liver injury. Herein, we report a polymer micelles-based ratiometric fluorescent probe (PM) based on the combination of intramolecular charge transfer (ICT) mechanism and fluorescence resonance energy transfer (FRET) principle for selective visualization of endogenous HClO in vivo. Upon the reaction of PM with HClO, the electron-donating thiourea moiety is oxidized and transformed into imidazoline moiety (electron-withdrawing group), resulting in a dramatic blue shift (˃100nm) in the fluorescence emission. The as-prepared PM shows good water dispersibility (100% aqueous media), fast response (<40s), high sensitivity (a detection limit of 1.75nM), and outstanding selectivity toward HClO over other ROS/RNS (50 equiv.). In addition, the vivo imaging experiments demonstrate that PM facilitates the visualization of endogenous HClO generation with LPS induced acute liver injury in zebrafish model.

A sensitive spectrofluorometric method for detection of berberine hydrochloride using Ag nanoclusters directed by natural fish sperm DNA

A novel DNA-directed AgNCs (DNA-AgNCs) was synthesized with economical raw material (natural fish sperm DNA) through a simple and rapid approach, and it first showed high and stable fluorescence emission as a AgNCs stabilized by natural DNA at about 635nm. Moreover, its emission intensity could be enhanced tremendously in acetic acid (HAc) medium. Whereas, when berberine hydrochloride (BRH) entered the solution system, it would interact and combine efficiently with DNA on the surface of AgNCs, which could lead to subtle change of charge distribution on its surface, and make it more lyophobic, inducing aggregation of DNA-AgNCs. As a result, fluorescence of the system was quenched visually; the process represented a color variance from yellow to hot pink under HAc medium, then back to yellowish-brown when BRH worked. Based on above phenomenon, a selective and accurate spectrofluorometric method for BRH detection was established. It can be applied to detect trace amounts of BRH in aqueous solution in the linear range from 1.0nM to 2000.0nM; and the detection limit (3σ/k) was 0.3nM, which is pretty lower compared to most reported spectral methods. Simultaneously, a semi-quantitative determination by visual evaluation from 5.0nM to 2000.0nM was also achieved. This method provided excellent selectivity for the detection of BRH in the presence of ten kinds of common natural amino acids and nine kinds of common mental ions. Furthermore, the BRH content in compound berberine tablets from drugstore was successfully investigated by this method and the results showed high accuracy.

One-pot fabrication of FRET-based fluorescent probe for detecting copper ion and sulfide anion in 100% aqueous media.

The design of effective tools for detecting copper ion (Cu2+) and sulfide anion (S2-) is of great importance due to the abnormal level of Cu2+ and S2- has been associated with an increase in risk of many diseases. Herein, we report on the fabrication of fluorescence resonance energy transfer (FRET) based fluorescent probe PF (PEI-FITC) for detecting Cu2+ and S2- in 100% aqueous media via a facile one-pot method by covalent linking fluorescein isothiocyanate (FITC) with branched-polyethylenimine (b-PEI). PF could selectively coordinate with Cu2+ among 10 metal ions to form PF-Cu2+ complex, resulting in fluorescence quenching through FRET mechanism. Furthermore, the in situ generated PF-Cu2+ complex can be used to selectively detect S2- based on the displacement approach, resulting in an off-on type sensing. There is no obvious interference from other anions, such as Cl-, NO3-, ClO4-, SO42-, HCO3-, CO32-, Br-, HPO42-, F- and S2O32-. In addition, PF was successfully used to determine Cu2+ and S2- in human serum and tap water samples. Therefore, the FRET-based probe PF may provide a new method for selective detection of multifarious analysts in biological and environmental applications, and even hold promise for application in more complicated systems.

Selective visualization of endogenous hydrogen sulfide in lysosomes using aggregation induced emission dots

The development of novel fluorescent probes for selective detection of endogenous hydrogen sulfide (H2S) in lysosomes is imperative for elucidating its functions associated with diseases. Nevertheless, the reported fluorescent probes for lysosomal H2S detection usually undergo an aggregation caused quenching (ACQ) phenomenon in the aggregate state and cause the self-quenching of fluorescence due to the “self-absorption” effect with a small Stokes shift, which largely limited their sensing application. To solve this problem, we present the aggregation induced emission dots (AIED) for selective visualization of endogenous H2S in lysosomes via a simple co-precipitation method. It is worth noting that AIED with excellent AIE properties and large Stokes shift (∼150 nm) could effectively prevent the notorious ACQ phenomenon and the self-quenching of fluorescence. In addition, the as-prepared AIED exhibits favorable features including good water dispersibility, high sensitivity (∼43.8 nM), outstanding selectivity toward H2S over other thiols, long-term stability (>15 weeks) and good biocompatibility. Significantly, the resultant AIED with good membrane permeability has been successfully employed to visualize the endogenous H2S in lysosomes in living cells. We expect that AIED can serve as a useful tool to investigate the roles of lysosomal H2S in H2S-associated diseases.

Real-time monitoring of endogenous cysteine levels in living cells using a CD-based ratiometric fluorescent nanoprobe

AbstractA simple and readily available fluorescent probe is needed for the real-time monitoring of endogenous cysteine (Cys) levels in living cells, as such a probe could be used to study the role of Cys in related diseases. Herein, we report the first fluorescent probe based on carbon dots (CDs-FITA) for the selective and ratiometric imaging of endogenous Cys in live cells. In this ratiometric fluorescent probe, a fluorescein derivative (FITA) that recognizes Cys is covalently linked to the surfaces of carbon dots (CDs); employing CDs greatly improves the water solubility of the probe. Acrylate on FITA is selectively cleaved by Cys in aqueous solution under mild conditions, leading to a dramatic increase in the fluorescence from fluorescein. The probe therefore allows the highly selective ratiometric fluorescent detection of Cys even in the presence of various interferents. The as-prepared CDs-FITA showed excellent performance when applied to detect Cys in blood serum. In addition, due to its negligible cytotoxicity, the CDs-FITA can also be utilized for the real-time monitoring of endogenous cysteine (Cys) levels in living cells. Graphical abstractIllustration of the CD-based probe for Cys imaging in living cells

Spectrofluorometric determination of berberine using a novel Au nanocluster with large Stokes shift

AbstractBerberine hydrochloride (BHC), a natural isoquinoline alkaloid, is widely applied as a an agent in traditional Chinese medicine. Almost all the traditional methods for BHC detection require complicated preprocessing steps or expensive instruments. In this article, we report a simple, rapid, sensitive, and selective method for BHC detection using fluorescent gold nanoclusters (F-AuNCs) as the fluorescent probe with a large Stokes shift of 237 nm. The F-AuNCs prepared with citrate-stabilized stannous chloride and hydrogen tetrachloroaurate(III) as raw materials in an aqueous medium display strong and stable fluorescence at 566 nm. When F-AuNCs are mixed with BHC, the fluorescence of F-AuNCs is effectively quenched. Under optimized conditions, this method allows sensitive and selective measurements of BHC in a concentration ranging from 1.0 × 10-6 to 1.0 × 10-4 mol L-1 with a detection limit of 7.5 × 10-8 mol L-1, which is relatively low among reported spectral methods. This method provides excellent selectivity for the detection of BHC against inorganic anions and natural amino acids. In addition, the BHC content in two different types of berberine tablets was successfully determined by this method and the results showed high accuracy. Graphical Abstractᅟ

Fabrication of Water-soluble Fluorescent Polymeric Micelles for Selective Detection of Hg2+ in Blood Serum

In this study, amphiphilic diblock copolymers were designed and synthesized via the incorporation of reversible addition-fragmentation chain transfer radical polymerization (RAFT) and a subsequent grafting technique. Subsequently, Hg2+-sensitive water-soluble fluorescent polymeric micelles (FNs) were prepared by a reprecipitation strategy. The spectroscopic characteristics demonstrate that the fluorescein isothiocyanate (FITC) was successfully linked into the polymer. Due to the promoted reaction of desulfurization cyclization by Hg2+, the fluorescence of fluorescein in FNs was obviously quenched. The as-prepared FNs showed admirable Hg2+-sensitivity (detection limit: 54 nM), excellent water-solubility and high selectivity. In addition, FNs were successfully used to determine Hg2+ in blood serum. We expected that the as-prepared FNs could perform potential applications in imaging, sensing, and bioanalytic chemistry.

Comparison of Full‐length Amino Acid Sequences of Chickens BF1 and BF2

To the Editor: The chicken MHC can determine striking resistance and susceptibility to infectious pathogens. MHC control of disease resistance has been especially well established for Marek’s disease (MD). The chicken MHC codes for three classes of cell surface antigens, BF and BL, which are equivalent to the mammalian class I and class II antigens, the MHC I/peptide complex is recognized by T cell receptors (TCR) on CD8 T lymphocytes, and BG which is absent from the mammalian MHC. BF is expressed on the surfaces of virtually all cells of the body and consists of two classical class Ia loci, BF1 (previously as BFa2 or B-FI or BF minor) and BF2 (previously as BFa1 or B-FIV or BF major). BF1 was poorly transcribed and BF2 well transcribed at a ratio varying from 1.5 to 33, supporting the ‘minimal essential MHC’ hypothesis that a single dominant class I molecule (BF2) determines MHC haplotype-associated differences in disease resistance. Previously, Wei [1] found that the expression modes of BF1 and BF2 genes might be related to traits of genetic resistance to MD in the chicken. However, variation in amino acid sequences between the full-length BF1 and BF2 mRNA transcripts has not been compared. Sample of cDNA extracted from peripheral blood leucocytes (PBL) of Xiayan chickens [2] was used in the experiment. The detailed steps of total RNA isolation and reverse transcription (RT) are described in the previous article [3]. All products of cDNA were stored at 80 °C until further usage. The forward primer (50-GAGAGTGCAGCGGTGCGAGGCGAT-30) and the reverse primer (50-AATGCTGGTGTGGACTGTTGGCTC-30) were designed by Dai [4] to amplify a 1 136 bp fragment of BF gene. Each 50 ll reaction mixtures contained Golden Easy PCR Mix 25 ll, 0.1 mM of each primer 1 ll, approximately 160 ng cDNA samples and ddH2O to 50 ll. The amplification process consisted of a 5-min initial denaturation at 95 °C, followed by 33 cycles of denaturation at 95 °C for 1 min, annealing at 67 °C for 1 min, elongation at 72 °C for 2 min and a final elongation at 72 °C for 10 min. The purified PCRproduct was cloned into pMD18-T vector and transformed into DH5 alpha cells. Nucleotide sequences of the positive clones were obtained by automated sequence analysis. The analysis of amino acid sequences alignment of theseBFswith the BFs ofB andB haplotypes was carried out usingDNAStar software (DNASTAR, Inc., Madison, WI, USA). An alignment of amino acid sequences in alpha 1/alpha 2/alpha 3/transmembrane (TM)/cytoplasmic (CP) domains of these BFs (GenBank accession numbers: KJ420534KJ420540 and KJ094479-KJ094494) is shown in Fig. 1, with phylogenetic BF1 sequences on top and BF2 on bottom. Sequence analysis showed that in all 19 new BF molecules, 9 BF molecules belong to BF1 molecules, while 10 BF molecules belong to BF2 molecules. Compared with BF2, BF1 alleles were less polymorphic and possessed a conserved locus-specific motif in the a1 helix. It is immediately apparent from the sequence alignment that all the 9 BF1 sequences are much more conserved in the Alpha1 helix between amino acids 65 and 82 than are all the 10 BF2 sequences. The 9 BF1 sequences share a motif, amino acids 8, 71–73, 75-76, 149 and 155, that differs from all 10 BF2 sequences at these positions, whereas the 10 BF2 sequences have a unique conserved amino acid at residue 8, 71, 72 and 82. Additionally, surprisingly, exon 7-deprived variant of BF molecules was detected in 3 of all 9 BF1 molecules while in only 3 of all 10 BF2 molecules.