Reversibly Migratable Fluorescent Probe for Precise and Dynamic Evaluation of Cell Mitochondrial Membrane Potentials.

The mitochondrial membrane potential (MMP, ΔΨmito) provides the charge gradient required for mitochondrial functions and is a key indicator of cellular health. The changes in MMP are closely related to diseases and the monitoring of MMP is thus vital for pathological study and drug development. However, most of the current fluorescent probes for MMP rely solely on the cell fluorescence intensity and are thus restricted by poor photostability, rendering them not suitable for long-term dynamic monitoring of MMP. Herein, an MMP-responsive fluorescent probe pyrrolyl quinolinium (PQ) which is capable of reversible migration between mitochondria and nucleolus is developed and demonstrated for dynamic evaluation of MMP. The fluorescence of PQ translocates from mitochondria to nucleoli when MMP decreases due to the intrinsic RNA-specificity and more importantly, the translocation is reversible. The cytoplasm to nucleolus fluorescence intensity ratio is positively correlated with MMP so that this method avoids the negative influence of photostability and imaging parameters. Various situations of MMP can be monitored in real time even without controls. Additionally, long-term dynamic evaluation of MMP is demonstrated for HeLa cells using PQ in oxidative environment. This study is expected to give impetus to the development of mitochondria-related disease diagnosis and drug screening.

Tri-functional SERS nanoplatform with tunable plasmonic property for synergistic antibacterial activity and antibacterial process monitoring.

Strategies integrating synergistic high-efficiency bacterial killing and antibacterial process monitoring capability are desirable. Herein, a tri-functional surface-enhanced Raman spectroscopy (SERS) nanoplatform, namely 4-mercaptobenzoic acid-encoded gold nanorods@silver coated with a layer of bovine serum albumin (AuNRs@Ag@4-MBA@BSA), with excellent biocompatibility, stability, tunable plasmonic property and activatable photothermal effect is introduced for Ag+/photothermal therapy (PTT) synergistic antibacterial activity and antibacterial process monitoring. An exogenous etchant is used to controllably model the physiological process of metallic silver biodegradation. Ag shell etching causes the surface plasmon resonance band of SERS nanotags to red-shift to near-infrared region, activates the photothermal conversion capability, and triggers PTT, which in turn accelerates Ag shell etching. The antibacterial rates for Staphylococcus aureus and Escherichia coli after 10 min treatment can achieve 99.5% and 99.9%, respectively. Furthermore, the near-field effect and ultrasensitive property render the SERS intensity decrease ratio is dependent on Ag shell etching as well as temperature rising and thus relevant to antibacterial activity. We have demonstrated a strong correlation between SERS signal and antibacterial effect, and have verified the possibility of antibacterial process monitoring in vitro using SERS-based methodology. We envision that our integrated strategy being used for in vivo high-efficiency bacterial killing and antibacterial process monitoring.

A Scalable Nickel-Cellulose Hybrid Metamaterial with Broadband Light Absorption for Efficient Solar Distillation.

Sophisticated metastructures are usually required to broaden the inherently narrowband plasmonic absorption of light for applications such as solar desalination, photodetection, and thermoelectrics. Here, nonresonant nickel nanoparticles (diameters < 20 nm) are embedded into cellulose microfibers via a nanoconfinement effect, producing an intrinsically broadband metamaterial with 97.1% solar-weighted absorption. Interband transitions rather than plasmonic resonance dominate the optical absorption throughout the solar spectrum due to a high density of electronic states near the Fermi level of nickel. Field solar purification of sewage and seawater based on the metamaterial demonstrates high solar-to-water efficiencies of 47.9-65.8%. More importantly, the solution-processed metamaterial is mass-producible (1.8 × 0.3 m2 ), low-cost, flexible, and durable (even effective after 7 h boiling in water), which are critical to the commercialization of portable solar-desalination and domestic-water-purification devices. This work also broadens material choices beyond plasmonic metals for the light absorption in photothermal and photocatalytic applications.

Large-Area 3D Hierarchical Superstructures Assembled from Colloidal Nanoparticles.

Assembling nanosized building blocks into macroscopic 3D complex structures is challenging. Here, nanosized metal and semiconductor building blocks with a variety of sizes and shapes (spheres, stars, and rods) are successfully assembled into a broad range of hierarchical (nanometer to micrometer) assemblies of functional materials in centimeter size using butterfly wings as templates. This is achieved by the introduction of steric hindrance to the assembly process, which compensates for attraction from the environmentally sensitive hydrogen bonds and prevents the aggregation of nanosized building blocks. Of these materials, Au nanostar assemblies show a superior enhancement in surface-enhanced Raman scattering (SERS) performance (rhodamine 6G, 1506 cm-1 ) under 532, 633, and 780 nm excitation-this is 3.1-4.4, 3.6-3.9, and 2.9-47.3 folds surpassing Au nanosphere assemblies and commercial SERS substrates (Q-SERS), respectively. This method provides a versatile route for the assembly of various nanosized building blocks into different 3D superstructures and for the construction of hybrid nanomaterials and nanocomposites.

Tumor marker detection using surface enhanced Raman spectroscopy on 3D Au butterfly wings.

Tumor markers are usually over-expressed in human body fluids during the development of cancers. Monitoring tumor markers' level is thus important for early diagnosis and screening of cancers. One way to achieve this is based on the surface enhanced Raman scattering (SERS) technique that can drastically amplify Raman signals of analytes on a plasmonic metal (e.g., Au, Ag, and Cu) surface. However, this promising method suffers from aggregation of plasmonic nanoparticles. Here we report a stable, reproducible, and facile SERS-based readout method to detect an important tumor marker, carcinoembryonic antigen (CEA). This route utilizes Au butterfly wings with natural three dimensional (3D) hierarchical sub-micrometer structures rather than relying on the aggregates of metal nanoparticles. The Au butterfly wings show excellent SERS property and are temperature (80 °C) and time (6 months) stable on a sub-micrometer scale. Thus, the detecting antibodies and enzyme-linked secondary antibodies that are usually applied in conventional enzyme-linked immunosorbent assay (ELISA) can be replaced by chemically synthesized CEA aptamers, significantly simplifying the whole detection process. We demonstrate the feasibility of this method via quantitative detection of clinical CEA level in human body fluids. This work thus demonstrates a promising tumor marker detection technique based on a hierarchical sub-micrometer SERS structure, which could be useful for the mass screening of early stage cancers.

A two-dimensional molecular beacon for mRNA-activated intelligent cancer theranostics.

Ideal theranostics should possess directly correlated imaging and therapy modalities that could be simultaneously activated in the disease site to generate high imaging contrast and therapeutic efficacy with minimal side effects. However, so far it still remains challenging to engineer all these characteristics into a single theranostic probe. Herein, we report a new type of photosensitizer (PS)-derived "two-dimensional" molecular beacon (TMB) that could be specifically activated within tumor cells to exhibit both high imaging contrast and therapeutic efficacy that outperforms conventional photosensitizers for cancer theranostics. The TMB is constructed by integrating a photosensitizer (chlorin e6 (Ce6)), a quantum dot (QD), and a dark quencher (BHQ3) into a hairpin DNA molecule to generate multiple synergistic FRET modes. The imaging modality and therapy modality, which are mediated by FRET between the QD and BHQ3 and FRET between the QD and Ce6 respectively, are interconnected within the TMB and could be simultaneously activated by tumor mRNA molecules. We show that highly effective cancer imaging and therapy could be achieved for cancer cell lines and xenografted tumor models. The reported TMB represents an unprecedented theranostic platform for intelligent cancer theranostics.

Photonic structure arrays generated using butterfly wing scales as biological units.

We report an effective process to transfer the scales of Morpho butterflies onto various substrates. Based on the difference in binding strength between molecular interactions and chemical bonds, this method provides photonic structure arrays with biological units, which are difficult to obtain otherwise.

A fluorescent probe for intracellular cysteine overcoming the interference by glutathione.

Cysteine (Cys) plays important roles in many physiological processes of eukaryotic cells and its detection in cells is of fundamental significance. However, glutathione (GSH), homocysteine, N-acetyl-L-cysteine and other thiols greatly hamper the detection of Cys. In particular, GSH strongly interferes with the detection of cellular Cys (30­200 µM) due to its high intracellular concentration (1­10 mM). In this work, an off­on fluorescent probe (HOTA) for the detection of Cys is presented. This probe possesses both excellent sensitivity and satisfactory selectivity for cellular Cys detection: with the addition of 200 µM Cys, the fluorescence intensity of the probe (10 µM) enhanced 117-fold and the detection limit was calculated to be 13.47 µM, which is lower than the cellular Cys concentration; the probe also selectively detected 30­200 µM cysteine over 1­10 mM glutathione. Consequently, cell imaging experiments were performed with probe HOTA. Furthermore, the results of the thiol-blocking and GSH synthesis inhibiting experiments confirmed that the intracellular emission mainly originates from the interaction between Cys and HOTA.

Novel fluorescent probes for highly selective two-photon imaging of mitochondria in living cells.

The synthesis and characterization of a pair of novel pyridine cation derivatives possessing two-photon excitation fluorescence (TPEF) properties and selectively staining mitochondria in living SiHa cells within 30 min, CAI and CAEI, were reported. And the green-emitting CAI displayed a much larger two-photon excitation fluorescence action absorption cross-section (δ × Φ) of 328 g at 860 nm in comparison with commercial MitoTracker Green (MTG) with maximum δ × Φ value of 2.18 g at 850 nm. As is known to all, δ × Φ is a crucial parameter to obtain a high-quality microscopic photo in living cells in two-photon microscopy (TPM). Moreover, the fact that the co-localization coefficient between CAI and conventional MitoTracker Red (MTR) was 0.95 in SiHa cells demonstrated specific staining performance of CAI to mitochondria. As biosensors, both CAI and CAEI possessed a number of beneficial properties such as large δ × Φ and Stokes shifts, good membrane permeability, long retention time, high photostability and excellent counterstain compatibility with different biosensors for instance Hoechst 33342 and D 307, which ranked them as one of the best TPEF mitochondrial probes. Furthermore, related mechanism research suggested that their localization properties were dependent on the mitochondrial membrane potential in living cells. And their remarkable properties can extend the investigation on mitochondria in a biological context.

Low molecular weight fluorescent probes with good photostability for imaging RNA-rich nucleolus and RNA in cytoplasm in living cells.

We have synthesized two low molecular weight organic molecules, PY and IN successfully, which selectively stain nucleolus and cytoplasm of living cells in 30 min, with a much lower uptake in the nucleus. Nucleic acids electrophoresis and digest test of ribonuclease indicate their markedly higher affinity for RNA, especially PY. Moreover their RNA localization in cells is further supported by digest test of ribonuclease, namely, the nucleolar fluorescence signal is distinctly lost upon treatment with RNase. And, the fact that live cells stained by PY and IN still possess physiological function can be confirmed: 1) MTT assay demonstrates that the mitochondria of cells stained remains its electron mediating ability, 2) Double assay of PY/IN and propidium iodide as well as trypan blue testing show that the membrane of cells stained still is intact. Importantly, compared with the only commercial RNA probe, SYTO RNA-Select, PY and IN exhibit much better photostability when continuously illuminated with 488 nm laser and mercury lamp. These results prove that PY and IN are very attractive staining reagents for visualizing RNA in living cells.

Lighting up cysteine and homocysteine in sequence based on the kinetic difference of the cyclization/addition reaction.

A novel one- and two-photon fluorescent probe CB1 has been developed for discriminating Cys and Hcy in a successive manner with high selectivity. The discrete time-dependent fluorescent responses enable us to sequentially detect Cys and Hcy in different time windows. Two-step reaction and kinetic modes were used to explain the sensing mechanism. As a promising biosensor for cell imaging, CB1 has been confirmed to exhibit membrane permeability to intact cells, low cytotoxicity to viable cells and photostability to ultraviolet light excitation. Furthermore, the results from the control assay have shown that the one- and two-photon fluorescence of CB1 within cells is associated with intracellular mercapto biomolecules but yet there is little interference with physiological pH value, viscosity and common bioanalytes. Finally one- and two-photon fluorescent images of CB1 within living SiHa cells have been presented.

Fluorescent imaging of acidic compartments in living cells with a high selective novel one-photon ratiometric and two-photon acidic pH probe.

Fluorescent imaging of acidic compartments in living cells was carried out successfully by a novel molecule (CAE) that contained a pyridine unit and a carbazole core. As the protonation of the pyridine N atom of CAE, pH-dependent absorption and fluorescence properties were shown with a pKa of 5.47 which matches the pH range of intracellular acidic compartments. Moreover, a large Stokes shift, a significant enhancement in ratios of I544 nm/I460 nm and a distinct two-photon turn-on character were exhibited in spectral analysis. Meanwhile, direct intracellular imaging and standard double-staining experiments of CAE and LTR (co-localization coefficient: 0.83) revealed that CAE is an effective one-photon ratiometric and two-photon acidic pH probe for imaging intracellular acidic compartments. The pH distribution pattern of intracellular acidic compartments can be obtained facilely by CAE. In especial, CAE possessed well membrane-permeability, brilliant selectivity among various bioanalyte and excellent counterstain compatibility with Hoechst 33342, MTR and LTR.

Highly selective red- and green-emitting two-photon fluorescent probes for cysteine detection and their bio-imaging in living cells.

Two highly selective two-photon fluorescent probes for cysteine over homocysteine, N-acetyl-L-cysteine, dithiothreitol, glutathione and other amino acids, and their fluorescent imaging in living cells have been shown.