Regulating self-assembly of spherical oligomers

In multistep reactions, stability of intermediates is critical to the rate of product formation and a significant factor in generating kinetic traps. The capsid protein of cowpea chlorotic mottle virus (CCMV) can be induced to assemble into spherical particles of 30, 60, and 90 dimers. Based on examining assembly kinetics and reaction end points, we find that formation of uniform, ordered structures is not always a result of reactions that reach equilibrium. Equilibration or, alternatively, kinetic trapping can be identified by a straightforward analysis. Altering the assembly path of "spherical" particles is a means of controlling the distribution of products, which has broad applicability to self-assembly reactions.     

Packaging of gold particles in viral capsids

In-vitro self-assembly conditions known to result in generating infectious virions have been used in vitro to reassemble bromovirus capsid proteins around negatively charged gold nanoparticles cores. We discuss here the optical properties (elastic light scattering) and the influence of the core size and of the functional moiety on the resulting virus-like particles. Our results indicate that the formation of a closed shell, as opposed to an amorphous protein coat, does occur and that the shell/core interactions can be tuned using different coatings on the nanoparticle core. Such studies may lead to real-time monitoring of viral traffic on the scale of a single virus, as well as to the possibility of chemical sensing along the intracellular and intercellular viral pathways and contribute to a better understanding of the virus transport and cellular compartmentalization.     

羟基磷灰石微纳结构对蛋白吸附的影响

本研究采用水热反应法, 在不同浓度环己烷六羧酸(H6E)模板调控作用下, 合成了具有不同表面微纳结构的羟基磷灰石(HAP)微粒, 并采用XRD、BET、FTIR和SEM对其进行表征。对HAP微粒进行了牛血清白蛋白(BSA)、纤维蛋白原(FN)和溶菌酶(LYS)的吸附及释放实验。结果表明: H6E能够在HAP微粒表面构建微纳结构, 不同微纳结构对不同蛋白质具有选择性吸附作用; 在H6E浓度为50 mmol/L的合成条件下制备的中空结构HAP微粒(HAP50)其载蛋白后体外释放具有明显的蛋白缓释性能。     

Reactive-electrospray-assisted laser desorption/ionization for characterization of peptides and proteins

Electrospray-assisted laser desorption/ionization (ELDI) is a soft ionization method for mass spectrometry (MS) and combines features of both electrospray ionization (ESI) and matrix-assisted laser desorption/ionization to generate ESI-like multiply charged molecules. The ELDI process is based on merging ESI-generated, charged droplets with particles UV laser desorbed from dried or wet sample deposits. We previously reported that ELDI is amenable for MS-based protein identification of large peptides and small proteins using top-down and bottom-up techniques (Peng, I. X.; Shiea, J.; Ogorzalek Loo, R. R.; Loo, J. A. Rapid Commun. Mass Spectrom. 2007, 21, 2541-2546). We have extended our studies by applying collisionally activated dissociation and electron-transfer dissociation MS ( n ) to protein analysis and show that ELDI is capable of multistage MS to MS (4) for top-down characterization of large proteins such as 29 kDa carbonic anhydrase. Multiply charged proteins generated by the ELDI mechanism can be shifted to higher charge by increasing the organic content in the ESI solvent to denature the protein molecules, or by adding m-nitrobenzyl alcohol to the ESI solvent. Furthermore, we introduce "reactive-ELDI", which supports chemical reactions during the ELDI process. Preliminary data for online disulfide bond reduction using dithiothreitol on oxidized glutathione and insulin show reactive-ELDI to be effective. These data provide evidence that the laser-desorbed particles merge with the ESI-generated charge droplets to effect chemical reactions prior to online MS detection. This capability should allow other chemical and enzymatic reactions to be exploited as online protein characterization tools, as well as extending them to flexible, spatially resolved tissue screening and imaging. Also, these reactive-ELDI disulfide reduction experiments enable direct top-down protein identification for proteomic study, side stepping laborious, time-consuming sample preparation steps such as in-solution reduction and alkylation.     

Controllable nanoreactor confined to atomic force microscopy tips and its application in low copy DNA ligation

Less molecules reaction, especially at the single molecule level, plays an important role in biochemical or chemical research. It is also significant to achieve low copy or single molecule DNA ligation during the whole genome project. In this paper, a new type of nanoreactor was constructed around atomic force microscopy (AFM) tips under certain humidity, where DNA molecules can be limited to a special space through water meniscus, so the probability of molecules collision was increased and the efficiency of DNA ligation was greatly enhanced. Combined with the nanomanipulation based on AFM, controllable nanoreactor may provide a new tool to single molecule reaction. Low copy DNA ligation was successfully achieved by this method. Results showed the number of DNA molecules involved in the nanoreactor can not be more than sixty. This method will found a base for the ultimate realization of single-molecule DNA ligation.     

Gold nanoparticles as selective and concentrating probes for samples in MALDI MS analysis

MALDI mass spectrometry is used widely in various fields because it has the characteristics of speed, ease of use, high sensitivity, and wide detectable mass range, but suppression effects between analyte molecules and interference from the sample matrix frequently arise during MALDI analysis. The suppression effects can be avoided if target species are isolated from complicated matrix solutions in advance. Herein, we proposed a novel method for achieving such a goal. We describe a strategy that uses gold nanoparticles to capture charged species from a sample solution. Generally, ionic agents, such as anionic or cationic stabilizers, encapsulate gold nanoparticles to prevent their aggregation in solution. These charged stabilizers at the surface of the gold particles are capable of attracting oppositely charged species from a sample solution through electrostatic interactions. We have employed this concept to develop nanoparticle-based probes that selectively trap and concentrate target species in sample solutions. Additionally, to readily isolate them from solution after attracting their target species, we used gold nanoparticles that are adhered to the surface of magnetic particles through S-Au bonding. A magnet can then be employed to isolate the Au@magnetic particles from the solution. The species trapped by the isolated particles were then characterized by MALDI MS after a simple washing. We demonstrate that Au@magnetic particles having negatively charged surfaces are suitable probes for selectively trapping positively charged proteins from aqueous solutions. In addition, we have employed Au@magnetic particle-based probes successfully to concentrate low amounts of peptide residues from the tryptic digest products of cytochrome c (10(-7) M).     

Native protein nanolithography that can write, read and erase

The development of systematic approaches to explore protein-protein interactions and dynamic protein networks is at the forefront of biological sciences. Nanopatterned protein arrays offer significant advantages for sensing applications, including short diffusion times, parallel detection of multiple targets and the requirement for only tiny amounts of sample. Atomic force microscopy (AFM) based techniques have successfully demonstrated patterning of molecules, including stable proteins, with submicrometre resolution. Here, we introduce native protein nanolithography for the nanostructured assembly of even fragile proteins or multiprotein complexes under native conditions. Immobilized proteins are detached by a novel vibrational AFM mode (contact oscillation mode) and replaced by other proteins, which are selectively self-assembled from the bulk. This nanolithography permits rapid writing, reading and erasing of protein arrays in a versatile manner. Functional protein complexes may be assembled with uniform orientation at dimensions down to 50 nm. Such fabrication of two-dimensionally arranged nano-objects with biological activity will prove powerful for proteome-wide interaction screens and single molecule/virus/cell analyses.     

Surface Plasmon Resonance Imaging Measurements of Electrostatic Biopolymer Adsorption onto Chemically Modified Gold Surfaces

A combination of in situ and ex situ surface plasmon resonance (SPR) imaging experiments is used to characterize the differential electrostatic adsorption of proteins and synthetic polypeptides onto photopatterned monolayers at gold surfaces. The nonspecific electrostatic adsorption of proteins onto negatively charged self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid (MUA) is found to depend on the protein pI, solution ionic strength, and solution pH. The pH dependence of the electrostatic adsorption of the protein avidin onto a MUA SAM indicates that a full monolayer adsorbs at a solution pH greater than 5.0, and an "effective pK(a)" of 3.6 is determined for the avidin adsorption. This effective pK(a) is a combination of the pK(a) of the MUA monolayer and the ion pairing adsorption coefficient for the avidin. Additional SPR imaging experiments show that the electrostatic adsorption of the synthetic polypeptide poly-l-lysine (PL) onto a MUA SAM varies with molecular weight, forming a full PL monolayer for polypeptides with more than 67 lysine residues.     

Modulating protein adsorption onto hydroxyapatite particles using different amino acid treatments

Hydroxyapatite (HA) is a material of choice for bone grafts owing to its chemical and structural similarities to the mineral phase of hard tissues. The combination of osteogenic proteins with HA materials that carry and deliver the proteins to the bone-defective areas will accelerate bone regeneration. The study investigated the treatment of HA particles with different amino acids such as serine (Ser), asparagine (Asn), aspartic acid (Asp) and arginine (Arg) to enhance the adsorption ability of HA carrier for delivering therapeutic proteins to the body. The crystallinity of HA reduced when amino acids were added during HA preparation. Depending on the types of amino acid, the specific surface area of the amino acid-functionalized HA particles varied from 105 to 149 m² g^–1. Bovine serum albumin (BSA) and lysozyme were used as model proteins for adsorption study. The protein adsorption onto the surface of amino acid-functionalized HA depended on the polarities of HA particles, whereby, compared with lysozyme, BSA demonstrated higher affinity towards positively charged Arg-HA. Alternatively, the binding affinity of lysozyme onto the negatively charged Asp-HA was higher when compared with BSA. The BSA and lysozyme adsorptions onto the amino acid-functionalized HA fitted better into the Freundlich than Langmuir model. The amino acid-functionalized HA particles that had higher protein adsorption demonstrated a lower protein-release rate.     

Characterization of functionalized nanoporous supports for protein confinement

Here we characterize a highly efficient approach for protein confinement and enzyme immobilization in NH(2)-?or HOOC-?functionalized mesoporous silica (FMS) with pore sizes as large as tens of nanometres. We observed a dramatic increase of enzyme loading in both enzyme activity and protein amount when using appropriate FMS in comparison with unfunctionalized mesoporous silica and normal porous silica. With different protein loading density in NH(2)-FMS, the negatively charged glucose oxidase (GOX) displayed an immobilization efficiency (I(e), the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in stock solution) in a range from 30% to 160%, while the same charged glucose isomerase (GI) showed an I(e) of 100% to 120%, and the positively charged organophosphorus hydrolase (OPH) exhibited I(e) of more than 200% in HOOC-FMS. The enzyme-FMS composite was stained with the charged gold nanoparticles and imaged by transmission electron microscopy (TEM). Fourier transform infrared (FTIR) spectroscopy showed no major secondary structural change for the enzymes entrapped in FMS. Thanks to the large, rigid, open pore structure of FMS, the reaction rate and K(m) of the entrapped enzymes in FMS were comparable to those of the free enzymes in solution. In principle, the general approach described here should be applicable to many enzymes, proteins, and protein complexes since both pore sizes and functional groups of FMS are controllable.     

In Vitro Stability of Poly(D,L-lactide) and Poly(D,L-lactide)/Poloxamer Nanoparticles in Gastrointestinal Fluids

Abstract

Poty(D,L-lactide) (PLA) nanoparticles of various surface and bulk properties were prepared by a nanoprecipitation procedure and evaluated for their physical and chemical in vitro stability in simulated gastrointestinal fluids of 37°C. The influence of polymer characteristics and poloxamer 188 (POL 188) adsorption was studied. Physical stability was followed by visual appearance, particle size, and zeta potential measurements. Molecular weight changes were analyzed by gel permeation chromatography (GPC). Due to a sharp decrease in their negative zeta potential, poloxamer-free nanoparticles flocculated in simulated gastric fluid, irrespective of the polymer properties. Their physical stability in protein-free intestinal fluids increased with an increase in carboxy end group concentration of the PLA and thus, with an increase in their negative zetapotential. Protein effects at pH 7.5 were rather complex indicating a stabilizing effect of negatively charged proteins and a destabilizing effect of positively charged proteins. Poloxamer 188 adsorption sterically stabilized the nanoparticles against flocculation in gastric fluid, irrespective of the PLA characteristics. Physical stability of the PLA/POL 188 nanoparticles in intestinal fluids was affected by the PLA characteristics. Poloxamer 188 increased the physical stability of nanoparticles composed of hydrophobic PLA, irrespective of the proteins present. A gradual particle size increase could, however, be observed for PLA/POL nanoparticles composed of PLA with a high content of carboxy end groups, especially in combination with positively charged proteins. This effect is most likely due to a decrease in PLA/POL interactions resulting from the ionization of the carboxy end groups located on the nanoparticle surface and leading to conformational changes and/or a distinct desorption of POL 188. The chemical stability of PLA and PLA/POL nanoparticles depended on the glass transition temperature (T_gH) of the hydrated polymer matrix. Enzymatic effects could not be detected. Nanoparticles with T_gH > 37°C were chemically stable in both gastric and intestinal fluids at 37°C over a time period of more than 48 hr.     

Multilayer-assembled microchip for enzyme immobilization as reactor toward low-level protein identification

A microchip reactor has been developed on the basis of a layer-by-layer approach for fast and sensitive digestion of proteins. The resulting peptide analysis has been carried out by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Natural polysaccharides, positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) were multilayer-assembled onto the surface of a poly(ethylene terephthalate) (PET) microfluidic chip to form a microstructured and biocompatible network for enzyme immobilization. The construction of CS/HA assembled multilayers on the PET substrate was characterized by AFM imaging, ATR-IR, and contact angle measurements. The controlled adsorption of trypsin in the multilayer membrane was monitored using a quartz crystal microbalance and an enzymatic activity assay. The maximum proteolytic velocity of the adsorbed trypsin was approximately 600 mM/min mug, thousands of times faster than that in solution. BSA, myoglobin, and cytochrome c were used as model substrates for the tryptic digestion. The standard proteins were identified at a low femtomole per analysis at a concentration of 0.5 ng/muL with the digestion time <5s. This simple technique may offer a potential solution for low-level protein analysis.     

Kinetic processes in the plasma formed in combustion of hydrocarbon fuels

An analysis of the basic kinetic processes responsible for the formation of ions, electrons, charged and neutral carbon clusters and particles of nanometer size in the combustion of hydrocarbon fuels has been made. It has been shown that the formation of a polydisperse ensemble of positively and negatively charged particles is mainly caused by the ion adhesion to primary particles and secondarily formed particles and also by particle coagulation. Account must be taken not only of the Coulomb interaction but also of the van der Waals and polarization interaction between particles. The distinstice features of the deposition of polar molecules on charged particles have been considered.     

pH‐Regulated Heterostructure Porous Particles Enable Similarly Sized Protein Separation

Porous particles are frequently used for various healthcare applications that involve protein separation processes. However, conventional porous particles, either homogeneous particles or those subjected to surface modification with a layer of specific molecules, often encounter bottlenecks in separating proteins with similar size. Here, it is reported that heterostructure‐enabled separation particles (HESP), synthesized by a double emulsion interfacial polymerization process, can effectively and rapidly separate similarly sized proteins. Double emulsion interfacial polymerization endows the HESP with a nanoscale carboxylic layer outside the particles and inside the pores, allowing pH‐regulated selective adsorption of proteins. Thus, by optimizing the environmental pH, proteins with similar size can be effectively and rapidly separated. These HESP are expected to show potential in widespread applications ranging from biomolecule adsorption, encapsulation, and separation to controlled release and other biomedical fields.     

Deciphering the kinetic mechanism of spontaneous self-assembly of icosahedral capsids

Self-assembly of viral proteins into icosahedral capsids is an interesting yet poorly understood phenomenon of which elucidation may aid the exploration of beneficial applications of capsids in materials science and medicine. Using molecular dynamics simulations of coarse-grained models for capsid proteins, we show that the competition between the formation of full capsids and nonidealized structures is strongly dependent upon the protein concentration and temperature, occurring kinetically as a cascade of elementary reactions in which free monomers are added to the growing oligomers on a downhill free-energy landscape. However, the insertion of the final subunits is the rate-limiting, energetically unfavorable step in viral capsid assembly. A phase diagram has been constructed to show the regions where capsids or nonidealized structures are stable at each concentration and temperature. We anticipate that our findings will provide guidance in identifying suitable conditions required for in vitro viral capsid assembly experiments.     

Goosebumps‐Inspired Microgel Patterns with Switchable Adhesion and Friction

A substrate mimicking the surface topography and temperature sensitivity of skin goosebumps is fabricated. Close‐packed arrays of thermoresponsive microgel particles undergo topographical changes in response to temperature changes between 25 and 37 °C, resembling the goosebump structure that human skin develops in response to temperature changes or other circumstances. Specifically, positively charged poly[2‐(methacryloyloxy)ethyltrimethylammonium chloride] (PMETAC) brushes serve as an anchoring substrate for negatively charged poly(NIPAm‐co‐AA) microgels. The packing density and particle morphology can be tuned by brush layer thickness and pH of the microgel suspension. For brush layer thickness below 50 nm, particle monolayers are observed, with slightly flattened particle morphology at pH 3 and highly collapsed particles at pH above 7. Polymer brush films with thickness above 50 nm lead to the formation of particle multilayers. The temperature responsiveness of the monolayer assemblies allows reversible changes in the film morphology, which in turn affects underwater adhesion and friction at 25 and 37 °C. These results are promising for the design of new functional materials and may also serve as a model for biological structures and processes.     

Membrane channels as a tool to control nanoreactors

We present an example of the use of self-assembly of biomolecules to create nanostructured building blocks. The resulting individual compartments can be tailored to fulfil specific functions: catalysis of a chemical reaction in a confined environment, detection on a molecular level and feedback with the outside. For example, such individually designed components can be assembled to build up macroscopic chemically active filters. The main component is membrane channels acting as molecular sieves, able to control the permeation across the capsule wall. We introduce briefly a new microdevice to characterise membrane channels with a future potential for high-throughput screening of channel properties based on automation, parallelisation and the use of microfluidics. Subsequently, we outline a possible application for channel-forming proteins: encapsulation of charged polymers or proteins into liposomes and restriction of diffusion through transmembrane channels to small ions, creating a Donnan potential. This Donnan potential can be used for external manipulation of nanocontainers by coupling of the capsule to an external electric field, or for the selective uptake of small charged molecules into the capsule.     

Directly created electrostatic micro-domains on hydroxyapatite: probing with a Kelvin Force probe and a protein

Micro-domains of modified surface potential (SP) were created on hydroxyapatite films by direct patterning by mid-energy focused electron beam, typically available as a microprobe of Scanning Electron Microscopes. The SP distribution of these patterns has been studied on sub-micrometer scale by the Kelvin Probe Force Microscopy method as well as lysozyme adsorption. Since the lysozyme is positively charged at physiological pH, it allows us to track positively and negatively charged areas of the SP patterns. Distribution of the adsorbed proteins over the domains was in good agreement with the observed SP patterns.     

碳纳米管和壳聚糖的层层静电自组装多层膜(英文)

将多壁碳纳米管(MWCNT)置于混酸(硝酸∶硫酸=1∶3)中,利用超声波振荡截短碳纳米管、并使其与羧基链接,而后基于阳离子聚合电解质壳聚糖(CS)和阴离子短切碳纳米管之间的静电作用,在玻璃衬底上通过层层的模式均匀稳定地自组装形成复合壳聚糖多层膜。UV-vis光谱显示:组装过程呈现均匀而连续的生长。AFM和SEM观察表明:CS/MWCNT多层膜具有良好的光学特性,在生物传感器方面具有潜在的应用前景。     

Self‐Assembly of Graphene Oxide at Interfaces

Due to its amphiphilic property, graphene oxide (GO) can achieve a variety of nanostructures with different morphologies (for example membranes, hydrogel, crumpled particles, hollow spheres, sack‐cargo particles, Pickering emulsions, and so on) by self‐assembly. The self‐assembly is mostly derived from the self‐concentration of GO sheets at various interfaces, including liquid‐air, liquid‐liquid and liquid‐solid interfaces. This paper gives a comprehensive review of these assembly phenomena of GO at the three types of interfaces, the derived interfacial self‐assembly techniques, and the as‐obtained assembled materials and their properties. The interfacial self‐assembly of GO, enabled by its fantastic features including the amphiphilicity, the negatively charged nature, abundant oxygen‐containing groups and two‐dimensional flexibility, is highlighted as an easy and well‐controlled strategy for the design and preparation of functionalized carbon materials, and the use of self‐assembly for uniform hybridization is addressed for preparing hybrid carbon materials with various functions. A number of new exciting and potential applications are also presented for the assembled GO‐based materials. This contribution concludes with some personal perspectives on future challenges before interfacial self‐assembly may become a major strategy for the application‐targeted design and preparation of functionalized carbon materials.