Nanomechanical properties of phospholipid microbubbles

This study uses atomic force microscopy (AFM) force-deformation (F-Δ) curves to investigate for the first time the Young's modulus of a phospholipid microbubble (MB) ultrasound contrast agent. The stiffness of the MBs was calculated from the gradient of the F-Δ curves, and the Young's modulus of the MB shell was calculated by employing two different mechanical models based on the Reissner and elastic membrane theories. We found that the relatively soft phospholipid-based MBs behave inherently differently to stiffer, polymer-based MBs [Glynos, E.; Koutsos, V.; McDicken, W. N.; Moran, C. M.; Pye, S. D.; Ross, J. A.; Sboros, V. Langmuir2009, 25 (13), 7514-7522] and that elastic membrane theory is the most appropriate of the models tested for evaluating the Young's modulus of the phospholipid shell, agreeing with values available for living cell membranes, supported lipid bilayers, and synthetic phospholipid vesicles. Furthermore, we show that AFM F-Δ curves in combination with a suitable mechanical model can assess the shell properties of phospholipid MBs. The "effective" Young's modulus of the whole bubble was also calculated by analysis using Hertz theory. This analysis yielded values which are in agreement with results from studies which used Hertz theory to analyze similar systems such as cells.     

In Situ Synchrotron Radiation X‐Ray Microspectroscopy of Polymer Microcontainers

Direct, real‐time analytical techniques that provide high‐resolution information on the chemical composition and submicrometer structure of various polymer micro‐ and nanoparticles are in high demand in a range of life science disciplines. Synchrotron‐based scanning transmission X‐ray microspectroscopy (STXM) combines both local‐spot chemical information (assessed via near‐edge X‐ray absorption fine structure spectroscopy) and imaging with resolution of several tens of nanometers, and thus can yield new insights into the nanoscale properties of these materials. Furthermore, this method allows in situ examination of soft‐matter samples in aqueous/gaseous environments and under external stimuli, such as temperature, pressure, ultrasound, and light irradiation. This Minireview highlights some recent progress in the application of the STXM technique to study the temperature‐dependent behavior of polymer core–shell microcapsules and to characterize the physicochemical properties of the supporting shells of gas‐filled microbubbles in their natural hydrated state.     

Probing the permeability of polyelectrolyte multilayer capsules via a molecular beacon approach

Application of polyelectrolyte multilayer (PEM) capsules as vehicles for the controlled delivery of substances, such as drugs, genes, pesticides, cosmetics, and foodstuffs, requires a sound understanding of the permeability of the capsules. We report the results of a detailed investigation into probing capsule permeability via a molecular beacon (MB) approach. This method involves preparing MB-functionalized bimodal mesoporous silica (BMSMB) particles, encapsulating the BMSMB particles within the PEM film to be probed, and then incubating the encapsulated BMSMB particles with DNA target sequences of different lengths. Permeation of the DNA targets through the capsule shell causes the immobilized MBs to open due to hybridization of the DNA targets with the complementary loop region of the MBs, resulting in an increase in the MB fluorescence. The assay conditions (BMSMB particle concentration, MB loading within the BMS particles, DNA target concentration, DNA target size, pH, sodium chloride concentration) where the MB-DNA sensing process is effective were first examined. The permeability of DNA through poly(sodium 4-styrenesulfonate) (PSS)/poly(allylamine hydrochloride) (PAH) multilayer films, with and without a poly(ethyleneimine) (PEI) precursor layer, was then investigated. The permeation of the DNA targets decreases considerably as the thickness of the PEM film encapsulating the BMSMB particles increases. Furthermore, the presence of a PEI precursor layer gives rise to less permeable PSS/PAH multilayers. The diffusion coefficients calculated for the DNA targets through the PEM capsules range from 10-19 to 10-18 m2 s-1. This investigation demonstrates that the MB approach to measuring permeability is an important new tool for the characterization of PEM capsules and is expected to be applicable for probing the permeability of other systems, such as membranes, liposomes, and emulsions.     

STXM研究氧元素在PAN预氧化纤维截面上的分布

采用扫描透射X射线显微镜( STXM),对聚丙烯腈(PAN)预氧化纤维截面进行具有30 nm空间分辨率的元素分布研究,结合将两幅吸收图上对应像素点的光密度相比求解元素分布的双能衬度分析法,获得氧元素在PAN预氧化纤维截面上的分布信息.STXM实验结果表明,氧元素在PAN原丝截面上分布是均匀的;经过预氧化后,PAN纤维皮...     

Interfacial Nanoprecipitation toward Stable and Responsive Microbubbles and Their Use as a Resuscitative Fluid

A new approach has been developed to prepare stable microbubbles (MBs) by interfacial nanoprecipitation of bioabsorbable polymers at air/liquid interfaces. This facile method offers robust control over the morphology and chemophysical properties of MBs by simple chemical modifications. This approach is amenable to large‐scale manufacturing, and is useful to develop functional MBs for advanced biomedical applications. To demonstrate this, a MB‐based intravenous oxygen carrier was created that undergoes pH‐triggered self‐elimination. Intravenous injection of previous MBs increased the risk of pulmonary vascular obstruction. However, we show, for the first time, that our current design is superior, as they 1) yielded no evidence of acute risks in rodents, and 2) improved the survival in a disease model of asphyxial cardiac arrest (from 0 to 100 %), a condition that affects more than 100 000 in‐hospital patients, and carries a mortality of about 90 %.     

Bulk and single-molecule characterization of an improved molecular beacon utilizing H-dimer excitonic behavior

Pairs of fluorophores in close proximity often show self-quenching of fluorescence by the well-known H-dimer mechanism. We use a pair of fluorophores in the new dicyanomethylenedihydrofuran (DCDHF) dye family in the design and characterization of a new fluorescent probe for nucleic acid detection, which we refer to as a self-quenched intramolecular dimer (SQuID) molecular beacon (MB). We obtain a quenching efficiency of 97.2%, higher than the only other reported value for a MB employing fluorophore self-quenching by H-dimer formation. Furthermore, the excellent single-molecule (SM) emitter characteristics of the DCDHF dyes allow observation of individual SQuID MB-target complexes immobilized on a surface, where the doubled SM emission intensity of our target-bound beacon ensures a higher signal-to-background ratio than conventional fluorophore-quencher MBs. Additional advantages of the SQuID MB are single-pot labeling, visible colorimetric detection of the target, and intrinsic single-molecule two-step photobleaching behavior, which offers a specific means of discriminating between functional MBs and spurious fluorescence.     

分子信标荧光探针用于抑癌基因ING1表达产物的定量测定

根据抑癌基因ING1基因的序列设计并合成了检测ING1转录产物的分子信标核酸探针,发展了一种快速测量从正常细胞系和鼻咽癌肿瘤细胞系提取的ING1转录产物的方法,所得结果与用逆转录结合PCR法(RT-PCR)得到的结果相吻合.并且,将能表达ING1基因的质粒转入肿瘤细胞进行培养后,再将分子信标转入肿瘤细胞,发现转导了质粒的肿瘤细胞比未转导质粒的肿瘤细胞内的荧光明显增强,从而进一步证实了所设计的分子信标核酸探针与ING1转录产物的结合.     

Molecular beacons for bioanalytical applications

Molecular beacons (MBs) are hairpin-shaped oligonucleotides that contain both fluorophore and quencher moieties. They act like switches and are normally in a closed state, when the fluorophore and the quencher are brought together to turn "off" the fluorescence. When prompted to undergo conformational changes that open the hairpin structure, the fluorophore and the quencher are separated, and fluorescence is turned "on." This Education will outline the principles of MBs and discuss recent bioanalytical applications of these probes for in vitro RNA and DNA monitoring, biosensors and biochips, real-time monitoring of genes and gene expression in living systems, as well as the next generation of MBs for studies on proteins, the MB aptamers. These important applications have shown that MBs hold great potential in genomics and proteomics where real-time molecular recognition with high sensitivity and excellent specificity is critical.     

Development of Flow Cytometric Assay for Detecting Papillary Thyroid Carcinoma Related hsa-miR-146b-5p through Toehold-Mediated Strand Displacement Reaction on Magnetic Beads

In this work, a simple enzyme-free flow cytometric assay (termed as TSDR-based flow cytometric assay) has been developed for the detection of papillary thyroid carcinoma (PTC)-related microRNA (miRNA), hsa-miR-146b-5p with high performance through the toehold-mediated strand displacement reaction (TSDR) on magnetic beads (MBs). The complementary single-stranded DNA (ssDNA) probe of hsa-miR-146b-5p was first immobilized on the surface of MB, which can partly hybridize with the carboxy-fluorescein (FAM)-modified ssDNA, resulting in strong fluorescence emission. In the presence of hsa-miR-146b-5p, the TSDR is trigged, and the FAM-modified ssDNA is released form the MB surface due to the formation of DNA/RNA heteroduplexes on the MB surface. The fluorescence emission change of MBs can be easily read by flow cytometry and is strongly dependent on the concentration of hsa-miR-146b-5p. Under optimal conditions, the TSDR-based flow cytometric assay exhibits good specificity, a wide linear range from 5 to 5000 pM and a relatively low detection limit (LOD, 3σ) of 4.21 pM. Moreover, the practicability of the assay was demonstrated by the analysis of hsa-miR-146b-5p amounts in different PTC cells and clinical PTC tissues.     

In situ characterization of aluminum-containing mineral-microorganism aqueous suspensions using scanning transmission X-ray microscopy

In situ characterization of colloidal particles under hydrous conditions is one of the key requirements for understanding their state of aggregation and impact on the transport of pollutants in aqueous environments. Scanning transmission X-ray microscopy (STXM) is one of the few techniques that can satisfy this need by providing element- and chemical-state-specific 2-D maps at a spatial resolution better than 50 nm using soft X-rays from synchrotron radiation wiggler or undulator sources tuned to the absorption edges of different elements. X-ray absorption near-edge structure (XANES) spectra can also be collected simultaneously at a similar spatial resolution and can provide phase identification in many cases. In this study, we report STXM images and XANES spectroscopy measurements at or above the Al K-edge (E = 1559.6 eV) of various Al-containing minerals and synthetic oxides [alpha-Al2O3 (corundum), gamma-Al2O3, gamma-AlOOH (boehmite), alpha-Al(OH)3 (bayerite), KAl2(AlSi3O10)(OH)2 (muscovite), (Al,Mg)8(Si4O10)4(OH)8.nH2O (montmorillonite), and Mg6Al2(OH)16CO3.4H2O (hydrotalcite)] and demonstrate the capability of this spectromicroscopic tool to identify different Al-containing mineral colloids in multiphase mixtures in aqueous solution. We also demonstrate that STXM imaging at or above the C K-edge (E = 284.2 eV) and Al K-edge can provide unique information on the interactions between bacteria and Al-containing nanoparticles in aqueous suspensions. STXM images of a mixture of Caulobacter crescentus and montmorillonite and corundum particles just above the C and Al K-edges show that the mineral particles and bacteria are closely associated in aggregates, which is likely due to the binding of bacteria to clay and corundum particles by extracellular polysaccharides.     

Recent trends in molecular beacon design and applications

A molecular beacon (MB) is a hairpin-structured oligonucleotide probe containing a photoluminescent species (PLS) and a quencher at different ends of the strand. In a recognition and detection process, the hybridization of MBs with target DNA sequences restores the strong photoluminescence, which is quenched before hybridization. Making better MBs involves reducing the background photoluminescence and increasing the brightness of the PLS, which therefore involves the development of new PLS and quenchers, as well as innovative PLS–quencher systems. Heavy-metal complexes, nanocrystals, pyrene compounds, and other materials with excellent photophysical properties have been applied as PLS of MBs. Nanoparticles, nanowires, graphene, metal films, and many other media have also been introduced to quench photoluminescence. On the basis of their high specificity, selectivity, and sensitivity, MBs are developed as a general platform for sensing, producing, and carrying molecules other than oligonucleotides.     

Covalent attachment of biotinylated molecular beacons via thiol-ene coupling. A study on conformational changes upon hybridization and streptavidin binding

The authors describe a method for the detection of DNA by using immobilized molecular beacons (MBs) on the surface of silicon, with a view on possible application in biosensing. MB hybridization and protein recognition were interrogated on silicon-on-insulator (SOI) surfaces by using fluorescently tagged probes. In order to better understand the conformational changes that occur to MBs upon hybridization, the process was studied by using dual polarization interferometry (DPI). A model system was developed that matches thickness, mass, and density parameters. The results experimentally demonstrate that hybridization promotes the displacement of a protein away from the surface. This finding may be further exploited in techniques such as photonic sensors, thereby paving the way to the design of more sensitive biosensors based on the use of MBs.

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Graphical abstract Schematic of a new DNA/RNA detection scheme by using immobilized molecular beacons (MBs) on silicon, with a view on possible application in biosensing. A study was performed on the conformational changes that occur to MBs upon hybridization by Dual Polarization Interferometry (DPI).

     

Enhancement of the Modulation Response of Quantum-Dot-Based Down-Converted Light through Surface Plasmon Coupling

In this paper, we first elaborate on the effects of surface plasmon (SP) coupling on the modulation responses of the emission of a light-emitting diode (LED) and its down-converted lights through colloidal quantum dots (QDs). The results of our past efforts for this subject are briefly discussed. The discussions lay the foundation for the presentation of the new experimental data of such down-converted lights in this paper. In particular, the enhancement of the modulation bandwidth (MB) of a QD-based converted light through SP coupling is demonstrated. By linking green-emitting QDs (GQDs) and/or red-emitting QDs (RQDs) with synthesized Ag nano-plates via surface modifications and placing them on a blue-emitting LED, the MBs of the converted green and red emissions are significantly increased through the induced SP coupling of the Ag nano-plates. When both GQD and RQD exist and are closely spaced in a sample, the energy transfer processes of emission-reabsorption and Förster resonance energy transfer from GQD into RQD occur, leading to the increase (decrease) in the MB of green (red) light. With SP coupling, the MB of a mixed light is significantly enhanced.     

Particle rearrangements during transitions between local minima of the potential energy landscape of a binary Lennard-Jones liquid

The potential energy landscape (PEL) of binary Lennard-Jones (BLJ) mixtures exhibits local minima, or inherent structures (IS), which are organized into metabasins (MBs). We study the particle rearrangements related to transitions between both successive IS and successive MB for a small 80:20 BLJ system near the mode-coupling temperature TMCT. The analysis includes the displacements of individual particles, the localization of the rearrangements, and the relevance of string-like motion. We find that the particle rearrangements during IS and MB transitions do not change significantly at TMCT. In particular, an onset of single particle hopping on the length scale of the interparticle distance is not observed. Further, it is demonstrated that IS and MB dynamics are spatially heterogeneous and facilitated by string-like motion. To investigate the mechanism of string-like motion, we follow the particle rearrangements during suitable sequences of IS transitions. We find that most strings observed after a series of transitions do not move coherently during a single transition, but subunits of different sizes are active at different times. Several findings suggest that, though string-like motion is of comparable relevance when the system explores a MB and when it moves from one MB to another, the occurrence of a successful string enables the system to exit a MB. Moreover, we show that the particle rearrangements during two consecutive MB transitions are basically uncorrelated. In particular, different groups of particles are highly mobile. We further find the positions of strings during successive MB transitions weakly but positively correlated, supporting the idea of dynamic facilitation. Finally, the relation between the features of the potential energy landscape and the relaxation processes in supercooled liquids is discussed.     

Quencher-free molecular beacons: a new strategy in fluorescence based nucleic acid analysis

Molecular beacons (MBs) have been used as viable fluorescent probes in nucleic acid analysis. Many researchers around the world continue to modify the MBs to suit their needs. As a result, a number of nucleic acid probing systems with close resemblance to the MBs are being reported from time to time. Quencher-free molecular beacons (QF-MBs) are a significant modification of the conventional MB; in QF-MBs the quencher part has been eliminated. Despite the absence of the quencher, the QF-MBs can identify specific target DNA. They can also be used in SNP typing and in real-time PCR analysis for quantification of DNAs. The design, factors behind functioning and applications of different types of QF-MBs and closely related quencher-free nucleic acid probing systems (QF-NAPs) have been described in this tutorial review.     

Molecular Engineering of DNA: Molecular Beacons

Molecular beacons (MBs) are specifically designed DNA hairpin structures that are widely used as fluorescent probes. Applications of MBs range from genetic screening, biosensor development, biochip construction, and the detection of single‐nucleotide polymorphisms to mRNA monitoring in living cells. The inherent signal‐transduction mechanism of MBs enables the analysis of target oligonucleotides without the separation of unbound probes. The MB stem–loop structure holds the fluorescence‐donor and fluorescence‐acceptor moieties in close proximity to one another, which results in resonant energy transfer. A spontaneous conformation change occurs upon hybridization to separate the two moieties and restore the fluorescence of the donor. Recent research has focused on the improvement of probe composition, intracellular gene quantitation, protein–DNA interaction studies, and protein recognition.     

Structure and electric properties of the hydration shell of a singly charged chloride ion in a nanopore with hydrophilic walls

The effect of hydrophilic walls on the structure of the hydration shell of a Cl^? ion is studied in terms of the model flat nanopore in contact with water vapors at room temperature by the Monte Carlo computerassisted simulations. In the field of hydrophilic walls, the hydration shell falls into two parts: the ion-enveloping part and the molecular-film spots spread over the wall surface above and under the ion. Both parts have the pronounced radial-layered structure. The three-dimensional scheme of distribution of the averaged local shell density represents a system of conical coaxial layers expanding in the direction from wall to ion. The effect of forcing out the ion from its own hydration shell is also observed for hydrophilic walls. The specific electric polarizability of the shell is strongly anisotropic. Its longitudinal component is several times larger than the transversal component and behaves nonmonotonically as the hydration shell grows, passing through the maximum. The molecular order near the walls is characterized by the preferential orientation of the molecule plane in parallel to the wall plane and the turn of symmetry axes of molecules in the direction parallel to the normal to the pore plane in the vicinity of the ion.     

Biosensors based on molecular beacons

A fundamental molecular beacon (MB) consists of a special short nucleic acid strand with a fluorophore-quencher pair attached to its ends. It provides a unique framework that is susceptible to conformational transitions between a hairpin (closed) conformation and an extended (open) conformation. These two conformations are readily discernible because of their differing fluorescence emission characteristics. The broad applicability of the robust MB sensing platform has attracted widespread interest, resulting in extensive research studies ranging from theoretical and bioanalytical chemistry to molecular biology and biomedical applications. In this paper, the principles of MB design and the modes and mechanisms of MB operation are reviewed, including MB modifications based on the utilisation of a thymidine-thymidine mismatch in hybridised MB stem, aptamers, peptides and locked nucleic acid strands in an MB loop, as well as plasmonic quenchers, quantum dots and interactions with graphene and graphene oxide. Specific applications of MBs in the analysis of enzymes, DNA mutation, phosphorylation, methylation and ligation, followed by the detection of pathogens and applications in cancer and other disease diagnostics and therapeutics are also reviewed. Molecular beacon-based sensing platforms are expanding rapidly and offer a promising bioanalytical tool for inexpensive and reliable analysis for research and field diagnostics.     

Adding Value in Production of Multifunctional Polylactide (PLA)–ZnO Nanocomposite Films through Alternative Manufacturing Methods

Due to the added value conferred by zinc oxide (ZnO) nanofiller, e.g., UV protection, antibacterial action, gas-barrier properties, poly(lactic acid) (PLA)–ZnO nanocomposites show increased interest for utilization as films, textile fibers, and injection molding items. The study highlights the beneficial effects of premixing ZnO in PLA under given conditions and its use as masterbatch (MB), a very promising alternative manufacturing technique. This approach allows reducing the residence time at high processing temperature of the thermo-sensitive PLA matrix in contact of ZnO nanoparticles known for their aptitude to promote degradation effects onto the polyester chains. Various PLA–ZnO MBs containing high contents of silane-treated ZnO nanoparticles (up to 40 wt.% nanofiller specifically treated with triethoxycaprylylsilane) were produced by melt-compounding using twin-screw extruders. Subsequently, the selected MBs were melt blended with pristine PLA to produce nanocomposite films containing 1–3 wt.% ZnO. By comparison to the more traditional multi-step process, the MB approach allowed the production of nanocomposites (films) having improved processing and enhanced properties: PLA chains displaying higher molecular weights, improved thermal stability, fine nanofiller distribution, and thermo-mechanical characteristic features, while the UV protection was confirmed by UV-vis spectroscopy measurements. The MB alternative is viewed as a promising flexible technique able to open new perspectives to produce more competitive multifunctional PLA–ZnO nanocomposites.