Ting-Ting Chen

A Dual-Emission Fluorescent Nanocomplex of Gold-Cluster-Decorated Silica Particles for Live Cell Imaging of Highly Reactive Oxygen Species

A novel nanocomplex displaying single-excitation and dual-emission fluorescent properties has been developed through a crown-like assembly of dye-encapsulated silica particles decorated with satellite AuNCs for live cell imaging of highly reactive oxygen species (hROS), including •OH, ClO(-) and ONOO(-). The design of this nanocomplex is based on our new finding that the strong fluorescence of AuNCs can be sensitively and selectively quenched by these hROS. The nanocomplex is demonstrated to have excellent biocompatibility, high intracellular delivery efficiency, and stability for long-time observations. The results reveal that the nanocomplex provides a sensitive sensor for rapid imaging of hROS signaling with high selectivity and contrast.

Fluorescence Activation Imaging of Cytochrome c Released from Mitochondria Using Aptameric Nanosensor

We have developed an aptameric nanosensor for fluorescence activation imaging of cytochrome c (Cyt c). Fluorescence imaging tools that enable visualization of key molecular players in apoptotic signaling are essential for cell biology and clinical theranostics. Cyt c is a major mediator in cell apoptosis. However, fluorescence imaging tools allowing direct visualization of Cyt c translocation in living cells have currently not been realized. We report for the first time the realization of a nanosensor tool that enables direct fluorescence activation imaging of Cyt c released from mitochondria in cell apoptosis. This strategy relies on spatially selective cytosolic delivery of a nanosensor constructed by assembly of a fluorophore-tagged DNA aptamer on PEGylated graphene nanosheets. The cytosolic release of Cyt c is able to dissociate the aptamer from graphene and trigger an activated fluorescence signal. The nanosensor is shown to exhibit high sensitivity and selectivity, rapid response, large signal-to-background ratio for in vitro, and intracellular detection of Cyt c. It also enables real-time visualization of the Cyt c release kinetics and direct identification of the regulators for apoptosis. The developed nanosensor may provide a very valuable tool for apoptotic studies and catalyze the fundamental interrogations of Cyt c-mediated biology.

Double-strand DNA-templated synthesis of copper nanoclusters as novel fluorescence probe for label-free detection of biothiols

In this work, we report a simple and sensitive method for the detection of biothiols including glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), using fluorescent copper nanoclusters synthesized by a double-stranded DNA template (DNA-CuNCs) as a probe. The photoluminescence intensity of DNA-CuNCs was found to be quenched effectively with the increase in the concentration of biothiols due to the formation of nonfluorescent coordination complexes between the DNA-CuNCs and biothiols, whereas the fluorescence of DNA-CuNCs was not changed in the presence of other amino acids at a 10-fold higher concentration. Satisfactory detection limits and linear relationships for the detection of GSH, Cys and Hcy were obtained The resulting calibration plots exhibited good linear correlations in the range from 2.0 × 10−6 to 1.0 × 10−4 mol L−1 for Cys, 2.0 × 10−6 to 8.0 × 10−5 mol L−1 for GSH, and 5.0 × 10−6 to 2.0 × 10−4 mol L−1 for Hcy. The detection limits of Cys, GSH and Hcy were 2 μmol L−1, 2 μmol L−1 and 5 μmol L−1, respectively. In addition, the method was successfully applied in the detection of biothiols in human plasma samples.

Nanoscale Zeolitic Imidazolate Framework-8 for Ratiometric Fluorescence Imaging of MicroRNA in Living Cells

MicroRNAs (miRNAs) play important roles in cell differentiation, proliferation, and apoptosis and have been recognized as valuable biomarkers for clinical disease diagnosis. Here, we adopt for the first time zeolitic imidazolate framework-8 (ZIF-8) as a nanocarrier to efficiently deliver a nucleic acid probe to living cells and develop a novel ratiometric fluorescence strategy based on DNAzyme for miRNA-21 imaging. A Cy5-labeled 8-17 DNAzyme strand and a Cy3-labeled substrate strand containing a segment complementary to the target miRNA-21 first form a duplex probe, and fluorescence resonance energy transfer (FRET) takes place. After adsorption on the ZIF-8 surface and cellular uptake, the probe/ZIF-8 nanocomplex degrades in acidic endosome and releases duplex probes and Zn2+, and the latter can act as an effective cofactor for 8-17 DNAzyme. The intracellular miRNA-21 hybridizes with the complementary segment of the substrate strand and results in dissociation from the DNAzyme-substrate duplex probe after DNAzyme cleaves the substrate into two fragments, accompanied by the change in the FRET signal. The proposed method has been applied to image miRNA-21 expression levels in MCF-7, HeLa, and L02 cells with high contrast and reliability. The fluctuation of miRNA-21 expression level induced by miRNA-21 mimic or inhibitor can also be monitored through the obvious imaging color change. Taken together, the proposed method provides a powerful tool for cancer diagnosis and miRNA-associated biological study.

An aptasensor based on cobalt oxyhydroxide nanosheets for the detection of thrombin

An aptasensor based on a fluorophore-labelled aptamer and cobalt oxyhydroxide (CoOOH) nanosheets was developed for determining the concentration of thrombin. We first found that the CoOOH nanosheets had the ability to distinguish a G-quadruplex from single-stranded DNA (ssDNA) due to the different abilities of the nanosheet to absorb these two DNA forms. The absorption of 6-carboxyfluorescein (6-FAM) labelled aptamer (ssDNA) on the surface of the CoOOH nanosheets resulted in the quenching of the fluorescence of the FAM through fluorescence resonance energy transfer (FRET) between the FAM and the CoOOH nanosheets. However, the binding of the aptamer to its target resulted in the formation of an antiparallel G-quadruplex complex, for which the CoOOH nanosheets had weak affinity, resulting in a recovery of fluorescence. We applied this strategy to the detection of thrombin. The intensity of the fluorescence recovery was found to be linear with the logarithm of the thrombin concentration in the range of 0.5 to 100 nM, and the limit of detection (LOD) was determined to be 0.5 nM. Because of the high selectivity of the aptamer and strong quenching ability of the CoOOH nanosheets, similar aptasensors but with different specific aptamers may potentially serve as platforms to detect a wide range of analytes, and may hence have promising applications in clinical diagnosis.

Biomineralized Metal–Organic Framework Nanoparticles Enable Intracellular Delivery and Endo-Lysosomal Release of Native Active Proteins

Efficient delivery and endo-lysosomal release of active proteins in living cells remain a challenge in protein-based theranostics. We report a novel protein delivery platform using protein-encapsulating biomineralized metal-organic framework (MOF) nanoparticles (NPs). This platform introduces an adapted biomimetic mineralization method for facile synthesis of MOF NPs with high protein encapsulation efficiency and a new polymer coating strategy to confer the NPs with long-term stability. In vitro results show that protein-encapsulating MOF NPs have the advantages of preserving protein activity for months and protecting proteins from enzyme-mediated degradation. Live cell studies reveal that MOF NPs enable rapid cellular uptake, efficient release and escape of proteins from endo-lysosomes, and preservation of protein activity in living cells. Moreover, the developed platform is demonstrated to enable easy encapsulation of multiple proteins in single MOF NPs for efficient protein co-delivery. To our knowledge, it is the first time that protein-encapsulating MOF NPs have been developed as a generally applicable strategy for intracellular delivery of native active proteins. The developed protein-encapsulating biomineralized MOF NPs can provide a valuable platform for protein-based theranostic applications.

Novel Sensitive Fluorometric Determination of Exonuclease I Using Polydopamine Nanospheres

ABSTRACT A novel fluorescence sensing platform based on polydopamine nanospheres and 6′-carboxyfluorescein labeled single-stranded DNA has been developed for monitoring the concentration of exonuclease I. Due to the interaction between single-stranded DNA and polydopamine nanospheres, the single-stranded DNA may be adsorbed on the surface of polydopamine nanospheres. The fluorescence of 6′-carboxyfluorescein was subsequently quenched by the polydopamine nanospheres through energy transfer or electron transfer. However, the 6′-carboxyfluorescein-labeled single-stranded DNA was specifically degraded by exonuclease I, producing mono or oligonucleotide fragments, which were not adsorbed by the polydopamine nanospheres, and thus the fluorescence signal was retained. The retained fluorescence of the sensing platform was found to be linear with the concentration of exonuclease I in the range of 0.15–10 U mL−1 with a detection limit of 0.05 U mL−1. In addition, the sensing platform was highly selective toward exonuclease I. Benefiting from the high efficiency and the simple design process, satisfactory performance has been successfully demonstrated for the determination of exonuclease I in complex samples.

CoOOH-induced synthesis of fluorescent polydopamine nanoparticles for the detection of ascorbic acid

As an important biological small molecule, ascorbic acid (AA) plays a key role in many bioprocesses. Here, we developed a novel method to evaluate AA based on the redox reaction between CoOOH and AA. In our work, a nanosystem was constructed with fluorescent polydopamine (PDA) nanoparticles and CoOOH nanosheets, which were used as signal indicators and an oxidant respectively. This is the first time that fluorescent PDA nanoparticles were synthesized through oxidation by CoOOH nanosheets. In the absence of AA, dopamine was oxidized to quinone derivatives and further spontaneously polymerized into fluorescent PDA nanoparticles which had strong fluorescence signals. When there was AA in the reaction system, CoOOH nanosheets would be reduced to Co2+, which would prevent the synthesis of fluorescent PDA nanoparticles due to the absence of oxidant CoOOH, resulting in weak fluorescence. We hence used the fluorescence intensity of the PDA nanoparticles to detect AA concentration. The fluorescence of this sensing platform was linear with the concentration of AA in the range of 0–500 μM with a detection limit of 4.8 μM. In addition, the sensor was simple, fast, label-free, and low cost, and may be used to detect other small molecules based on the redox reaction.

2D g-C3N4–MnO2 nanocomposite for sensitive and rapid turn-on fluorescence detection of H2O2 and glucose

The accurate determination and quantification of H2O2 and glucose are necessary for diagnosis and bioengineering. Herein, we have reported a novel method for the rapid and selective detection of H2O2 and glucose using g-C3N4–MnO2 nanocomposite. In this method, the fluorescence of graphitic-C3N4 (g-C3N4) was quenched by MnO2, which was attributed to fluorescence resonance energy transfer (FRET) between g-C3N4 nanosheets and the deposited MnO2. When H2O2 was introduced, the MnO2 was reduced to Mn2+, which led to the elimination of FRET and the recovery of the quenched fluorescence. This H2O2 detection system was also suitable for glucose detection since H2O2 is one of the main products in the oxidation reaction of glucose catalyzed by glucose oxidase. Therefore, our method has a great application prospect in diabetes research and clinical diagnosis. Additionally, the fluorescence was linear with the concentration of H2O2 in the range of 0 to 130 μM, with the detection limit of 1.5 μM, and also showed a good linear relationship with glucose in the range of 0 to 150 μM, with the detection limit of 1.5 μM. In addition to a wide linear response, the proposed sensor showed advantageous characteristics, such as easy preparation, low cost, high selectivity, rapid detection, and turn-on fluorescence response. Therefore, our proposed strategy would provide a new general strategy to develop low-cost, sensitive biological and clinical diagnostic methods.

In Situ Synthesis of Ultrathin ZIF-8 Film-Coated MSNs for Codelivering Bcl 2 siRNA and Doxorubicin to Enhance Chemotherapeutic Efficacy in Drug-Resistant Cancer Cells

Multiple drug resistance is a persistent obstacle for efficient chemotherapy of cancer. Herein, we report a novel drug delivery platform. A zeolitic imidazole framework-8 (ZIF-8) film with a few nanometer thickness was in situ synthesized on the surface of carboxylated mesoporous silica (MSN-COOH) nanoparticles (NPs) for pore blocking and efficient loading of small interfering RNAs to fabricate a pH-responsive drug delivery system. The ZIF-8 film could convert the charge of MSN-COOH from negative to positive for efficient loading of siRNA via electrostatic interactions and protect siRNA from nuclease degradation. The positively charged ZIF-8 film facilitates cellular uptake and endo-lysosome escape of the NPs. In addition, the ultrathin ZIF-8 film can decompose in the acidic endo-lysosome and trigger the intracellular release of siRNAs and chemotherapeutic drugs, leading to a significantly enhanced chemotherapeutic efficacy for multidrug-resistant cancer cells including MCF-7/ADR and SKOV-3/ADR cells as demonstrated by the confocal laser scanning microscopy image, cell viability assay, Annexin V&PI staining, and flow cytometry. This approach provides a promising strategy for pH-triggered, stimuli-responsive delivery of nucleic acid drugs and chemotherapeutic agents with remarkably enhanced chemotherapeutic efficacy.