Nanoscale Assembly Research Articles

Membrane protein reconstitution into liposomes guided by dual-color fluorescence cross-correlation spectroscopy

Proteoliposomes represent nanoscale assemblies of indispensable value for studying membrane proteins in general and membrane transporters in particular. Since no universal protocol exists, conditions for proteoliposome formation must be determined on a case-by-case basis. This process will be significantly expedited if the size and composition of the assemblies can be analyzed in a single step using only microliters of sample. Here we show that dual-color fluorescence cross-correlation spectroscopy (FCCS) is of great value for optimizing the reconstitution process, because it distinguishes micelles, liposomes and aggregates in heterogeneous mixtures and permits direct monitoring of the co-localization of proteins and lipids in the diffusing assemblies. As proof-of-principle, liposomes containing the functional multidrug resistance transporter NorA from Staphylococcus aureus were prepared, demonstrating that FCCS is an excellent tool to guide the development of reconstitution protocols.

A Ring of Rings and Other Multicomponent Assemblies of Cages

Ring a ring of roses: Spectacular nanoscale molecular assemblies have been created by design of individual polymetallic components that are then linked together through simple reactions. These include an assembly where six octametallic rings surround a dodecametallic central ring.

Catalyst nanoscale assembly from the vapor phase on corrosion resistant supports

The synthesis process, reactive spray deposition technology (RSDT), utilized a jet-flame to produce Pt nanoparticles. The RSDT process bypasses traditional wet chemical routes by simultaneously nucleating the catalyst on a support and sequential deposition of catalyst layer via the gas phase. Pt nanoparticles were attached, in the process gas during the time-of-flight, to the surface of several supports. The supports show promising corrosion resistance under the cathode conditions of a proton exchange membrane fuel cell (PEMFC). The supported Pt catalysts were then studied in regards to structure, stability and electrochemical behavior toward the oxygen reduction reaction (ORR) in perchloric acid. Transmission electron microscopy studies showed that the average Pt particle diameter is ∼2.5nm. The average diameter and distribution of the Pt particles are independent of the support type and a high degree of catalyst dispersion has been achieved on all supports. The greatest surface area and electrochemical mass activity were obtained using Vulcan XC-72R, while a graphitized carbon support produced the highest specific activity. Based on X-ray photoelectric spectroscopy (XPS) measurements, approximately 30% of the surface of the Pt particles is comprised of Pt2+. This oxide coverage does not extend into the bulk and is below the detection limits of X-ray diffraction (XRD). The electrochemical reduction of oxygen exhibits a typical Tafel slope of −65 to −71mV/dec.

Nanoscale Superstructures Assembled by Polymerase Chain Reaction (PCR): Programmable Construction, Structural Diversity, and Emerging Applications

Polymerase chain reaction (PCR) is an essential tool in biotechnology laboratories and is becoming increasingly important in other areas of research. Extensive data obtained over the last 12 years has shown that the combination of PCR with nanoscale dispersions can resolve issues in the preparation DNA-based materials that include both inorganic and organic nanoscale components. Unlike conventional DNA hybridization and antibody-antigen complexes, PCR provides a new, effective assembly platform that both increases the yield of DNA-based nanomaterials and allows researchers to program and control assembly with predesigned parameters including those assisted and automated by computers. As a result, this method allows researchers to optimize to the combinatorial selection of the DNA strands for their nanoparticle conjugates. We have developed a PCR approach for producing various nanoscale assemblies including organic motifs such as small molecules, macromolecules, and inorganic building blocks, such as nanorods (NRs), metal, semiconductor, and magnetic nanoparticles (NPs). We start with a nanoscale primer and then modify that building block using the automated steps of PCR-based assembly including initialization, denaturation, annealing, extension, final elongation, and final hold. The intermediate steps of denaturation, annealing, and extension are cyclic, and we use computer control so that the assembled superstructures reach their predetermined complexity. The structures assembled using a small number of PCR cycles show a lower polydispersity than similar discrete structures obtained by direct hybridization between the nanoscale building blocks. Using different building blocks, we assembled the following structural motifs by PCR: (1) discrete nanostructures (NP dimers, NP multimers including trimers, pyramids, tetramers or hexamers, etc.), (2) branched NP superstructures and heterochains, (3) NP satellite-like superstructures, (4) Y-shaped nanostructures and DNA networks, (5) protein-DNA co-assembly structures, and (6) DNA block copolymers including trimers and pentamers. These results affirm that this method can produce a variety of chemical structures and in yields that are tunable. Using PCR-based preparation of DNA-bridged nanostructures, we can program the assembly of the nanoscale blocks through the adjustment of the primer intensity on the assembled units, the number of PCR cycles, or both. The resulting structures are highly complex and diverse and have interesting dynamics and collective properties. Potential applications of these materials include chirooptical materials, probe fabrication, and environmental and biomedical sensors.

A method to study in vivo stability of DNA nanostructures

DNA nanostructures are rationally designed, synthetic, nanoscale assemblies obtained from one or more DNA sequences by their self-assembly. Due to the molecularly programmable as well as modular nature of DNA, such designer DNA architectures have great potential for in cellulo and in vivo applications. However, demonstrations of functionality in living systems necessitates a method to assess the in vivo stability of the relevant nanostructures. Here, we outline a method to quantitatively assay the stability and lifetime of various DNA nanostructures in vivo. This exploits the property of intact DNA nanostructures being uptaken by the coelomocytes of the multicellular model organism Caenorhabditis elegans. These studies reveal that the present fluorescence based assay in coelomocytes of C. elegans is an useful in vivo test bed for measuring DNA nanostructure stability.

Nanoscale assemblies and their biomedical applications

Nanoscale assemblies are a unique class of materials, which can be synthesized from inorganic, polymeric or biological building blocks. The multitude of applications of this class of materials ranges from solar and electrical to uses in food, cosmetics and medicine. In this review, we initially highlight characteristic features of polymeric nanoscale assemblies as well as those built from biological units (lipids, nucleic acids and proteins). We give special consideration to protein nanoassemblies found in nature such as ferritin protein cages, bacterial microcompartments and vaults found in eukaryotic cells and designed protein nanoassemblies, such as peptide nanofibres and peptide nanotubes. Next, we focus on biomedical applications of these nanoscale assemblies, such as cell targeting, drug delivery, bioimaging and vaccine development. In the vaccine development section, we report in more detail the use of virus-like particles and self-assembling polypeptide nanoparticles as new vaccine delivery platforms.

Nanoscale assembly into extended and continuous structures and hybrid materials

Harnessing synthetic power to create novel materials with unique assembly properties is of fundamental interest for pushing the limits of molecular and macromolecular assembly, while further holding the potential for improving electronic and medical device performance. Controlling the assembly of aromatic molecules into nanostructures provides routes towards continuous and extended structures with performance that is improved markedly over that of their individual molecular components, opening possibilities for the formation of composites that have tailored interactions with other materials and thus improved applications. This review covers recent advances in the synthesis of conjugated materials, their controlled assembly into nanoscale architectures and implementation into devices. Specifically, we discuss the solution-based assembly of polythiophene into nanowire fibrils, the formation of columnar stacks of hexabenzocoronene liquid crystals and the preparation of functionalized solution processible graphene for nanocomposite formation. When polymers are mentioned, it is ‘plastics’ that typically spring to mind. Yet the assembly of conjugated polymers or aromatic molecules into hierarchical and hybrid structures also brings about materials that are attractive for electronic and medical devices. Emily Pentzer and Todd Emrick from the University of Massachusetts review recent advances into the preparation of well-defined molecular building blocks, their assembly into complex architectures, and the properties emerging from these arrangements. This approach is illustrated by the variety of assemblies featuring conjugated polythiophene chains, such as nanowires, fibrils that can further aggregate into ribbons, or composites with quantum dots. These higher-order structures display a variety of electronic properties, useful in semiconductor and photovoltaic applications among others. Another example of the potential of this approach is the construction of carbon nanotubes or graphene flakes from aromatic molecules. Pentzer and Emrick also highlight the remaining challenges that relate to controlling the structures obtained, their stability, and their integration into devices. Poly(3-hexylthiophene) nanowires, microns in length and nanometers in diameter, are stably suspended in solution, and crystallize by adjusting solvent strength. Cocrystalization of P3HT with P3HT-covered CdSe nanorods gave hybrid nanostructures in which the p-type P3HT fibrils were flanked by the n-type CdSe nanorods. The close proximity and arrangement of the two materials provides direct, continuous pathways suitable for charge transport in photovoltaic devices.

Nano-encrypted Morse code: a versatile approach to programmable and reversible nanoscale assembly and disassembly.

While much work has been devoted to nanoscale assembly of functional materials, selective reversible assembly of components in the nanoscale pattern at selective sites has received much less attention. Exerting such a reversible control of the assembly process will make it possible to fine-tune the functional properties of the assembly and to realize more complex designs. Herein, by taking advantage of different binding affinities of biotin and desthiobiotin toward streptavidin, we demonstrate selective and reversible decoration of DNA origami tiles with streptavidin, including revealing an encrypted Morse code "NANO" and reversible exchange of uppercase letter "I" with lowercase "i". The yields of the conjugations are high (>90%), and the process is reversible. We expect this versatile conjugation technique to be widely applicable with different nanomaterials and templates.

Interfacial Manipulations: Controlling Nanoscale Assembly in Bulk, Thin Film, and Solution Block Copolymer Systems

Nanostructured soft materials from self-assembled block copolymers (BCP)s and polymer blends can enable the reliable, high-throughput, and cost-effective generation of nanoscale structural motifs for many emerging technologies. Our research group has studied the phase behavior of BCPs in bulk, thin film, and solution environments with a particular focus on using interfacial manipulations to control self-assembly and to access a vast array of nanoscale morphologies and orientations. These interfacial manipulations can be synthetic alterations that are directly incorporated into the BCP chain to modify polymer-polymer interactions, post-polymerization and non-synthetic modifications that affect block interactions, or changes to the polymer specimen's external surroundings to control self-assembly in a confining environment. Herein, we describe methods that we have employed to manipulate BCP self-assembly for various application targets, and we discuss the key effects of such manipulations on the resulting nanoscale morphologies.

Hollow Block Copolymer Nanoparticles through a Spontaneous One-step Structural Reorganization

The spontaneous one-step synthesis of hollow nanocages and nanotubes from spherical and cylindrical micelles based on poly(acrylic acid)-b-polylactide (P(AA)-b-P(LA)) block copolymers (BCPs) has been achieved. This structural reorganization, which occurs simply upon drying of the samples, was elucidated by transmission electron microscopy (TEM) and atomic force microscopy (AFM). We show that it was necessary to use stain-free imaging to examine these nanoscale assemblies, as the hollow nature of the particles was obscured by application of a heavy metal stain. Additionally, the internal topology of the P(AA)-b-P(LA) particles could be tuned by manipulating the drying conditions to give solid or compartmentalized structures. Upon resuspension, these reorganized nanoparticles retain their hollow structure and display significantly enhanced loading of a hydrophobic dye compared to the original solid cylinders.

Nanoscale Assembly of Paramagnetic Organic Radicals on Au(111) Single Crystals

The successful thin-film deposition of a pyrene-substituted nitronyl nitroxide radical under controlled conditions has been demonstrated. The electronic properties, chemical environment at the interface, and morphology of the thin films have been investigated by a multitechnique approach. Spectroscopic and morphological analyses indicate a Stranski-Krastanov growth mode and weak physisorption of molecules onto the metallic surface. Electron spin resonance (ESR) spectroscopy shows that evaporation processes and deposition do not affect the paramagnetic character of the molecules. Useful concepts for the engineering of new, purely organic-based magnets, which may open the way to fruitful exploitation of organic molecular-beam deposition for assembly on solid surfaces in view of future technological applications, are presented.

Nanoscopic Amyloid Pores Formed via Stepwise Protein Assembly.

Protein aggregation leading to various nanoscale assemblies is under scrutiny due to its implications in a broad range of human diseases. In the present study, we have used ovalbumin, a model non-inhibitory serpin, to elucidate the molecular events involved in amyloid assembly using a diverse array of spectroscopic and imaging tools such as fluorescence, laser Raman, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images revealed a progressive morphological transition from spherical oligomers to nanoscopic annular pores that further served as templates for higher-order supramolecular assembly into larger amyloid pores. Raman spectroscopic investigations illuminated in-depth molecular details into the secondary structural changes of the protein during amyloid assembly and pore formation. Additionally, Raman measurements indicated the presence of antiparallel β-sheets in the amyloid core. Overall, our studies revealed that the protein conformational switch in the context of the oligomers triggers the hierarchical assembly into nanoscopic amyloid pores. Our results will have broad implications in the structural characterization of amyloid pores derived from a variety of disease-related proteins.

Practical applications of small-angle neutron scattering

Recent improvements in beam-line accessibility and technology have led to small-angle neutron scattering (SANS) becoming more frequently applied to materials problems. SANS has been used to study the assembly, dispersion, alignment and mixing of nanoscale condensed matter, as well as to characterise the internal structure of organic thin films, porous structures and inclusions within steel. Using time-resolved SANS, growth mechanisms in materials systems and soft matter phase transitions can also be explored. This review is intended for newcomers to SANS as well as experts. Therefore, the basic knowledge required for its use is first summarised. After this introduction, various examples are given of the types of soft and hard matter that have been studied by SANS. The information that can be extracted from the data is highlighted, alongside the methods used to obtain it. In addition to presenting the findings, explanations are provided on how the SANS measurements were optimised, such as the use of contrast variation to highlight specific parts of a structure. Emphasis is placed on the use of complementary techniques to improve data quality (e.g. using other scattering methods) and the accuracy of data analysis (e.g. using microscopy to separately determine shape and size). This is done with a view to providing guidance on how best to design and analyse future SANS measurements on materials not listed below.

Co-continuous nanoscale assembly of Nafion–polyacrylonitrile blends within nanofibers: a facile route to fabrication of porous nanofibers

A facile route to fabrication of porous high surface area nanofibers is demonstrated via electrospinning of a polymer blend followed by selective removal of one of the components. First, electrospinnability of Nafion–polyacrylonitrile (PAN) solutions in N,N-dimethylformamide is studied over a wide range of blend compositions. Pure Nafion solutions are not electrospinnable by themselves owing to the presence of aggregates in solution due to ionic interactions, thereby resulting in low solution viscosities. Addition of only 5 wt% PAN in Nafion allows us to successfully electrospin nanofibers, possibly due to the enhancement of entanglements within the solution. An extensional rheology study is conducted to quantify and understand non-electrospinnability. In the second step, the nanoscale internal morphology of Nafion–PAN nanofibers is studied using transmission electron microscopy of microtomed nanofiber sections. The electrospinning process allows us to develop the co-continuous morphology of PAN and Nafion within the nanofibers. To further corroborate our findings and develop high surface area nanofibers, we selectively remove Nafion or PAN to form pure mesoporous carbon (after calcination) and mesoporous Nafion nanofibers respectively. Interestingly, even with an initial composition of 80 : 20 (wt/wt) Nafion : PAN, the nanofibers retain their overall shape after selective Nafion removal, indicating the presence of a continuous PAN phase. The resultant porous carbon nanofibers exhibit a specific surface area of >1500 m2 g−1. While mesoporous carbon nanofibers can have a wide range of applications such as in supercapacitors, high surface area porous Nafion nanofibers will facilitate the development of super-sensitive gas sensors.

A versatile approach for the functionalization of gold nanorods and nanoparticles

A robust functionalized procedure for gold nanorods (Au NR) with mercaptoundecanoic acid (MUA), which are assembled with nanoparticles, has been reported in the present work. The self-assembled nanostructures were characterized by UV–Vis-NIR spectrophotometer and transmission electron microscopy (TEM). Self-assembly procedures, governed by surface chemistry, indicated that chemical reactivity of nanomaterial may contribute in construction of potential nanoscale assemblies. These nanostructures have striking applications as single-molecule detection, plasmonics, and sensing.

Construction of Snowflake-Shaped Dendritic Covalent Assemblies with Rigid Conjugated Networks

A convergent method for the construction of shape-persistent nanoscale assemblies with conjugated backbones was developed. The copper-free Sonogashira coupling reaction was successfully applied to the formation of multiple covalent connections between conjugated terminals (iodide substituted aryl groups) of AB(2)-type outer components and four conjugated terminals (acetylenic bonds) of an inner A(4)-type core dendrimer. The conjugated networks in the starting components are expanded during the assembly process to afford nanoscale dendritic conjugated networks of the type A(4)(AB(2))(4), which have a porphyrin core, and longer (3.9 or 4.5 nm) and shorter (1.6 nm) conjugated chains. Fluorescence measurements revealed that singlet energy is effectively transferred in the assemblies from peripheral benzyl ether units and conjugated chains to the free base porphyrin core.

Modular-DNA-Programmed Construction of Permanent Nanoscale Cyclic Assemblies by Reaction of Covalently linked 2,5-bis(2-Thienyl)pyrrole Monomers

DNA modules that contain complementary recognition units and covalently linked 2,5-bis(2-thienyl)pyrrole (SNS) monomers spontaneously assemble in aqueous buffer solution into cyclic structures. Ligation of the DNA modules is readily accomplished by an oxidative reaction with horseradish peroxidase (HRP)/H2O2, which results in covalent bond formation between the SNS monomers to form permanent nanoscale assemblies. The flexibility of the cyclic assemblies, and their ability to form SNS-to-SNS bonds, was assessed by varying the number and location of SNS monomers on each DNA module. Efficient ligation is observed when each module contains at least two SNS monomers. Ligation efficiency is diminished when the single stranded regions of the assembly are converted to duplex DNA. The efficient ligation of the DNA structures is attributed to the self-association of the monomers due to their hydrophobicity and is enabled by the flexibility of the single-stranded regions. It was found by systematically controlling the position and number of SNS monomers that these DNA modules are efficiently ligated into cyclic assemblies when each nucleobase module contains at least two SNS monomers. This provides a method for formation of unique covalently linked DNA structures and a process that can readily ligate or cross-link DNA chemically.

Plasmon Transmutation: Inducing New Modes in Nanoclusters by Adding Dielectric Nanoparticles

Planar clusters of coupled plasmonic nanoparticles support nanoscale electromagnetic "hot spots" and coherent effects, such as Fano resonances, with unique near and far field signatures, currently of prime interest for sensing applications. Here we show that plasmonic cluster properties can be substantially modified by the addition of individual, discrete dielectric nanoparticles at specific locations on the cluster, introducing new plasmon modes, or transmuting existing plasmon modes to new ones, in the resulting metallodielectric nanocomplex. Depositing a single carbon nanoparticle in the junction between a pair of adjacent nanodisks induces a metal-dielectric-metal quadrupolar plasmon mode. In a ten-membered cluster, placement of several carbon nanoparticles in junctions between multiple adjacent nanoparticles introduces a collective magnetic plasmon mode into the Fano dip, giving rise to an additional subradiant mode in the metallodielectric nanocluster response. These examples illustrate that adding dielectric nanoparticles to metallic nanoclusters expands the number and types of plasmon modes supported by these new mixed-media nanoscale assemblies.

Catalytic Activity of Macroion–Porphyrin Nanoassemblies

A new type of catalytically active self-assembled nanostructures in aqueous solution is presented. Polyelectrolyte-porphyrin nanoscale assemblies consisting of anionic cylindrical poly(styrene sulfonate) brush molecules and cationic tetravalent meso-tetrakis(4-N-methyl-pyridinium)porphyrin (TMPyP) or meso-tetrakis(4-(trimethyl-ammonium)phenyl)-porphyrin (TAPP), respectively, exhibit up to 8-fold higher catalytic activity with regard to light induced iodide oxidation than the corresponding porphyrins without polymeric template. This is particularly interesting because a general concept rather than a specific binding motif is exploited. The approach introduced here hence is attractive due to its facility and versatility and bears potential, for example, in light harvesting and energy conversion.

Nanoscale programmable sequence-specific patterning of DNA scaffolds using RecA protein

Molecular self-assembly inherent to many biological molecules, in conjunction with suitable molecular scaffolds to facilitate programmable positioning of nanoscale objects, offers a promising approach for the integration of functional nanoscale complexes into macroscopic host devices. Here, we report the use of the protein RecA as a means of highly efficient programmable patterning of double-stranded (ds)DNA molecules with molecular-scale precision at specific locations along the DNA strand. RecA proteins form nucleoprotein filaments with single-stranded (ss)DNA molecules, which are chosen to be of sequence homologous to the desired binding region on the dsDNA scaffold. We show that the patterning yield can be in excess of 85% and we demonstrate that concurrent patterning of multiple locations on the same dsDNA scaffold can be achieved with separation between the assembled nucleoprotein filaments of less than 4 nm. This is an important prerequisite for this programmable and flexible DNA scaffold patterning technique to be employed in molecular- and nanoscale assembly applications.