Macromolecular self-assembly refers to the assembly of synthetic polymers, biomacromolecules and supra-molecular polymers. Through macromolecular self-assembly, the fabrication of ordered structures at different scales, the control of the dynamic assembly process and the integrations of advanced functions can be realized. Macromolecular self-assembly and nanotechnology research in China has developed rapidly, from the early periods of follow-up at low to high level and progress into a stage of innovation and creation. This review selects some representative progresses achieved recently, aiming to reflect the current status of macromolecular self-assembly and nanotechnology research in China.
Self-assembly is the autonomous organization of components into patterns or structures without human intervention via non-covalent interactions. Examples of self-assembly in materials science include the formation of colloids, lipid bilayers, self-assembled monolayers and the folding of polypeptide chains into proteins. Self-assembly extends the scope of traditional chemistry aiming at the construction of individual molecules (1–100 Å length scale) from atoms, dealing with the construction of organized molecular assemblies on all length scales. One characteristic of self-assembly is that self-assembled structures are thermodynamically more stable than the single, unassembled building blocks. Another property often found in self-assembled systems is the sensitivity to perturbations exerted by the external environment. Self-assembly has been an important method to prepare new materials and create new functions [1–5].
Self-assembly is a significant aspect of bottom-up approaches to nanotechnology, because the building blocks are not only atoms and molecules but also a wide range of nano- and mesoscopic structures, with different chemical compositions, shapes and functionalities. Self-assembly can meet the demand for fabrication of novel, stable, functional and technologically important materials [1–5]. Macromolecular self-assembly refers to the assembly of synthetic polymers, biomacromolecules and supramolecular polymers. It aims at the study of the interaction between polymers and polymers, polymers and small molecules, polymers and molecular aggregates, polymers and interfaces. Through macromolecular self-assembly, the fabrication of ordered structures at different scales, the control of the dynamic assembly process and the integrations of advanced functions can be realized .
Macromolecular self-assembly and nanotechnology research in China can be dated back to the late 1980s, from the early periods of follow-up at low to high level and progress into a stage of innovation and creation [7,8]. This review aims to reflect the current status of macromolecular self-assembly and nanotechnology research in China. It should be noted that one review cannot cover all of the recent progress of the achievement of Chinese scientists. In this review, we will select a few representative researches and hope it can reflect our current effort to advance the research on molecular self-assembly and nanotechnology in China, including the fabrication of non-covalently connected micelles from homopolymers, polymeric supramolecular amphiphiles, self-assembly of hyperbranched polymers (HBPs), responsive selenium-containing polymer assemblies, design of layered nanocomposites, single-molecule force spectroscopy study of driving force for macromolecular assembly and hybrid macromolecular assemblies for sensing.
2. Non-covalently connected polymeric micelles
Polymeric micelles are widely studied polymeric nano-objects that have important applications in diverse theoretical and applied research fields . Usually, polymeric micelles are prepared via micellization of block copolymers, of which the chemically different polymer blocks are covalently connected, in selective solvents that dissolve only one of the blocks. The resultant polymeric micelles are simply core–shell structured, with the core and the shell being covalently connected [6,9]. Jiang et al. at Fudan University, based on their research over a long period on inter-polymer complexation via hydrogen bonding, made important contributions to the field by developing ‘block-copolymer-free’ strategy using pairs of complementary polymers, rather than block copolymers as the building blocks for preparing polymeric micelles [10,11]. They established two major approaches in their early work. The first approach involves localization of the binding sites of hydrogen bonds of homopolymers (or random copolymers) as building blocks. As shown in figure 1, A is a proton-donating polymer with the donor at the polymer chain end only, and B is the proton-accepting polymer, on which the acceptors randomly distribute. Then, in a common solvent, hydrogen-bonding graft polymer is formed. After switching the medium to a selective solvent, polymeric micelles, with the core and the shell being non-covalently connected, form by self-assembly. The micelles are stable for a long period of time, and more importantly, the composition, structural factors and size of the non-covalently connected micelles (NCCMs) can be tuned by adjusting the composition, structure and concentration of the corresponding building blocks in solution. For example, low molecular weight, carboxyl-ended polystyrene (CPS) as polymer A and poly(4-vinyl pyridine) (P4VP) as polymer B are mixed in the common solvent chloroform. After switching the medium to toluene, a selective solvent for CPS, stable polymeric micelles with P4VP as the core and CPS as the shell formed; the core and the shell are connected by hydrogen bonding. This approach is applicable to polymers of diverse compositions and architectures, leading to NCCMs of different compositions and structures.
The second approach is a solvent/non-solvent process: in this approach, there are no restrictions to the distribution of the proton donors and acceptors on the polymer chains. The key point here is to control the process of two polymers contacting each other and then the assembly process. The two polymers A and B containing hydrogen bonding sites are dissolved in their own solvents, respectively. The solvent of B should be the precipitant for A. When the solution of A is added to that of B dropwise, polymer A will aggregate immediately and polymer B will gather around the aggregates of A driven by hydrogen bonding. Thus, NCCMs with A as the core and B as the shell come into being (figure 2). This approach is more convenient to achieve when compared with the first. Based on this approach, various NCCMs have been prepared from a series of polymer pairs, including lightly sulfonated polystyrene/P4VP, poly(styrene-co-methacrylic acid)/poly(vinyl pyrrolidone), hydroxyl-containing polystyrene/P4VP, carboxyl-ended polybutadiene/poly(vinyl alcohol), poly(3-caprolactone)/poly(acrylic acid), etc.
Taking advantage of their unique character that the core and shell are connected by hydrogen bonding, the NCCMs can be conveniently converted into hollow spheres (sometimes called nanocages), by cross-linking the shell and then dissolving the core. It was confirmed that the as-prepared hollow spheres show perfect stimuli responsive properties when the hollow spheres were constructed of sensitive polymeric components. Clearly, this simple method for preparing hollow spheres is inapplicable to the micelles of ordinary block copolymers, for which the transition to hollow spheres requires chemical or biological degradation of the core component. In addition, they found that rigid polymer chains and their complementary homopolymers can directly assemble in their common solvent into large hollow spheres, owing to the propensity for radial packing of the rigid chains. They have demonstrated that a broader range of material sources are available for use as building blocks to self-assemble into NCCMs and the corresponding hollow spheres.
Recently, they have further extended their NCCM study by introducing supramolecular interactions, including inclusion complexation and biological recognition to replace hydrogen bonding, as the driving forces to form NCCMs [12,13]. This not only opens a new avenue for preparing NCCMs but also brings unique properties to NCCMs. For example, the hydrophobic copolymer poly(tert-butyl acrylate) (PtBA)-ADA with adamantine (ADA) side groups, and a hydrophilic polymer of poly(glycidyl methacrylate) (PGMA)-CD with β-CD (cyclodextrin) side groups were prepared. Driven by the inclusion interaction between β-CD and ADA, the two polymers self-assembled into NCCMs in water with PtBA-ADA as the core and PGMA-CD as the shell. The resultant micelles contain both a hydrophobic PtBA-ADA core on a scale of several hundred nanometres and a large number of hydrophobic cavities of size 0.7 nm in the shell, which can be used for further modification with functional species. Similarly, after the shell is cross-linked, and the core is dissolved, the NCCMs can be converted into the hollow spheres that have a central hole of submicrometre diameter and many β-CD cavities of 0.7 nm radius in the shell.
3. Polymeric supra-amphiphiles
Amphiphilicity is one of the molecular bases for supramolecular assemblies. By tuning the amphiphilicity of the building blocks, controllable self-assembly and disassembly can be realized, leading to fabrication of new functional supramolecular assemblies and materials. Recently, Zhang et al. from Tsinghua University developed the idea of supramolecular amphiphiles, in short supra-amphiphiles. In contrast to conventional amphiphiles, supra-amphiphiles are constructed on the basis of non-covalent interactions or dynamic covalent bonds. In supra-amphiphiles, the functional groups can be attached to the amphiphiles by non-covalent synthesis, greatly speeding their construction. The building blocks for supra-amphiphiles can be either small organic molecules or polymers [14–16].
When polymers are used to construct supra-amphiphiles, the resulting molecules are known as polymeric supra-amphiphiles. Polymeric supra-amphiphiles can be fabricated by the non-covalent interactions between polymers, or between polymers and small organic molecules. Owing to the different functionalities of the used building blocks, supra-amphiphiles allow for tuning of their amphiphilicity by external stimuli, leading to controlled self-assembly and disassembly. For polymeric supra-amphiphiles, different responsive small molecules can be introduced to tune the amphiphilicity of the complex by external stimuli.
Among all the stimuli, light is one of the most attractive for rapid and clean control of solution properties with specific direction and position. Zhang et al. have developed the concept of photo-controllable polymeric supra-amphiphiles through the electrostatic association between an azobenzene-containing surfactant and the double-hydrophilic poly(ethylene glycol)-block-poly(acrylic acid) (PEG-b-PAA; figure 3). They can self-assemble in aqueous solution to form vesicle-like aggregates. The photoisomerization of azobenzene moieties in the polymeric supra-amphiphiles can reversibly tune the amphiphilicity of the supra-amphiphiles, inducing disassembly of the vesicles . In another system, they reported the fabrication of a UV responsive polymeric supra-amphiphile formed by an anionic malachite green derivative and the cationic double hydrophilic block copolymer poly(ethylene glycol)-block-poly(l-lysine hydrochloride) (PEG-b-PLKC) .
Enzyme-responsive polymeric assemblies are particularly attractive because of their good biocompatibility and high degree of selectivity, because overexpression of enzymes has frequently been implicated in the diseased state of cells. Zhang et al. have prepared a series of enzyme-responsive aggregates formed by polymeric supra-amphiphiles for controlled cargo release. For example, they used multi-negatively charged adenosine triphosphate (ATP) and positively charged double hydrophilic PEG-b-PLKC to form a supra-amphiphile (PEG-b-PLKC/ATP; figure 3). The supra-amphiphiles can self-assemble in water to form spherical aggregates. Upon treatment with alkaline phosphatase (e.g. CIAP), the spherical aggregates will disassemble and release the loaded guest molecules . In addition, chitosan and ATP are used as building blocks to fabricate polymeric supra-amphiphiles based on electrostatic interactions, which can self-assemble to form spherical aggregates. The spherical aggregates inherit the phosphatase responsiveness of ATP . They have also reported the fabrication of phosphatase and acetylcholinesterase responsive polymeric supra-amphiphiles [21,22]. Therefore, this is a general method for fabrication of enzyme-responsive assemblies.
Besides the various intermolecular interactions, dynamic covalent bonding can also be used to drive the formation of polymeric supra-amphiphiles. Zhang et al. also demonstrated the fabrication of a polymeric supra-amphiphile based on imine bonds, which is responsive to physiological pH. PEG-b-PLKC and 4-(decyloxy)benzaldehyde (DBA) are chosen as the two components of the polymeric supra-amphiphile. At pH 7.4, the hydrophobic DBA molecules are attached to the PLKC blocks by the dynamic imine bonds formed between amine groups and benzaldehyde groups, leading to the formation of a brush-type supra-amphiphile. The supra-amphiphiles can self-assemble into spherical aggregates. However, when pH changes to 6.5, which is about extracellular pH of tumour cells, the imine bonds are broken, and the super-amphiphilic spherical aggregates are disassembled, releasing the encapsulated guest molecules . Similar idea can also be used to fabricate oxidation responsive polymeric supra-amphiphiles by the complexation of PEG-b-PAA and selenium-containing positively charged surfactants .
4. Self-assembly of hyperbranched polymers
Previously, the reported building blocks used in self-assembly often possess well-defined molecular structures, such as small amphiphiles, dendrimers and linear block copolymers. Through the molecular self-assembly of these building blocks, many elaborate microscopic or mesoscopic supramolecular objects have been observed over the past two decades [1–5]. Dendritic polymers, including dendrimers and HBPs, are the fourth major polymer architecture following the linear, branched and cross-linking polymers. HBPs, comprising dendritic units, linear units and terminal units, are highly branched macromolecules with three-dimensional dendritic globular architecture. Owing to the random distribution of these structure units along the polymer backbone, HBP is rather irregular when compared with its dendrimer analogue which is perfectly branched and monodisperse, while it has a unique advantage of facile one-pot fabrication. In addition, similar to dendrimer, HBP has demonstrated several characteristics when compared with linear polymer, including a large population of terminal functional groups, lower solution or melt viscosity and better solubility [25,26]. Thus, in the past 20 years, HBPs have already demonstrated great potential in nanotechnology, sensors and some typical polymer fields such as coatings and resins. However, little attention has been paid to the molecular self-assembly of HBPs, especially the macroscopic self-assembly behaviour of HBPs.
In 2004, Zhou and Yan et al. from Shanghai Jiaotong University reported macroscopic multi-walled tubes of millimetres in diameter and centimetres in length through the self-assembly of an amphiphilic hyperbranched multi-arm copolymer with a hydrophobic hyperbranched poly(3-ethyl-3-oxetanemethanol) (HBPO) core and many hydrophilic poly(ethylene glycol) (PEO) arms (HBPO-star-PEO) in acetone. HBPO-star-PEOs spontaneously aggregated into macroscopic membranes with a lamellar structure consisting of an alternate packing of a hydrophobic HBPO core layer and hydrophilic PEO arm layer induced by phase separation, which further folded into macroscopic multi-wall tubes. In addition, the tubes could be cross-linked by epichlorohydrin to form super-macromolecules with unusually large molecular weight . Since this groundbreaking work, there has been an explosive growth of interest in supramolecular self-assembly of HBPs. Many impressive molecular aggregates at all scales and dimensions, such as physical gels, micro- or nano-vesicles, fibres, spherical micelles, honeycomb films and large compound vesicles, have been reported by direct solution self-assembly, interfacial self-assembly and hybrid self-assembly (figure 4) [28–31]. These works have extended the research of molecular self-assembly into the macroscopic scale and the irregular molecule field. In addition, compared with the linear block copolymers, HBPs have demonstrated several advantages or specialities in self-assembly behaviours, including controllable morphologies and structures, special properties, characteristic self-assembly mechanism and facile functionalization process . Especially, a small change in the molecular architecture of HBPs will give rise to a big change in topology owing to an enlarged effect of the highly branched structure, which leads to a variety of designable self-assembly morphologies and structures . Such a characteristic is called ‘topological amplification effects’ .
In addition, the self-assembly of HBPs has displayed great potential in biomedical applications: for example, the model membranes to mimic cell morphologies, structures and functions; intelligent drug carriers and nanoreactors; hierarchical self-assembly and so on [34–39]. In short, although still being at the early stage, self-assembly of HBPs has provided a new avenue for the development of supramolecular chemistry.
5. Selenium-containing polymers
Selenium is a necessary element in the human body with potential antioxidant property. In chemical activity and physical properties, it resembles sulfur and tellurium . Redox-responsive polymers have attracted wide interest for their promising applications in controllable encapsulation and delivery in physiological environments, where the redox process is constantly and widely present. In recent decades, sulfur-containing polymers have been widely studied biomaterials as redox-responsive nanocarriers for active intracellular anti-cancer drug release or self-healing materials. However, the reports on selenium-containing polymers for bioapplication are scarce.
Xu and Zhang et al. from Tsinghua University recently synthesized a series of selenium-containing polymers for controlled self-assembly and disassembly under various responsive stimuli [41–46]. They successfully synthesized a dual redox-responsive block copolymer with diselenide bonds located at the polymer main chains and poly(ethylene glycol) chains at both ends (PEG–PUSeSe–PEG) which can be used as drug delivery vehicles in a controlled manner . PEG–PUSeSe–PEG, a typical amphiphilic block copolymer, can self-assemble in an aqueous environment to form micelles (figure 5). The micelles exhibit unique disassembly behaviour upon the addition of oxidant (i.e. H2O2) or reductant (i.e. glutathione, GSH). The encapsulated cargos can be released within 5 h in response to a very dilute concentration of oxidant (H2O2, 0.01% v/v or 2.9 mM) or reductant (GSH, 0.01 mg ml−1 or 30 μM). Because oxidative stress is often caused predominantly by accumulation of H2O2 and thought to be involved in the development of many diseases, H2O2 can potentially be useful as a stimulus for targeted drug delivery to diseased tissue. However, current polymeric systems are not sensitive enough to biologically relevant concentrations of H2O2 (50–100 μM). This work may open a new avenue for the preparation of block copolymer micelles capable of undergoing backbone cleavage and thus release of loaded cargoes upon exposure to such low concentrations of hydrogen peroxide. It should be further noted that intracellular reduction-response is exceedingly fast and efficient due to the presence of a high concentration of GSH (approx. 1–10 mM) in the cytosol and cell nucleus. Thus, PEG–PUSeSe–PEG block copolymer micelles could be an ideal burst release system in physiological environment of cells .
One important feature of the aggregates formed by PEG–PUSeSe–PEG is their sensitivity to gamma-radiation of low dosage. The anti-cancer drug doxorubicin (Dox) loaded in the polymer aggregates showed a controlled release profile with different radiation doses. It should be noted that even with a small dose (5 Gy) of gamma-radiation, the PEG–PUSeSe–PEG aggregates can still release about 45% of the loaded Dox molecules. This is important because this small dose of 5 Gy is close to the radiation dose that patients receive during a single radiotherapy treatment. Thus, this greatly enhances the possibility of biological and medical applications for these diselenide-containing block copolymer aggregates. It is anticipated greatly that this line of work may open an avenue for the combination of radiotherapy and chemotherapy .
They have reported the fabrication of a coordination-responsive system for controlled release of Dox, by using a mediator of Pt2+ that performs coordination to selenium-containing polymer micelles and subsequent release of Pt2+ by competitive coordination with GSH. The PEG–PUSe–PEG polymers can coordinate with Pt2+ and form spherical micelles in aqueous media. In the presence of GSH, the platinum can be released from the micelles in a controlled manner through competitive coordination with GSH. The coordination-responsive micelles can be used to load Dox and release it under the stimuli of GSH. With platinum-containing drugs coordinated with the block copolymer, for example, cisplatin, multi-drug systems for cooperative chemotherapy may be achieved by encapsulating other anti-cancer drugs simultaneously [43–45].
Besides selenide-containing main-chain polymers, there are side-chain block copolymers which can be fabricated in a covalent or supramolecular approach. Xu and Zhang et al. reported the synthesis and study of a series of side-chain selenium-containing amphiphilic poly(ethylene glycol-b-acrylic acid) block copolymers (PEG-b-PAA-g-Se). These block copolymers can self-assemble in aqueous solution and form spherical micellar aggregates. The selenide group of PEG-b-PAA-g-Se can change into hydrophilic selenoxide under mild oxidation of 0.1% hydrogen peroxide, leading to the disassembly of the spherical micellar aggregates and release of the encapsulated cargo. More interestingly, the oxidation state of selenoxide can be reversed to selenide under reduction of vitamin C, thus recovering the spherical aggregates. The reversible oxidation and reduction process shows good reversibility and can be repeated at least seven times .
6. Layered polymer composite films
Polymer films which can protect the underlying substrates and endow them with desired functions are widely used in daily life. A prominent method widely used in the past two decades to fabricate polymer composite films is layer-by-layer (LbL) assembly [47–49]. The LbL assembly involves the alternate deposition of species with complementary non-covalent interactions. The LbL assembly allows a broad range of materials, including synthetic polymers, particles, biomacromolecules, oligo-charged organic molecules, polymeric complexes and so forth, to be alternately assembled on various substrates in a predesigned fashion [47–49]. Compared with other film preparative methods, the LbL assembly is unique in precisely controlling the chemical composition and structure of the films on micro- and nanoscales and enables large-area film fabrication on non-flat surfaces.
The LbL assembly was originally developed as a group of methods for ultrathin nanocomposite film fabrication (i.e. films with thickness less than 100 nm), because the deposition of one layer of film usually takes several tens of minutes, and several hundreds of layers are often required to reach a thickness of 1 μm. Recently, great efforts have been devoted to speed up the LbL assembly process for the fabrication of micrometre-thick films. Sun et al. from Jilin University used polymeric complexes such as polyelectrolyte–polyelectrolyte complexes (PECs), polyelectrolyte–surfactant complexes, uncharged polymer–polymer complexes and polymeric–inorganic hybrid complexes for LbL assembly, which have larger dimensions than uncomplexed polymers and conveniently tailored structure and composition in solution. The LbL assembly of polymeric complexes not only provides a way for rapid fabrication of polymer composite films but also enables fine tailoring of structures and functionalities of the resultant films [50–52]. Novel functions are expected to be derived from LbL-assembled thick polymeric films because they possess good mechanical stability, allowing an increased amount of materials to be incorporated and film structures to be tailored on the micrometre scale.
The LbL assembly of PECs of poly(allylamine hydrochloride) (PAH) and sulfonated poly(ether ether ketone) (SPEEK; sulfonation degree approx. 82%) with poly(acrylic acid) (PAA) produces porous polyelectrolyte coatings containing micro- and nanoscale hierarchical structures . Upon thermal cross-linking of carboxylate/amine groups and chemical vapour deposition of 1H,1H,2H,2H-perfluorooctyltriethoxysilane (POTS), self-healing superhydrophobic (PAH-SPEEK/PAA)*60.5 films were fabricated, because POTS molecules not only deposited on film surface but also penetrated into the whole films through their interconnected porosity . O2 plasma etching of the superhydrophobic (PAH-SPEEK/PAA)*60.5 film removed the surface POTS and caused it to become superhydrophilic. However, the film regained its original superhydrophobicity after being transferred to an ambient environment with a relative humidity of 40% for 4 h, demonstrating that the destroyed superhydrophobicity was self-healed. The self-healing is driven by the migration of the preserved POTS molecules to the film surface to lower the surface energy as the surface with destroyed superhydrophobicity becomes hydrophilic and has increased surface energy. This etching–healing process can be repeated many times without decreasing the superhydrophobicity of the self-healed films, because the thick porous films can preserve an abundance of healing agents of POTS molecules.
The LbL assembly is also promising in fabricating intrinsic self-healing films capable of repairing deep cuts or scratches on the films [54,55]. Taking advantage of exponential LbL assembly to rapidly fabricate thick polyelectrolyte multi-layer films and adjust polyelectrolyte inter-diffusion, intrinsic self-healing films composed of alternately deposited branched poly(ethylenimine) (bPEI) and PAA were fabricated. An exponentially grown (PEI/PAA)*30 film with an average thickness of 34.1±3.3 μm can automatically repair cuts several tens of micrometres deep and wide when water is sprayed on the film. The self-healing ability of the bPEI/PAA films originates from the high flow ability of polyelectrolytes in the presence of water .
LbL-assembled polymeric thick films can be released from substrates to produce free-standing films. Without the restriction of underlying substrates, free-standing polymeric films can be designed to fabricate various actuators, which can reversibly and efficiently undergo bending/unbending movements in response to different external stimuli. In particular, LbL-assembled polyelectrolyte multi-layer films can be used to fabricate humidity-responsive actuators [56,57]. A powerful bilayer actuator was fabricated by spin-coating a flexible non-water-adsorbing layer of commercial UV-cured Norland optical adhesive 63 (NOA 63) on top of an exponentially grown (PAA/PAH)*30 film which was thermally cross-linked . Because of the large difference in water-adsorbing ability of (PAA-PAH)*30 and NOA 63 layers, the (PAA/PAH)*30/NOA 63 actuator can perform quick bending/unbending movements when environmental humidity changes. An energetic walking device was built by connecting two poly(ethylene terephthalate) plates as claws at opposite ends of the actuator (figure 6a). The actuator drove unidirectional walking of the device on a ratchet substrate when the RH was alternated between high and low values (figure 6b,c). Impressively, the actuator can drive a walking device carrying a load 120 times heavier than the actuator to walk steadily on a ratchet substrate under a periodic alternation of the RH between 11% and 40%. Theoretical analysis revealed that the powerful (PAA/PAH)*30/NOA 63 actuators originate from the combination of the high Young’s modulus and large linear coefficient of moisture expansion of the PAA/PAH layer which can be optimized by controlling the inter-diffusion of polyelectrolytes during their LbL assembly process.
7. To reveal the driving force for macromolecular assembly by single-molecule force spectroscopy
The study of the driving force (or polymer interactions) for macromolecular assembly is of crucial importance for a better understanding of the formation mechanism of those elegant naturally occurring systems (such as virus particle) as well as for the building of novel assembly systems. One of the effective ways towards such an objective is to simplify the real system and extract those interacting (ligand and receptor) pairs for single-molecule study. However, owing to the limitation of detecting methods, the study of molecular interactions at individual molecule level was impossible before the appearance of elegant single-molecule manipulation techniques (or single-molecule force spectroscopy, SMFS), such as atomic force microscopy (AFM) [58,59], optical tweezers , magnetic beads  and so on. These techniques can measure the force ranging from entropic forces at several femtonewtons to the strength of covalent bonds at several nanonewtons. Among these single-molecule manipulation techniques, AFM has become one of the most widely used techniques due to its friendly working interface and better commercialization.
Zhang et al. from Tsinghua University have made important progress in the investigation of some basic interactions such as π–π interactions between pyrene and graphite, the intercalating interactions between acridine and double-stranded (ds) DNA, and the multi-valency interactions between C60 and porphyrin using AFM-based SMFS, and valuable information, which is not available by conventional methods, has been obtained [62–64]. Apart from the study of fundamental interactions in synthetic systems, progress has also been made in the understanding of polymer interactions in biological systems. For example, the nature of force-induced conformation transition of dsDNA has been revealed at single-molecule level, and the investigation of interactions between single-stranded binding (SSB) protein and single-stranded DNA (ssDNA) has been demonstrated based on the force fingerprint of dsDNA . In addition, by using hetero bifunctional PEG as a cross-linker and introduction of cysteine at the terminus of SSB protein by genetic engineering, mild protein immobilization on solid substrate has been realized and quantitative information on ssDNA and SSB protein interactions has been obtained .
The investigation of polymer interactions in real assembly systems will provide more direct and useful information for the better design of novel assembly systems. On the basis of the study on simplified systems, AFM–SMFS has been extended to the study of polymer interactions in polymer single crystals and intact virus particles—two model systems of macromolecule assemblies [67,68]. The mechanical properties of polymer materials are governed by their chain composition/structure as well as the polymer chain interactions. However, due to the complexity of the system, the single-molecule investigation of inter-macromolecular interactions in their condensed states was difficult to realize. Zhang et al. at Jilin University have made the first successful attempt of the investigation of macromolecular interactions in a polymer single crystal by a good combination of AFM imaging and SMFS . They used a polyethylene oxide (PEO) with a sulfur-containing end group as a model polymer system. PEO single crystals were prepared from dilute solution by using a self-seeding method. Gold nanoparticles (GNPs) were then used to label the thiol-terminus of the PEO chain by gold-thiol chemistry. AFM imaging was firstly used to locate the specific GNP, then thiol-group functionalized AFM tip was brought to interact with the GNP forming the bridge structure. During the separation of AFM tip from the sample, the specific PEO chain was extracted from its single crystal, and the interaction strength between folded polymer fragments was measured quantitatively, as shown in figure 7. This study extends the usefulness of AFM-based SMFS to the investigations of polymer interactions in their condensed states (e.g. in polymer single crystals). The method established here can be used to study crystallization of various polymers at the single-molecule level.
A good understanding of the mechanism of nucleic acid–protein interactions will help us gain control in many important biological processes, such as virus infection and cancer cell growth. Furthermore, the investigation of nucleic acid–protein interactions in real biological systems will provide more accurate information and direct guidance in practice. Owing to the limitation of detection method, such study was quite difficult to carry out. As a proof-of-concept study, the same group has chosen tobacco mosaic virus (TMV) as a model system to study the interactions between genetic RNA and its coat proteins. By subtle experimental design/control, TMV particles were immobilized perpendicularly on a gold substrate leaving their 5′ openings (especially the 5′ end of RNA) exposed for picking up by the AFM tip. The genetic RNA was pulled step-by-step out of the TMV particle by the AFM tip via physical adsorption, and unbinding forces between RNA and protein coat were measured directly at the single-molecule level . It has been found that the unbinding force increases with the increase of stretching speed, and decreases with the increase in pH. In addition, the detached RNA can find its way back to the protein coat with the help of intact RNA protein complexes during relaxation. In doing this, they have extended AFM-based SMFS to study protein–nucleic acid interactions in more complicated biological systems, and the established method may provide a new gateway towards investigations of the mechanism of virus infection.
Other similar studies on the understanding of driving force for macromolecular assembly in real systems have been demonstrated successfully by Zhang et al. at Tsinghua University in the investigation of polymer interactions in polymer micelles formed by block copolymers [40,69], as well as in the study of supramolecular polymers .
8. Macromolecular assembly for sensing
Conjugated polymers (CPs) have demonstrated themselves as useful optical platforms to sensitively detect chemical and biological molecules due to the signal amplification by a collective system response [71,72]. In this optical response system, a single-molecular binding event quenches the fluorescence of many chromophores, amplifying the chemosensory response by orders of magnitude. The signal amplification property of CPs imparts to the sensor high sensitivity, and therefore offers a key advantage over sensors based on small molecules. They exhibit high fluorescence brightness, excellent photostability and lower toxicity in live-cell imaging, showing promising application as new generation of fluorescent probes [73,74]. Wang et al. from Institute of Chemistry, CAS, have prepared a series of semi-synthetic, bio-optical conjugates to provide a means of coupling the sensitivity and specificity of biomolecules with the robust physical properties and large optical responses of CP materials [71–74]. They have developed a novel technique for preparing encoded multi-colour nano/microparticles based on the self-assembly of microorganisms and CPs . Three types of red, green and blue (RGB)-emissive cationic CPs based on polyfluorene structure were synthesized and characterized. The novel approaches for preparing encoded multi-colour nano/microparticles were demonstrated by the self-assembly or controlled growth of CPs with microorganisms as scaffold. They could tune the multi-colour emissions by tuning fluorescence resonance energy transfer (FRET) efficiencies among three CPs under single excitation wavelength and obtain large Stokes shifts. These multi-colour particles exhibit low toxicity toward cells and have great potential applications in biorecognition, multiplexed bioassays, optical barcoding and molecular imaging (figure 8).
They also explored the suggestion that the macromolecular assemblies of cationic water-soluble CPs with biomacromolecules can be used to develop biosensors for DNA, RNA and protein. Alterations in the methylation of promoters of cancer-related genes are promising biomarkers for the early detection of disease. Recently, they reported a novel and simple method for sensitive detection of regional DNA methylation of a specific gene. The method is based on FRET technique devoid of labelling DNA, implementing water-soluble cationic-conjugated polyelectrolytes. As a result in the study of methylation status in the promoters of three genes for five cancer cells, genes p16, HPP1 and GALR2 are considered to be candidate molecular markers for colon cancer [76,77]. Compared with single methylation alteration, assessing combined methylation alterations can provide higher association with specific cancer. They used cationic CP-based FRET to quantitatively analyse DNA methylation levels of seven colon-cancer-related genes in a Chinese population . Through a stepwise discriminant analysis and cumulative detection of methylation alterations, they acquired high accuracy and sensitivity for colon cancer detection (86.3 and 86.7%) and for differential diagnosis (97.5 and 94%). Moreover, they identified a correlation between the CpG island methylator phenotype and clinically important parameters in patients with colon cancer. The cumulative analysis of promoter methylation alterations by the cationic CP-based FRET may be useful for the screening and differential diagnosis of patients with colon cancer, and for performing clinical correlation analyses. This system can also be used for simple, rapid and sensitive detection of human disease-related single nucleotide polymorphism (SNP) . The method can detect as low as 2% allele frequency (the proportion of all copies of a gene that is made up of a particular gene variant). The SNP genotypes of 76 individuals of Chinese ancestry were clearly discriminated. They also developed a new design of an electrostatic complex consisting of cationic polyfluorene with negatively charged Y-DNA, respectively labelled at the 5′ termini with fluorescein, TexRed and Cy5 to generate an energy-transfer cascade [80,81]. Multi-step FRET processes regulate the fluorescence intensities of the CP and three dyes. Different types of logic gate can be operated by observing the emission wavelengths of different dyes with multiplex nucleases as inputs. The technique gives rise to a new method for the simultaneous detection of multiplex nucleases.
Multi-functional materials that simultaneously provide therapeutic action and image the results provide new strategies for the treatment of various diseases [82,83]. They showed that water-soluble CPs with a molecular design centred on the polythiophene–porphyrin dyad are effective for killing neighbouring cells. Following photoexcitation, energy is efficiently transferred from the polythiophene backbone to the porphyrin units, which readily produce singlet oxygen (1O2) that is toxic for the cells. Owing to the light-harvesting ability of the electronically delocalized backbone and the efficient energy transfer among optical partners, the polythiophene–porphyrin dyad shows higher 1O2 generation efficiency than a small molecule analogue. They also showed that CPs can specifically inactivate proteins under light irradiation through their assemblies with proteins [84,85]. Upon light irradiation, the polymer produces reactive oxygen species (ROS) that react with and inactivate adjacent proteins. In comparison with typical chromophore-assisted light inactivation technique based on organic dyes, this technique takes advantage of the light-harvesting property of CPs that is beneficial for ROS generation. This review adds a new dimension to the function of CPs.
9. Conclusions and outlook
Although great progress has been made during past decades for the macromolecular assembly and nanotechnology research in China, a lot of challenges still remain: to understand the mechanisms underlying macromolecular self-assembly has been a main focus during the past decade, in particular, with respect to multiple interactions, cooperative effects and dynamic behaviour of the self-assembly process. Another aspect is to control the properties in distinguished functions emerging from self-assembled macromolecular systems, and demonstrate their advanced and enhanced functionalities, particularly in responsive molecular systems, by increasing participation of theoretic and simulation support.
Particularly, Chinese scientists need to pay more attention to develop new detection techniques with high sensitivity and specificity such as gene chips, to develop novel smart drug delivery systems that can work efficiently in vivo, to develop novel materials with better biocompatibility and cooperative functions, eventually leading to the innovation of high science and technology in the combined field of life science and material science. The long-term goals are developing functional materials at all scales with tailored electronic, optical and sensing properties through their organization and collective behaviour, and accomplishing multiple or complex functions which individual macromolecules cannot perform.
One contribution of 17 to a Theme Issue ‘Molecular nanostructure and nanotechnology’.
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