Switchable Fluorescent Imaging of Intracellular Telomerase Activity Using Telomerase-Responsive Mesoporous Silica Nanoparticle

This work designs a telomerase-responsive mesoporous silica nanoparticle (MSN) to realize in situ “off-on” imaging of intracellular telomerase activity. In the wrapping DNA (O1) sealed MSN probe, a black hole fluorescence quencher is covalently immobilized on the inner walls of the mesopores, while fluorescein is loaded in the mesopores. In the presence of telomerase and dNTPs, the designed O1 can be extended and then moves away from the MSN surface via forming a rigid hairpin-like DNA structure. Thus the O1 can act as a “biogate” to block and release fluorescein for “off-on” switchable fluorescent imaging. The MSN probe exhibits good performance for sensitive in situ tracking of telomerase activity in living cells. The practicality of this protocol has been verified by monitoring the change of cellular telomerase activity in response to telomerase-related drugs

Smart Vesicle Kit for In Situ Monitoring of Intracellular Telomerase Activity Using a Telomerase-Responsive Probe

A smart vesicle kit was designed for in situ imaging and detection of cytoplasmic telomerase activity. The vesicle kit contained a telomerase primer (TSP) and a Cy5-tagged molecular beacon (MB) functionalized gold nanoparticle probe, which were encapsulated in liposome for intracellular delivery. After the vesicle kit was transfected into cytoplasm, the released TSP could be extended in the presence of telomerase to produce a telomeric repeated sequence at the 3′ end, which was just complementary with the loop of MB assembled on probe surface. Thus, the MB was opened upon hybridization to switch the fluorescent state from “off” to “on”. The fluorescence signal depended on telomerase activity, leading to a novel strategy for in situ imaging and quantitative detection of the cytoplasmic telomerase activity. The cytoplasmic telomerase activity was estimated to be 3.2 × 10^–11, 2.4 × 10^–11, and 8.6 × 10^–13 IU in each HeLa, BEL tumor and QSG normal cell, respectively, demonstrating the capability of this approach to distinguish tumor from normal cells. The proposed method could be employed for dynamic monitoring of the cytoplasmic telomerase activity in response to a telomerase-based drug, suggesting the potential application in discovery and screening of telomerase-targeted anticancer drugs

Functional Dual-Color Indicator To Achieve in Situ Visualization of Intracellular Glycosylation

A functional dual-color indicator is designed for in situ visualization of intracellular glycosylation. Using O-GlcNAcylation as model, the indicator is constructed on a poly-GlcNAc-coated gold nanoparticle (AuNP) by assembling dye labeled lectin (FSWGA) and then another dye-labeled GlcNAc (FGlcNAc) through the two opposite subunits of FSWGA. These dyes possess negligible overlapping emission and can be quenched by AuNP. In the presence of intracellular dissociated GlcNAc residue and O-GlcNAcylated proteins, the assembled FGlcNAc and the conjugate of FSWGA with FGlcNAc are released from AuNP through the dynamic competitive conjugation, which lights up the fluorescence of two dyes, respectively, and provides a simple technique for simultaneously monitoring the level of O-GlcNAcylated proteins and the total amount of GlcNAc groups in living cells. The practicality of the protocol for visually monitoring the biological pathway between intracellular O-GlcNAcylation and cell surface differentiation-related proteins demonstrates a convenient and powerful tool for research of glycosylation equilibrium and related biological processes

Arrayed Profiling of Multiple Glycans on Whole Living Cell Surfaces

An array-based method for profiling and quantification of multiple glycans on whole living cell surfaces was developed through combining DNA encoding technology with DNA microarray. Using four kinds of lectins as the model to recognize four types of cell surface glycans, the specific barcode-lectin probes that contained the endonuclease cutting site were designed. The barcode-lectin probes had the DNA sequences complementary to four sequences immobilized on a DNA microarray, respectively. After the living cells were incubated with the mixture of four barcode-lectin probes, these probes could bind to cell surface through the specific interaction between the lectins and corresponding glycans. Thus, the glycans and their amounts could be profiled by releasing the barcodes from cell surface with endonuclease cleaving, binding the barcodes to DNA microarray with specific hybridization, and then producing the amplified fluorescence signal with hybridization chain reaction (HCR). The HCR was performed with two kinds of Cy5 labeled hairpins. The average amount of mannose, <i>N</i>-acetylgalactosamine, <i>N</i>-acetylglucosamine, and <i>N</i>-acetylneuraminic acid on BGC cell was obtained to be 6.8 × 10⁷, 3.8 × 10⁷, 2.1 × 10⁸, and 1.1 × 10⁷ moieties per cell, respectively. The proposed method possessed whole cell surface accessibility, powerful distinguishing capability, fast recognition kinetics, easy miniaturization, and high throughput without need of cell pretreatment or labeling. It could become a powerful tool for elucidation of the complex glycan-related biological processes

Thiol-Functionalized Zr-Based Metal–Organic Framework for Capture of Hg(II) through a Proton Exchange Reaction

Rational design and facile synthesis of thiol-modified metal–organic frameworks (MOFs) for the efficient capture of highly toxic mercuric ions from water has attracted great attention. However, the corresponding adsorption mechanism is not well understood. In this paper, a thiol-modified Zr-based MOF (Zr-DMBD) with free-standing and accessible thiol groups was prepared. It exhibited remarkable performance in the capture of Hg­(II), and its maximum adsorption capacity was 171.5 mg·g^–1, approximately 9 times that of the pristine UiO-66. Impressively, the maximum value of the selective coefficient was as high as 28899.6. Additionally, 99.64% of Hg­(II) could be eliminated by Zr-DMBD from the actual wastewater, rendering the concentration of Hg (II) below 0.05 ppm (Emission Standard of Mercury (GB30770-2014)). The excellent adsorption capacity and outstanding selectivity were ascribed to the remarkable coordination between S^2– and Hg­(II), as supported by the results of FT-IR and XPS. Unexpectedly, a good correlation (R² = 0.982) between the increased H⁺ concentration after adsorption and its corresponding adsorption capacity was obtained. This result suggested that the thiol groups’ sulfur atoms coordinated with Hg­(II) while the hydrogen atoms in thiol groups were replaced and released as hydrogen ions in the solution, thus extending a proton exchange reaction mechanism for Hg­(II) adsorption

Liberation of Protein-Specific Glycosylation Information for Glycan Analysis by Exonuclease III-Aided Recycling Hybridization

A strategy for information liberation of protein-specific glycosylation is designed via an exonuclease III-aided recycling “hybridization and cleavage” process with glycan and protein probes, which achieves homogeneous quantification of cell surface glycan. The protein probe contains matching and spacer DNA sequences and an aptamer specific to target protein. The glycan probe contains a complementary sequence modified with neighboring fluorescein and quencher, a spacer sequence, and a dibenzocyclooctyne-amine end to bind azide-tagged glycan. Upon sequential binding to their targets, the complementary sequences of two probes approach enough for their hybridization, which leads to the cleavage of hybridized glycan probe by exonuclease III and followed recycling “hybridization and cleavage” process of protein probe with other adjacent glycan probes to release the labeled fluorescein for obtaining the information on protein-specific glycosylation. This protocol has been used to in situ quantify EpCAM-specific sialic acid on MCF-7 cell surface and monitor its variation during drug treatment. This work demonstrates a powerful quantification tool for research of glycosylation

Enhancing the Hg(II) Removal Efficiency from Real Wastewater by Novel Thymine-Grafted Reduced Graphene Oxide Complexes

In this study, the reduced graphene oxide was modified by grafting thymine on its surface. The resultant reduced graphene oxide-thymine composite (rGO-Thy) exhibits a higher Hg­(II) adsorption capacity and selectivity compared with rGO as the functional group of thymine shows a strong affinity toward Hg­(II) and forms the thymine-Hg­(II)-thymine complex. The relative selectivity coefficients of rGO-Thy for Hg­(II)/Pb­(II), Hg (II)/Ni­(II), Hg (II)/Co­(II), Hg (II)/Cu­(II), and Hg­(II)/Cd­(II) are 21.72, 7.08, 5.37, 4.37, and 10.51, respectively. This is mainly attributed to the thymine-specific binding with Hg­(II). In addition, the adsorption capacity of rGO-Thy for Hg­(II) is almost 2 times higher than reduced graphene oxide (rGO). Kinetics studies indicate that the adsorption process fits well with the pseudo-second-order model, and the adsorption kinetic constant is 0.02 g·mg^–1·min^–1. Moreover, the practical application of rGO-Thy achieves almost 100% removal efficiency, and the treatment volumes of actual industrial wastewater using a fixed bed column are as high as 390 BV for Hg­(II), which indicates that rGO-Thy has great potential in advanced wastewater treatment

Folate Receptor-Targeted and Cathepsin B‑Activatable Nanoprobe for <i>In Situ</i> Therapeutic Monitoring of Photosensitive Cell Death

The integration of diagnostic and therapeutic functions in a single system holds great promise to enhance the theranostic efficacy and prevent the under- or overtreatment. Herein, a folate receptor-targeted and cathepsin B-activatable nanoprobe is designed for background-free cancer imaging and selective therapy. The nanoprobe is prepared by noncovalently assembling phospholipid-poly­(ethylene oxide) modified folate and photosensitizer-labeled peptide on the surface of graphene oxide. After selective uptake of the nanoprobe into lysosome of cancer cells via folate receptor-mediated endocytosis, the peptide can be cleaved to release the photosensitizer in the presence of cancer-associated cathepsin B, which leads to 18-fold fluorescence enhancement for cancer discrimination and specific detection of intracellular cathepsin B. Under irradiation, the released photosensitizer induces the formation of cytotoxic singlet oxygen for triggering photosensitive lysosomal cell death. After lysosomal destruction, the lighted photosensitizer diffuses from lysosome into cytoplasm, which provides a visible method for <i>in situ</i> monitoring of therapeutic efficacy. The nanoprobe exhibits negligible dark toxicity and high phototoxicity with the cell mortality rate of 0.06% and 72.1%, respectively, and the latter is specific to folate receptor-positive cancer cells. Therefore, this work provides a simple but powerful protocol with great potential in precise cancer imaging, therapy, and therapeutic monitoring

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