Chiral nanoprobes for targeting and long-term imaging of the Golgi apparatus.

The Golgi apparatus is an essential subcellular organelle. Targeting and monitoring the Golgi change at the single-cell level over a long time scale are critical but are challenges that have not yet been tackled. Inspired by the precise Golgi positioning ability of galactosyltransferase and protein kinase D, due to their cysteine residues, we developed a method for long-term Golgi imaging. Fluorescent molecules, carbon quantum dots (CQDs) and silica nanoparticles could target the Golgi when they are modified with l-cysteine. l-Cysteine-rich chiral carbon quantum dots (LC-CQDs), which have the benefits of a high Golgi specificity from l-cysteine and excellent photostability and biocompatibility from the CQDs, are proven to be highly suitable for long-term in situ imaging of the Golgi. Investigation of the mechanism showed that free thiol groups and the l-type stereo configuration of LC-CQDs are essential for specific targeting of the Golgi. With the aid of the as-prepared LC-CQDs, the dynamic changes of the Golgi in the early stage of viral infection were visualized. The Golgi targeting and imaging strategy used in this work is beneficial for Golgi-targeted drug delivery and early diagnosis and therapy of Golgi diseases.

Surface-engineered quantum dots/electrospun nanofibers as a networked fluorescence aptasensing platform toward biomarkers.

A membrane-based fluorescent sensing platform is a facile, point-of-care and promising technique in chemo/bio-analytical fields. However, the existing fluorescence sensing films for cancer biomarkers have several problems, with dissatisfactory sensitivity and selectivity, low utilization of probes encapsulated in films as well as the tedious design of membrane structures. In this work, a novel fluorescence sensing platform is fabricated by bio-grafting quantum dots (QDs) onto the surface of electrospun nanofibers (NFs). The aptamer integrated into the QDs/NFs can result in high specificity for recognizing and capturing biomarkers. Partially complementary DNA-attached gold nanoparticles (AuNPs) are employed to efficiently hybridize with the remaining aptamer to quench the fluorescence of QDs by nanometal surface energy transfer (NSET) between them both, which are constructed for prostate specific antigen (PSA) assay. Taking advantage of the networked nanostructure of aptamer-QDs/NFs, the fluorescent film can detect PSA with high sensitivity and a detection limit of 0.46 pg mL-1, which was further applied in real clinical serum samples. Coupling the surface grafted techniques to the advanced network nanostructure of electrospun NFs, the proposed aptasensing platform can be easily extended to achieve sensitive and selective assays for other biomarkers.

A dynamic cell entry pathway of respiratory syncytial virus revealed by tracking the quantum dot-labeled single virus.

Studying the cell entry pathway at the single-particle level can provide detailed and quantitative information for the dynamic events involved in virus entry. Indeed, the viral entry dynamics cannot be monitored by static staining methods used in cell biology, and thus virus dynamic tracking could be useful in the development of effective antiviral strategies. Therefore, the aim of this work was to use a quantum dot-based single-particle tracking approach to monitor the cell entry behavior of the respiratory syncytial virus (RSV) in living cells. The time-lapse fluorescence imaging and trajectory analysis of the quantum dot-labeled RSV showed that RSV entry into HEp-2 cells consisted of a typical endocytosis trafficking process. Three critical events during RSV entry were observed according to entry dynamic and fluorescence colocalization analysis. Firstly, RSV was attached to lipid rafts of the cell membrane, and then it was efficiently delivered into the perinuclear region within 2 h post-infection, mostly moving and residing into the lysosome compartment. Moreover, the relatively slow velocity of RSV transport across the cytoplasm and the formation of the actin tail indicated actin-based RSV motility, which was also confirmed by the effects of cytoskeletal inhibitors. Taken together, these findings provided new insights into the RSV entry mechanism and virus-cell interactions in RSV infection that could be beneficial in the development of antiviral drugs and vaccines.

Color resolution improvement of the dark-field microscopy imaging of single light scattering plasmonic nanoprobes for microRNA visual detection.

Imaging of light scattering plasmonic nanoparticles (PNPs) with the aid of the dark-field microscopy imaging (iDFM) technique has attracted wide attention owing to its high signal-to-noise ratio, but to improve the color resolution and contrast of dark-field microscopy (DFM) images of single light scattering PNPs in a small spectral variation environment is still a challenge. In this study, a new color analytical method for resolving the resolution and contrast in DFM images has been developed and further applied for colorimetric analysis using the digital image processing technique. The color of single light scattering PNP images is automatically coded at first with the hue values of the HSI color model, and then amplified using the MATLAB program even for marginal spectral changes, leading to significant improvement of the color resolution of DFM images and easy detection with the naked eye. As a proof of concept, this method is then applied to distinguish single PNPs with various sizes and to visually detect hepatocellular carcinoma-associated microRNA. As it greatly improved the color resolution of iDFM and its detection sensitivity, this method shows promise to serve as a better alternative for sensitive visual analysis and spectrometer-based spectral analysis.

His-tag based in situ labelling of progeny viruses for real-time single virus tracking in living cells.

Tracking virus infection events in live cells is useful for understanding the mechanism of virus infection, and fluorescent labelling is a critical step. Herein a noninvasive strategy for labelling viruses with His-tags was developed by in situ modifying the cell surface proteins with polypeptides containing His-tags during progeny virus assembly. The His-tagged viruses were further conjugated with Ni2+-nitrilotriacetate complex modified quantum dots, and retained their infectivity for real-time single virus tracking in living cells.

HSI colour-coded analysis of scattered light of single plasmonic nanoparticles.

Single plasmonic nanoparticles (PNPs) analysis with dark-field microscopic imaging (iDFM) has attracted much attention in recent years. The ability for quantitative analysis of iDFM is critical, but cumbersome, for characterizing and analyzing the scattered light of single PNPs. Here, a simple automatic HSI colour coding method is established for coding dark-field microscopic (DFM) images of single PNPs with localized surface plasmon resonance (LSPR) scattered light, showing that hue value in the HSI system can realize accurate quantitative analysis of iDFM and providing a novel approach for quantitative chemical and biochemical imaging at the single nanoparticle level.

A magnetic nanoparticle-based aptasensor for selective and sensitive determination of lysozyme with strongly scattering silver nanoparticles.

Qualitative and quantitative determination of lysozyme concentrations in urine and serum with high selectivity and sensitivity is important for diagnosing the progression of several diseases. In this report, we devised an improved method for specifically detecting lysozyme by combining magnetic nanoparticles (for separation and enrichment), an aptamer (for selective binding of lysozyme) and strongly scattering silver nanoparticles (AgNPs, for detection by light scattering, but also providing another level of selectivity due to their electrostatic binding with lysozyme). In this system, 0.4-30 nM lysozyme could be simply detected owing to the decreased light scattering of AgNPs in solution after magnetic separation, with a detection limit of 100 pM. In addition, lysozyme was also able to be semi-quantified by using the dark-field light scattering images of AgNPs after enrichment by the MNP-apt-lysozyme complex. Moreover, this design shows great promise for the robust and reliable detection of lysozyme in real samples, with a recovery rate ranging from 98.6% to 101.3% in human serum samples. Therefore, this assay provided robust measurements with good specificity, sensitivity, and tolerance of changes in the sample matrix. We expect that this MNP-based aptasensor may find utility in the accurate diagnosis of lysozyme-related diseases.

DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding.

Viral infections have caused numerous diseases and deaths worldwide. Due to the emergence of new viruses and frequent virus variation, conventional antiviral strategies that directly target viral or cellular proteins are limited because of the specificity, drug resistance and rapid clearance from the human body. Therefore, developing safe and potent antiviral agents with activity against viral infection at multiple points in the viral life cycle remains a major challenge. In this report, we propose a new modality to inhibit viral infection by fabricating DNA conjugated gold nanoparticle (DNA-AuNP) networks on cell membranes as a protective barrier. The DNA-AuNPs networks were found, via a plaque formation assay and viral titers, to have potent antiviral ability and protect host cells from human respiratory syncytial virus (RSV). Confocal immunofluorescence image analysis showed 80 ± 3.8% of viral attachment, 91.1 ± 0.9% of viral entry and 87.9 ± 2.8% of viral budding were inhibited by the DNA-AuNP networks, which were further confirmed by real-time fluorescence imaging of the RSV infection process. The antiviral activity of the networks may be attributed to steric effects, the disruption of membrane glycoproteins and limited fusion of cell membrane bilayers, all of which play important roles in viral infection. Therefore, our results suggest that the DNA-AuNP networks have not only prophylactic effects to inhibit virus attachment and entry, but also therapeutic effects to inhibit viral budding and cell-to-cell spread. More importantly, this proof-of-principle study provides a pathway for the development of a universal, broad-spectrum antiviral therapy.

Coomassie brilliant blue R-250 as a new surface-enhanced Raman scattering probe for prion protein through a dual-aptamer mechanism.

Surface-enhanced Raman scattering (SERS) spectra, which can provide large information about trace amount of chemical and biological species have been widely performed as a well-established tool in complex biological system. In this work, coomassie brilliant blue (R-250) with high affinity to proteins and high Raman activity was employed as a Raman reporter to probe prion protein (PrP) through a dual-aptamer mechanism, and thus an original strategy for PrP determination was proposed, which showed great potential to turn on the SERS response through specific recognition of anti-prion aptamers towards the target protein. Aptamers (Apt1 and Apt 2) recognizing distinct epitopes of PrP with high affinity were first conjugated to Ag@Si NPs, and Ag@Si-PrP/R-250-Ag@Si conjugates were obtained in the presence of PrP/R-250, inducing dramatically enhanced Raman signal. SERS responses enhanced with increasing amount of PrP and a linear equation of ISERS=6729.7+3091.2 cPrP was obtained in the range of 3.0-12.0×10(-9)M with the determination coefficient of 0.988. The proposed strategy is simple, rapid, and high specificity to probe protein-aptamer recognition in the solution.

In situ labelling chemistry of respiratory syncytial viruses by employing the biotinylated host-cell membrane protein for tracking the early stage of virus entry.

An in situ labelling strategy was proposed to produce quantum dot-labelled respiratory syncytial viruses (RSVs) by incorporating the biotinylated membrane protein of the host cells into mature virions, followed by conjugation with streptavidin modified quantum dots (SA-QDs), which has the advantages such as convenience, efficiency and minor influence on viral infectivity and thus could be successfully applied to track the early stage of virus entry.

Selective and sensitive colorimetric detection of stringent alarmone ppGpp with Fenton-like reagent.

Stringent alarmone, namely, guanosine 3'-diphosphate-5'-diphosphate (ppGpp), is a global regulator that plays a critical role in the survival, growth, metabolism, and many other vital processes of microorganisms. Because of its structural similarity to normal nucleotides, it is also a challenge for the selective and sensitive detection of ppGpp nowadays. Herein, we developed a colorimetric method for the selective detection of ppGpp by inhibiting the redox reaction between Fenton-like reagent (composed of Fe(3+) and H2O2) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). Owing to the strong coordination affinity between ppGpp and Fe(3+), the chromogenic reaction between ABTS and Fenton-like reagent, occurred in aqueous medium at 37 °C and resulted in a bluish-green solution, which was inhibited with the addition of ppGpp. This phenomenon forms the basis for the colorimetric detection of ppGpp, with a detection limit of 0.19 µM and good selectivity for ppGpp over other nucleotides and anions. Furthermore, the results could be visualized by the naked eye, and the sensitivity of the naked-eye observation could even be further improved with the aid of the introduction of a background color.

Real-time light scattering tracking of gold nanoparticles- bioconjugated respiratory syncytial virus infecting HEp-2 cells.

Real-time tracking of virus invasion is crucial for understanding viral infection mechanism, which, however, needs simple and efficient labeling chemistry with improved signal-to-noise ratio. For that purpose, herein we investigated the invasion dynamics of respiratory syncytial virus (RSV) through dark-field microscopic imaging (iDFM) technique by using Au nanoparticles (AuNPs) as light scattering labels. RSV, a ubiquitous, non-segmented, pleiomorphic and negative-sense RNA virus, is an important human pathogen in infants, the elderly, and the immunocompromised. In order to label the enveloped virus of paramyxoviridae family, an efficient streptavidin (SA)-biotin binding chemistry was employed, wherein AuNPs and RSV particles modified with SA and biotin, respectively, allowing the AuNP-modified RSVs to maintain their virulence without affecting the native activities of RSV, making the long dynamic visualization successful for the RSV infections into human epidermis larynx carcinoma cells.

A general quantitative pH sensor developed with dicyandiamide N-doped high quantum yield graphene quantum dots.

A general quantitative pH sensor for environmental and intracellular applications was developed by the facile hydrothermal preparation of dicyandiamide (DCD) N-doped high quantum yield (QY) graphene quantum dots (GQDs) using citric acid (CA) as the carbon source. The obtained N-doped GQDs have excellent photoluminesence (PL) properties with a relatively high QY of 36.5%, suggesting that N-doped chemistry could promote the QY of carbon nanomaterials. The possible mechanism for the formation of the GQDs involves the CA self-assembling into a nanosheet structure through intermolecular H-bonding at the initial stage of the reaction, and then the pure graphene core with many function groups formed through the dehydration between the carboxyl and hydroxyl of the intermolecules under hydrothermal conditions. These N-doped GQDs have low toxicity, and are photostable and pH-sensitive between 1.81 to 8.96, giving a general pH sensor with a wide range of applications from real water to intracellular contents.

DNA-templated Ag nanoclusters as fluorescent probes for sensing and intracellular imaging of hydroxyl radicals.

We have developed a simple, rapid and label-free sensor for the essential biological OH radicals based on the fluorescence quenching of DNA-templated Ag nanoclusters (DNA-Ag NCs). The OH radicals generated from the Fenton reagent attack and cleave the DNA template, which disturbs the microenvironments around Ag NCs, resulting in spontaneous aggregation due to the lack of stabilization and further the quenching of the Ag NCs fluorescence. These changes in fluorescence intensity allow sensing of OH radicals with good sensitivity and selectivity under optimal conditions. The sensor can be also applied for quantifying the radical scavenging action of antioxidants. Various characterizations including absorption spectra, fluorescence lifetimes, light scattering (LS) spectra, transmission electron microscopy (TEM), dark field light scattering imaging, and circular dichroism (CD) spectrometry have been employed to illustrate the proposed sensing mechanism. Further investigations demonstrate that the fluorescent probe could penetrate into intact cell membranes to selectively detect intracellular OH radicals induced by the phorbol myristate acetate (PMA) stimulation. These advantageous characteristics make the fluorescent DNA-Ag NCs potentially useful as a new candidate to monitor OH in broad biosystems.

Aptamer-mediated nanocomposites of semiconductor quantum dots and graphene oxide as well as their applications in intracellular imaging and targeted drug delivery.

Fluorescent semiconductor quantum dot-graphene oxide (QD-GO) nanocomposites with unique optical properties can be prepared by a facile decoration of aptamer-labelled CdSe@ZnS QDs on GO nanosheets. The formation of such nanocomposites is based on the π-π stacking between the DNA bases on the QD surfaces and the GO. TEM and AFM were used to study the morphologies and distribution of the QDs on the GO surfaces. Steady-state fluorescence spectra, time-resolved fluorescence experiments and fluorescence imaging were employed to study the optical properties of the prepared aptamer-QD-GO nanocomposites. Furthermore, we investigate the potential applications of the nanocomposites in bio-imaging and cell-targeted drug delivery. The QDs decorated on the surfaces of GO could serve as fluorescent labeling probes for tracking the intracellular transport, while the GO combined with the aptamer conjugated on the outside of the nanocomposites facilitates the targeted drug delivery with enhanced loading capability. It is believed that the present aptamer-QD-GO nanocomposite-based nanomedicine would permit the development of more effective means for diagnosing and treating malignancies compared to the currently used methods.

Metal-enhanced fluorescence of nano-core-shell structure used for sensitive detection of prion protein with a dual-aptamer strategy.

Metal-enhanced fluorescence (MEF) as a newly recognized technology is widespread throughout biological research. The use of fluorophore-metal interactions is recognized to be able to alleviate some of fluorophore photophysical constraints, favorably increase both the fluorophore emission intensity and photostability. In this contribution, we developed a novel metal-enhanced fluorescence (MEF) and dual-aptamer-based strategy to achieve the prion detection in solution and intracellular protein imaging simultaneously, which shows high promise for nanostructure-based biosensing. In the presence of prion protein, core-shell Ag@SiO2, which are functionalized covalently by single stranded aptamer (Apt1) of prions and Cyanine 3 (Cy3) decorated the other aptamer (Apt2) were coupled together by the specific interaction between prions and the anti-prion aptamers in solution. By adjusting shell thickness of the pariticles, a dual-aptamer strategy combined MEF can be realized by the excitation and/or emission rates of Cy3. It was found that the enhanced fluorescence intensities followed a linear relationship in the range of 0.05-0.30 nM, which is successfully applied to the detection of PrP in mice brain homogenates.

A new type of pH-responsive coordination polymer sphere as a vehicle for targeted anticancer drug delivery and sustained release.

A new type of coordination polymer sphere prepared by combining 1,1'-(1,4-butanediyl)bis(imidazole) (bbi) and ferrous ions has been demonstrated as a targeted delivery system for in situ encapsulating anticancer drugs. These stable coordination polymer spheres can be fabricated simply by a deposition method. Drugs, doxorubicin hydrochloride (DOX·HCl) for example, can be easily in situ encapsulated by simply mixing the drug with bbi ligand through the deposition method and results in a high drug loading efficiency up to 98% and a drug loading content of nearly 40%, which is remarkably high for not only metal-organic but also other materials. A noticeable feature of the drug loaded coordination polymer spheres is that they show sustainable drug release for several days due to their superior stability, and are sensitive to external pH owing to the coordination bonds. The drug can be released faster in mild acidic conditions in comparison to physiological acidity. By conjugating folic acid to the surface of the coordination polymer spheres, the vehicles can be taken up selectively by cancer cells through cell surface receptor-mediated mechanisms. Cell viability experiments with HeLla cells demonstrated the low toxicity of the delivery vehicles and the good anticancer efficacy of the drug-loaded coordination polymer spheres.