4 research outputs found

    Multifunctional Probe Based on Cationic Conjugated Polymers for Nitroreductase-Related Analysis: Sensing, Hypoxia Diagnosis, and Imaging

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    Nitroreductase (NTR) is overexpressed in hypoxic tumors. Moreover, hypoxia is usually considered as the most important feature of various diseases. Thus, it is important to build a sensitive and selective method for NTR detection and hypoxia diagnosis. Herein, a new cationic conjugated polymer (PBFBT-NP) with <i>p</i>-nitrophenyl group in the side chain was designed and synthesized as a fluorescent probe for the detection of NTR. In the absence of NTR, the fluorescence of PBFBT-NP was quenched due to photoinduced electron transfer (PET). On the contrary, in the presence of NTR, NTR can specifically react with <i>p</i>-nitrophenyl group to form <i>p</i>-aminophenyl group, which leads to the PET being inhibited and the polymer’s fluorescence significantly increasing (>110-fold). The sensitive and selective NTR sensing method in vitro is thus constructed with a low detection limit of 2.9 ng/mL. Moreover, the hypoxic status of tumor cells can be visualized by fluorescence bioimaging with very low cytotoxicity. Interestingly, the probe was successfully used for imaging an NTR-expressed microorganism, such as <i>E. coli</i>, and showed excellent antibacterial activity against <i>E. coli</i> under white light irradiation. In brief, this multifunctional probe is promising for widespread use in NTR-related biological analysis

    Precise Quantitation of MicroRNA in a Single Cell with Droplet Digital PCR Based on Ligation Reaction

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    MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA, respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate a single-base difference among miRNAs with simple operations. After the miRNA-templated ligation, the ddPCR partitions individual ligated products into a water-in-oil droplet and digitally counts the fluorescence-positive and negative droplets after PCR amplification for quantification of the target molecules, which possesses the power of precise quantitation and robustness to variation in PCR efficiency. By integrating the advantages of the precise quantification of ddPCR and the simplicity of the ligation-based PCR, the proposed method can sensitively measure let-7a miRNA with a detection limit of 20 aM (12 copies per microliter), and even a single-base difference can be discriminated in let-7 family members. More importantly, due to its high selectivity and sensitivity, the proposed method can achieve precise quantitation of miRNAs in single-cell lysate. Therefore, the ligation-based ddPCR assay may serve as a useful tool to exactly reveal the miRNAs’ actions in a single cell, which is of great importance for the study of miRNAs’ biofunction as well as for the related biomedical studies

    Electrospun Fibrous Scaffolds Promote Breast Cancer Cell Alignment and Epithelial–Mesenchymal Transition

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    In this work we created electrospun fibrous scaffolds with random and aligned fiber orientations in order to mimic the three-dimensional structure of the natural extracellular matrix (ECM). The rigidity and topography of the ECM environment have been reported to alter cancer cell behavior. However, the complexity of the in vivo system makes it difficult to isolate and study such extracellular topographical cues that trigger cancer cells’ response. Breast cancer cells were cultured on these fibrous scaffolds for 3–5 days. The cells showed elongated spindle-like morphology in the aligned fibers, whereas they maintained a mostly flat stellar shape in the random fibers. Gene expression profiling of these cells post seeding showed up-regulation of transforming growth factor β-1 (TGFβ-1) along with other mesenchymal biomarkers, suggesting that these cells undergo epithelial–mesenchymal transitions in response to the polymer scaffold. The results of this study indicate that the topographical cue may play a significant role in tumor progression

    <i>In Vivo</i> Virus-Based Macrofluorogenic Probes Target Azide-Labeled Surface Glycans in MCF‑7 Breast Cancer Cells

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    Chemical addressability of viral particles has played a pivotal role in adapting these biogenic macromolecules for various applications ranging from medicine to inorganic catalysis. Cowpea mosaic virus possesses multiple features that are advantageous for the next generation of virus-based nanotechnology: consistent multimeric assemblies dictated by its genetic code, facile large scale production, and lack of observable toxicity in humans. Herein, the chemistry of the viral particles is extended with the use of Cu-free strain-promoted azide–alkyne cycloaddition reaction, or SPAAC reaction. The elimination of Cu, its cocatalyst and reducing agent, simplifies the reaction scheme to a more straightforward approach, which can be directly applied to living systems. As a proof of concept, the viral particles modified with the azadibenzylcyclooctyne functional groups are utilized to trigger and amplify a weak fluorescent signal (azidocoumarin) in live cell cultures to visualize the non-natural sugars. Future adaptations of this platform may be developed to enhance biosensing applications