Atomic Force Microscopy for Molecular Structure Elucidation

Using scanning probe microscopy techniques, at low temperatures and in ultrahigh vacuum, individual molecules adsorbed on surfaces can be probed with ultrahigh resolution to determine their structure and details of their conformation, configuration, charge states, aromaticity, and the contributions of resonance structures. Functionalizing the tip of an atomic force microscope with a CO molecule enabled atomic‐resolution imaging of single molecules, and measurement of their adsorption geometry and bond‐order relations. In addition, by using scanning tunneling microscopy and Kelvin probe force microscopy, the density of the molecular frontier orbitals and the electric charge distribution within molecules can be mapped. Combining these techniques yields a high‐resolution tool for the identification and characterization of individual molecules. The single‐molecule sensitivity and the possibility of atom manipulation to induce chemical reactions with the tip of the microscope open up unique applications in chemistry, and differentiate scanning probe microscopy from conventional methods for molecular structure elucidation. Besides being an aid for challenging cases in natural product identification, atomic force microscopy has been shown to be a powerful tool for the investigation of on‐surface reactions and the characterization of radicals and molecular mixtures. Herein we review the progress that high‐resolution scanning probe microscopy with functionalized tips has made for molecular structure identification and characterization, and discuss the challenges it will face in the years to come.     

Self-assembled monolayers of peptide nucleic acids on gold surfaces: a spectroscopic study

We have characterized self-assembled monolayers (SAMs) of thiol-derivatized peptide nucleic acid (PNA) chains adsorbed on gold surfaces by using reflection absorption infrared spectroscopy (RAIRS) and X-ray photoemission spectroscopy (XPS) techniques. We have found that the molecular orientation of PNAs strongly depends on surface coverage. At low coverage, PNA chains lie flat on the surface, while at high coverage, PNA molecules realign their molecular axes with the surface normal and form SAMs without the need of co-immobilization of spacers or other adjuvant molecules. The change in the molecular orientation has been studied by infrared spectroscopy and it has been confirmed by atomic force microscopy (AFM). PNA immobilization has been followed by analyzing the N(1s) XPS core-level peak. We show that the fine line shape of the N(1s) core-level peak at optimal concentration for biosensing is due to a chemical shift. A combination of the above-mentioned techniques allow us to affirm that the structure of the SAMs is stabilized by molecule-molecule interactions through noncomplementary adjacent nucleic bases.     

Factors Affecting Extracellular Vesicles Based Drug Delivery Systems

Extracellular vesicles (EVs) play major roles in intracellular communication and participate in several biological functions in both normal and pathological conditions. Surface modification of EVs via various ligands, such as proteins, peptides, or aptamers, offers great potential as a means to achieve targeted delivery of therapeutic cargo, i.e., in drug delivery systems (DDS). This review summarizes recent studies pertaining to the development of EV-based DDS and its advantages compared to conventional nano drug delivery systems (NDDS). First, we compare liposomes and exosomes in terms of their distinct benefits in DDS. Second, we analyze what to consider for achieving better isolation, yield, and characterization of EVs for DDS. Third, we summarize different methods for the modification of surface of EVs, followed by discussion about different origins of EVs and their role in developing DDS. Next, several major methods for encapsulating therapeutic cargos in EVs have been summarized. Finally, we discuss key challenges and pose important open questions which warrant further investigation to develop more effective EV-based DDS.     

Nanoscale analyses of modified polypropylene microtubes internal surface: an approach covering topographical and force spectroscopic parameters

A nanoscale characterization of modified and unmodified polypropylene (PP) microtubes internal surface was performed to investigate their structural, chemical, and physical properties. Nanoroughness, stiffness, elasticity, attraction behavior, adhesion forces, and chemical environment were investigated to test some manufacturer statements regarding Axygen MAXYMum Recovery® products. They announced that this class of material presented special features, originated from a modification to the original PP resin and by using a diamond polished mould, providing lower retention and minor interference on laboratorial tests, such as low roughness and little interaction tendency. Then, in this study, modified and control internal surfaces of PP microtubes were compared by atomic force microscopy, scanning electron microscopy, and Fourier transform infrared spectroscopy. Nanoroughness and force spectroscopy parameters assessed by atomic force microscopy showed out as a sensible and high‐resolution technique, crucial to discriminate differences between the surfaces. This type of investigation can be considered as a promising approach that can be applied to other polymeric systems, considering nanoscale properties, physical/chemical modifications, and as an alternative route for quality control checking concerning polymeric surfaces. Copyright © 2013 John Wiley & Sons, Ltd.     

Inhibitory Action of Funtumia elastica Extracts on the Corrosion of Q235 Mild Steel in Hydrochloric Acid Medium: Experimental and Theoretical Studies

The inhibiting effect of aqueous extracts of Funtumia elastica (FE) on mild steel corrosion in 1 M HCl solution was investigated using electrochemical and surface characterization techniques. The results revealed that FE effectively inhibited the corrosion reaction. Polarization data reveal that the extract functioned as a mixed-type inhibitor, while impedance results show that the extract organic matter gets adsorbed on the metal/solution interface. Fourier transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy results confirmed the formation of a protective layer of extract adsorbed on the mild steel surface. Adsorption of some organic constituents of FE on mild steel was theoretically described by quantum chemical computations and molecular dynamics simulations, in the framework of the density functional theory.     

Phosphate-Based Self-Immolative Linkers for Tuneable Double Cargo Release

Phosphorus-based self-immolative (SI) linkers offer a wide range of applications, such as smart materials and drug-delivery systems. Phosphorus SI linkers are ideal candidates for double-cargo delivery platforms because they have a higher valency than carbon. A series of substituted phosphate linkers was designed for releasing two phenolic cargos through SI followed by chemical hydrolysis. Suitable modifications of the lactate spacer increased the cargo release rate significantly, from 1 day to 2 hours or 5 minutes, as shown for linkers containing p-fluoro phenol. In turn, double cargo linkers bearing p-methyl phenol released their cargo more slowly (4 days, 4 hours, and 15 minutes) than their p-fluoro analogues. The α-hydroxyisobutyrate linker released both cargos in 25 minutes. Our study expands the current portfolio of SI constructs by providing a double cargo delivery option, which is crucial to develop universal SI platforms.     

Theragnostic Applications of Mammal and Plant-Derived Extracellular Vesicles: Latest Findings,Current Technologies,and Prospects

The way cells communicate is not fully understood. However, it is well-known that extracellular vesicles (EVs) are involved. Researchers initially thought that EVs were used by cells to remove cellular waste. It is now clear that EVs function as signaling molecules released by cells to communicate with one another, carrying a cargo representing the mother cell. Furthermore, these EVs can be found in all biological fluids, making them the perfect non-invasive diagnostic tool, as their cargo causes functional changes in the cells upon receiving, unlike synthetic drug carriers. EVs last longer in circulation and instigate minor immune responses, making them the perfect drug carrier. This review sheds light on the latest development in EVs isolation, characterization and, application as therapeutic cargo, novel drug loading techniques, and diagnostic tools. We also address the advancement in plant-derived EVs, their characteristics, and applications; since plant-derived EVs only recently gained focus, we listed the latest findings. Although there is much more to learn about, EV is a wide field of research; what scientists have discovered so far is fascinating. This paper is suitable for those new to the field seeking to understand EVs and those already familiar with it but wanting to review the latest findings.     

Patterns of surface immobilized block copolymer vesicle nanoreactors

The immobilization and positioning of ultra small reaction vessels on solid supports open new pathways in applications such as lab-on-a-chip, sensors, microanalyses and microreactors. In our work block copolymer vesicles made from polystyrene-block-polyacrylic acid (PS-b-PAA) were immobilized from aqueous medium onto 3-amino propyl trimethoxysilane functionalized silicon surfaces exploiting electrostatic interactions. The immobilization of the vesicles was investigated by Fourier transform infrared (FTIR) spectroscopy, as well as fluorescence optical and atomic force microscopy (AFM). In addition, the influence of pH and ionic strength on the surface coverage of vesicles bound to the surface was elucidated. Finally micro-molding in capillaries (MIMIC) was utilized to create line patterns of the vesicles containing the enzyme trypsin and the fluorogenic substrate rhodamine 110 bisamide. The selective positioning of vesicle nanoreactors in conjunction with electrostatic immobilization serves as a proof of principle for potential applications in real-time observation of confined chemical reaction inside vesicles as nanocontainers and for the fabrication of integrated microarray systems.     

Self-assembling peptide as a potential carrier of hydrophobic compounds

Microcrystals of a hydrophobic cargo were stabilized by EAK16 II, a self-assembling oligopeptide, and suspended in aqueous solution. Pyrene was used as a model hydrophobic compound. Egg phosphatidylcholine (EPC) vesicles were prepared to mimic a cell membrane. Pyrene was released from its EAK16 II coating into EPC vesicles. The excimer decay profiles were acquired. They showed that pyrene is present in the crystalline form when stabilized by EAK16 II, it is molecularly dispersed in EPC vesicles, and it is completely released from its EAK16 II coating into the membrane bilayers. The release of pyrene from the microcrystals coated with EAK16 II into the EPC membrane was followed by fluorescence as a function of time. The amount of pyrene released into the EPC vesicles at a given time was quantified using a calibration curve. The concentration of pyrene released was determined as a function of time, and the concentration-versus-time profile was fitted with one exponential. The rate of pyrene release was found to depend on the peptide-to-pyrene molecular ratio. Higher peptide-to-pyrene ratios lead to slower transfer of pyrene to the lipophilic environment. Scanning electron micrographs demonstrated that a thicker coating on the pyrene crystals results in a slower release. The data presented in this work demonstrate that the self-assembling EAK16 II can stabilize a hydrophobic cargo in aqueous solution and deliver it into a lipophilic environment, and that the rate of transfer can be adjusted by tuning the peptide-to-pyrene ratio.     

Inclusion and fractionated release of nucleic acids using microcapsules made from plant cells.

The encapsulation and fractionated release of nucleic acids on vesicular packing (VP) materials have been investigated. The earlier described dependence of the permeation of nucleic acid molecules through the vesicle membranes on the salt concentration is a necessary precondition for both encapsulation and fractionation. Encapsulation is achieved by applying a suitable sample onto a VP column that has been equilibrated with a high-salt buffer. In that buffer the sample molecules are permeable. Immediately after sample application, elution is started with a low-salt buffer, from which the sample molecules are excluded. At the front between the two buffers the permeability changes, and some of the sample molecules distributed inside the vesicles cannot pass through the membranes. These encapsulated molecules can be released by increasing the salt concentration in the eluent. If the encapsulated nucleic acid sample is polydisperse, a stepwise or linear increase in the salt concentration leads to a fractionated release. The fractions obtained differ in their molecular size composition.     

Probing the local structure and mechanical response of nanostructures using force modulation and nanofabrication

Nanostructures of self-assembled monolayers (SAMs) are designed and produced using coadsorption and nanografting techniques. Because the structures of these artificially engineered domains are predesigned and well-characterized, a systematic investigation is possible to study the mechanical responses to force modulation under atomic force microscope tips. Force modulation imaging reveals characteristic contrast sensitivity to changes in molecular-level packing, molecule chain lengths, domain boundaries, and surface chemical functionalities in SAMs. By means of actively tuning the driving frequency, the resonances at the tip-surface contact are selectively activated. Therefore, specific surface features, such as the edges of the domains and nanostructures or desired chemical functionalities, can be selectively enhanced in the amplitude images. These observations provide a new and active approach in materials characterization and the study of nanotribology using atomic force microscopy.     

外泌体分离技术及其临床应用研究进展

外泌体是细胞通过胞吐过程分泌的一类粒径为30~200 nm的囊泡，其组成包括脂质双分子层以及其内部包裹的细胞来源的蛋白质、核糖核苷酸（RNA）和脱氧核糖核苷酸（DNA）等生物分子。作为一种细胞间交流的重要方式，外泌体在一系列生理和病理过程中起着至关重要的作用。由于体液环境复杂，加之自身体积小、密度低，外泌体的富集与分离对于其后续分析和功能研究至关重要。该文介绍了外泌体的研究策略、表征手段及生物学功能和临床应用研究进展，特别对外泌体的提取方法进行了详细介绍，并加以系统评述。     

Layer-by-layer self-assembled polyelectrolyte multilayers with embedded liposomes: immobilized submicronic reactors for mineralization

The development of chemical reactions in nanospaces is of paramount importance for the development of active nanodevices, particularly in nanofluidics. It has been shown in a previous paper that phospholipid vesicles can be incorporated without spontaneous bilayer rupture into poly-L-glutamic acid/poly(allylamine) (PGA/PAH) multilayered polyelectrolyte films. The aim of the present study was to use such a system as an "embedded submicronic reactor" able to trigger precipitation of calcium phosphates within closed spaces through an enzymatic reaction, the enzyme also being encapsulated in the vesicle interior. To this aim, large unilamellar vesicles (LUVs) were produced containing calcium ions as active ions in the mineralization process, spermine as an activator of crystal growth, and alkaline phosphatase as a catalyst to convert phosphate esters into phosphates. After stabilization by adding a layer of poly-(D-lysine), these vesicles were embedded in a (PGA-PAH)n film. A paranitrophenyl phosphate containing solution was then put in contact with this film. It is shown by means of infrared spectroscopy in the attenuated total reflection mode that, consecutively to this contact, calcium phosphates are growing inside the embedded vesicles. By using scanning near-field fluorescence microscopy, it is demonstrated that the alkaline phosphatase enzymes are most probably located inside the vesicles after their embedding. In addition, atomic force microscopy was used to show, after chemical removal of the organic top layer of the film, that the inorganic platelets produced after the precipitation reaction are localized in volumes of similar size and shape as that of the vesicles into which the phosphate ester hydrolysis and subsequent precipitation reaction did occur.     

Microfluidics-based technologies for the analysis of extracellular vesicles at the single-cell level and single-vesicle level

Extracellular vesicles(EVs) are membrane vesicles secreted by cells, playing critical roles in mediating intercellular communications for various physiological and pathological processes. Most of the EV analysis is currently performed at the bulk level, obscuring the origin of the EVs and diverse characteristics of the individual extracellular vesicle. Technologies to analyze the extracellular vesicles at the single-cell and single-vesicle levels are needed to evaluate EV comprehensively and dec...     

Measurement of the elastic modulus of single bacterial cellulose fibers using atomic force microscopy

The ability of the atomic force microscope to measure forces with subnanonewton sensitivity at nanometer-scale lateral resolutions has led to its use in the mechanical characterization of nanomaterials. Recent studies have shown that the atomic force microscope can be used to measure the elastic moduli of suspended fibers by performing a nanoscale three-point bending test, in which the center of the fiber is deflected by a known force. We extend this technique by modeling the deflection measured at several points along a suspended fiber, allowing us to obtain more accurate data, as well as to justify the mechanical model used. As a demonstration, we have measured a value of 78 +/- 17 GPa for Young's modulus of bacterial cellulose fibers with diameters ranging from 35 to 90 nm. This value is considerably higher than previous estimates, obtained by less direct means, of the mechanical strength of individual cellulose fibers.     

Adhesion of bacterial exopolymers to alpha-FeOOH: inner-sphere complexation of phosphodiester groups

Extracellular polymeric substances (EPS) constitute a heterogeneous mixture of polyelectrolytes that mediate biomineralization and bacterial adhesion and stabilize biofilm matrixes in natural and artificial environments. Although nucleic acids are exuded extracellularly and are purported to be required for biofilm formation, direct evidence of the active mechanism is lacking. EPS were extracted from both Bacillus subtilis (a gram-positive bacterium) and Pseudomonas aeruginosa (a gram-negative bacterium) and their interaction with the goethite (alpha-FeOOH) surface was studied using attenuated total internal reflection infrared spectroscopy. Correspondence between spectral data and quantum chemical calculations demonstrate that phosphodiester groups of nucleic acids mediate the binding of EPS to mineral surfaces. Our data indicate that these groups emerge from the EPS mixture to form monodentate complexes with Fe centers on the goethite (alpha-FeOOH) surface, providing an energetically stable bond for further EPS or cell adhesion.     

Vesicular release dynamics are altered by the interaction between the chemical cargo and vesicle membrane lipids

The release of the cargo from soft vesicles, an essential process for chemical delivery, is mediated by multiple factors. Among them, the regulation by the interaction between the chemical cargo species and the vesicular membrane, widely existing in all vesicles, has not been investigated to date. Yet, these interactions hold the potential to complicate the release process. We used liposomes loaded with different monoamines, dopamine (DA) and serotonin (5-HT), to simulate vesicular release and to monitor the dynamics of chemical release from isolated vesicles during vesicle impact electrochemical cytometry (VIEC). The release of DA from liposomes presents a longer release time compared to 5-HT. Modelling the release time showed that DA filled vesicles had a higher percentage of events where the time for the peak fall was better fit to a double exponential (DblExp) decay function, suggesting multiple kinetic steps in the release. By fitting to a desorption–release model, where the transmitters adsorbed to the vesicle membrane, the dissociation rates of DA and 5-HT from the liposome membrane were estimated. DA has a lower desorption rate constant, which leads to slower DA release than that observed for 5-HT, whereas there is little difference in pore size. The alteration of vesicular release dynamics due to the interaction between the chemical cargo and vesicle membrane lipids provides an important mechanism to regulate vesicular release in chemical and physiological processes. It is highly possible that this introduces a fundamental chemical regulation difference between transmitters during exocytosis.

The release of the cargo from soft vesicles, an essential process for chemical delivery, is mediated by multiple factors.     

Self-assembled monolayer of alkanephosphoric acid on nanotextured Ti

Surface modification of titanium and its alloys is of great importance for their practical application as biomedical implants. We have studied and compared assembly of dodecylphosphoric acid on commercial polished and on nanostructured titanium disks. The latter were produced by chemical etching that created nanoscale pits of typical size of about 20 nm. Enhanced hydrophobicity and high molecular density were obtained after functionalization of the nanotextured substrate. Aging tests showed a lifetime of the organic films of about one month in phosphate buffer. The samples were characterized by means of infrared spectroscopy, contact angle measurements, ellipsometry, and atomic force and scanning tunneling microscopies.     

低介电常数聚喹啉衍生物薄膜的合成与表征

采用等离子体聚合技术合成了一种新型的低介电常数聚喹啉衍生物薄膜: 聚3-氰基喹啉(PP3QCN)薄膜. 借助于傅里叶变换红外光谱(FT-IR)、紫外-可见(UV-Vis)吸收光谱、X光电子能谱(XPS)和原子力显微镜(AFM)对薄膜结构进行了系统表征. 结果表明, 等离子体聚合条件对沉积膜的化学结构、表面组成、膜形态以及介电性能均有影响. 在较低的等离子体放电功率(10 W)条件下, 可得到具有较高芳环保留率和较大π-共轭体系的高质量聚3-氰基喹啉薄膜材料; 而在较高功率(25 W)条件下, 聚合过程中会出现比较严重的单体分子破碎, 形成较多非π-共轭体系的聚合物, 从而导致聚3-氰基喹啉的共轭度降低. 聚3-氰基喹啉薄膜的介电性能测试结果表明, 低放电功率(10 W)条件下制得的聚3-氰基喹啉薄膜具有比较低的介电常数值, 仅为2.45.     

Atomic force microscopy assisted immobilization of lipid vesicles

We report on a new approach to direct the immobilization of unilamellar lipid vesicles on substrate-supported lipid bilayers in a spatially confined manner. The adsorption of vesicles from solution is limited to areas of disorder in the bilayers, which is induced by scanning a pattern in situ with an atomic force microscopy (AFM) tip using high imaging forces. Lines of vesicles with a length exceeding 25 microm and a width corresponding to that of a single surface-immobilized vesicle have been fabricated. The adsorbed vesicles are effectively immobilized and do not desorb spontaneously. However, AFM with forces of several nanoNewtons allows one to displace vesicles selectively. The novel methodology described, which may serve as a platform for research on proteins incorporated in the lipid bilayers comprising the vesicles, does not require chemical labeling of the vesicles to guide their deposition.