2D bio-nanostructures fabricated by supramolecular self-assembly of protein, peptide, or peptoid
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Biomolecular self-assembly is a promising strategy for fabricating two-dimensional (2D) nanostructures such as sheets, films, lattices, or membranes. In this paper, we summarize the recent development of 2D bio-nanostructures that are formed by supramolecular self-assembly of protein, peptide, or peptoid, respectively. Specific focus is given on the formation mechanisms and the structures as well as functionality of the 2D bio-nanostructures. Besides, some typical applications of 2D bio-nanostructures have been listed. At last, the potential research direction of 2D bio-nanostructures is discussed.
Keywords2D materials Bio-nanostructures Amyloid assembly Protein Peptide/peptoid
2D nanostructures are emerging as an important material class in recent years. They have broad application prospect in various fields of electronics, optics, sensing, actuating, catalysis, energy, etc. [1, 2, 3, 4, 5]. Compared to the low dimensional and three-dimensional nanostructures, 2D nanostructures possess higher superficial area to volume ratios resulting from their extreme aspect ratio. Thus, they present a variety of interesting properties and can provide a general platform on which to display an enormous diversity of functionalities [6, 7, 8].
In contrast to common inorganic or organic nanostructures formed by limited source, biomacromolecule-derived 2D bio-nanostructures, for instance, lattices, sheets, arrays, membranes, films, and ribbons by self-assembly of proteins, peptides, or peptoids, have attracted interests of many researchers [9, 10, 11].
Herein, we describe the recent development of proteins, peptide, or peptoid as building blocks to from 2D bio-nanostructures through supramolecular self-assembly and highlight the formation mechanisms and the structures as well as functionality of the 2D bio-nanostructures.
2 2D bio-nanostructures derived from self-assembly of proteins
Complex but highly ordered various architectures derived from protein self-assembly are the best form of supreme wisdom of nature. In the field of biological materials, well-defined 2D architectures derived from designed assembly of proteins can contribute to tailor materials with new physical and chemical properties.
Surface layer (S-layer) proteins are promising building blocks in nanotechnology for they not only create a cell-surface layer architecture in both archaea and bacteria but also can easily self-assemble into a single layer of crystalline nanostructure in vitro.
Moll et al.  fused streptavidin to a crystalline bacterial cell S-layer protein and exploited these S-layer-streptavidin fusion proteins as building blocks to fabricate 2D protein lattice or sheets. Furthermore, chimeric S-layer with the ability to bind with biotinylated ferritin was designed.
Similarly, Wang et al.  genetically fused the S-layer protein of Bacillus anthracis EA1 with methyl parathion hydrolase (MPH) and constructed a new EA1-based 2D nanostructure through self-assembly in vitro. When applied in detecting anthrax-specific antibodies in serum samples, the new nanostructure of S-layer–enzyme conjugation showed excellent performances in both detection sensitivity and enzymatic stability of MPH.
Based on the understanding that the nanostructures of self-assembling proteins were strongly influenced by the solution conditions, Rad and co-workers  established the diagrams coupled with modeling of the self-assembly process of SbpA, a S-layer protein from the insect pathogen Lysinibacillus sphaericus, in a large range of concentrations of SbpA and Ca2+. The diagrams mapped by high-throughput light scattering showed that both nanosheet yield and size varied as a function of time and Ca2+ concentration. Moreover, calcium ions were found to mediate specific as well as nonspecific interactions during the course of self-assembly.
Besides, the self-assembly behaviors of many other kind of proteins had been investigated in-depth. Some valuable research efforts are introduced as follows.
By taking advantage of both directional metal-protein coordination and nonspecific protein-protein interactions, Bai et al.  developed a solution to control the self-assembly behaviors of protein accurately. A series of highly ordered S-transferase (GST) nanorings with different diameters had been obtained through altering the Ni2+ strength of the solution in a certain range.
Amyloid fibrils generally possess very high mechanical strength and good adhesion capability to various substrates. By using simple assembly techniques, amyloid fibrils can create 2D amyloid nanostructures in diversity. For instance, the manipulation of the assembly of a classical globular protein, lysozyme, could serve as a kind of typical amyloid aggregation structure.
In recent years, much significant progress on the theory and applications of amyloid-like protein assembly has been made by Yang’s group [20, 21, 22, 23, 24, 25, 26]. With deeply understanding the mechanism of the novel phase transition process of lysozyme, they discovered that the amyloid-like assembly of typical globular proteins, e.g., lysozyme could be rapidly achieved after efficiently reducing their disulfide bonds by Tris (2-Carboxyethyl) phosphine (TCEP) under quasi-physiological condition. TCEP is known for its good reduction ability of disulfide bonds of biomolecules in various environments . When mixing TECP with lysozyme in a neutral buffer, TCEP could effectively break down the disulfide bond of lysozyme chain; meanwhile, α-helix of native lysozyme was unfolded into β-sheet structure , resulting in the formation of insoluble amyloid nanospheres. Therefore, with stepwise nucleation and fusion, 2D assembled nanofilm of phase-transitioned lysozyme (PTL) will be formed gradually if the concentrations of lysozyme and TECP are enough.
As a result, HAp on the PTL coating acted as intermediate adhesion layer and presented excellent bonding stability to meet general load requirements when be applied on artificial bone and tooth. Cytotoxicity assays indicated that PTL nanofilm as well as HAp on the PTL nanofilm supports good biocompatibility and cytocompatibility toward rat bone marrow mesenchymal stem cells (rBMSCs). Subsequent animal tests on subcutaneous implantation in a rat model indicated that this biomaterial presented a favorable in vivo osteoconductivity.
3 2D bio-nanostructures derived from self-assembly of peptides
Just like proteins, a peptide is another major molecular scaffold material in fabricating 2D bio-nanostructures by self-assembly. Moreover, benefit from shorter chains and smaller molecular weights, peptides are more likely to spontaneously self-assemble into nanoarchitectures than proteins. Importantly, peptides can be synthesized on a large scale by conventional chemical techniques.
As early in 1993, Zhang et al.  found that a 16-residue peptide [(Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)2] could spontaneously assemble to form a macroscopic membrane in aqueous solution upon the addition of salts. And the stability of the membrane was so high that it did not dissolve in hot, acidic, or alkaline solutions as well as some frequently-used solvents, nor did it dissolve upon addition of enzymes.
Similarly, Hamley and coworkers  reported on the formation of free-floating nanosheets with 3 nm thick by an amphiphilic peptide (Ala)6(Arg) in aqueous solution. They discovered that the concentration of peptide was the decisive factor in the course of self-assembly, that is, ultrathin sheets without β-sheet would form at low concentration (below 2 wt%), whereas helically wrapped ribbons coexisting with nanotubes would appear at 15 wt% and above accompanying with β-sheet formation. Whereafter, the self-assembly behavior of surfactant-like peptide (Ala)6-(Asp) (A6D) in aqueous solution especially when hexamethylene diamine was added into the system at different molar ratio was investigated by Hamley’s research group . They found that acid-coated multiple ordered nanostructures, such as nanosheet and bicontinuous network structures, could be obtained by adding appropriate amount of diamine. However, the self-assembled structure would be lost completely at 2:1 M ratio of diamine to A6D. So, addition of a diamine is an effective means to modulate the self-assembly behavior of A6D.
Two years later, the design and self-assembly performance of another peptide sequence YFCFY, where Y represents tyrosine, F represents phenylalanine, and C refers to cysteine, was reported by the same research group . Different from YYACAYY could self-assemble only in the buffering conditions; YFCFY can assemble even in pure water. Moreover, the final assembly form of YFCFY at air/water interface is always flat sheet regardless of the initial status if it is a fibril-aggregated, rough film or uniformly packed.
Except for the intensive study on the formation mechanisms and basic application of self-assembling 2D peptide nanostructure, some researchers used the unique 2D nanostructure to solve the problems which existed in other areas. Some examples are listed below.
4 2D bio-nanostructures derived from self-assembly of peptoids
Peptoid is a novel class of highly designable and biomimetic heteropolymers . Different from the structure of peptides, peptoids are poly(N-substituted glycines) in which the side chains are attached to the nitrogen rather than the α-carbon . They can fold into higher order–specific protein-like structures but with the ability of resisting to proteolysis . Peptoids are designed to act as typical representative of versatile biomimetic materials in the field of nanobioscience due to the synthetic flexibility, robustness, and ordering at the atomic level . So, peptoid is one of the ideal candidates to create 2D bio-nanostructures.
A number of processes for different peptoid synthesis and self-assembly into 2D nanosheets have been investigated [44, 45, 46, 47, 48, 49, 50, 51, 52]. Tran et al.  described the overall synthesis process of sequence-specific peptoid polymers by solid-phase sub-monomer synthesis method in detail. And the protocol to form highly ordered nanosheets from an amphiphilic 36-mer peptoid was presented as well.
Based on designing five kinds of lipid-like peptoids, named Pep1-Pep5, stable membrane-mimetic 2D nanomaterials were achieved through self-assembly of the peptoids by Jin  and Jiao et al.  Just like real bilayer cell membranes, the peptoid membranes could respond to external stimuli, coat surfaces in single layers as well. More excitingly, the heavily scratched 2D peptoid membranes could self-repair when they were in certain conditions, such as introduction peptoid-containing solution and suitable pH. A number of factors which concern the rate of repair, such as charge state and wettability of the surface, peptoid headgroups, the concentration of the solution used for repair, were studied systematically by the authors.
Kudirka et al.  designed two kinds of single-chain peptoid with different sequences; the charged residues of them were configurated according to alternating patterning and block patterning, respectively. Then, nanosheets with higher order structures have been obtained by using the same vial-rocking device to Sanii et al.’s work . The two nanosheets were confirmed to display different sensitivities to pH and chemical denaturants due to their different block charge design.
Differing from general knowledge that nanosheets prefer to form at the air-water interface, Robertson et al.  confirmed that peptoid nanosheets could be formed at oil-water interface. And the detection results of vibrational sum frequency spectroscopy demonstrated that an ordered peptoid monolayer formed at oil-water interface mainly by means of electrostatic interactions.
Based on the above systematic study, Sanii et al.  proposed a detailed mechanism model that peptoid polymers self-assembled into nanosheets at an air-water or oil-water interface. Moreover, the sequential order of several of structure-determining steps was pointed out one by one. Their results contributed to the insight into the general strategy of accelerating the assembly of 2D nanomaterials by using planar fluid interfaces.
Another typical practical application of peptoid nanosheets has been attempted by Olivier and coworkers . They designed and synthesized a kind of antibody-mimetic materials based on functionalized nanosheets that were fabricated by self-assembling aggregates of peptoid polymers with specific sequences. Compared with native antibody, peptoid nanosheets possessed the advantages of higher stability, cheap raw material sources, additional functionalities, and so on.
Until now, peptoids belong to a side-chain-dominated system, and thus lack both chirality and the hydrogen bond donor in their backbone as well. So, more opportunities and possibilities can be offered to design new molecular structures and diverse properties. Compared with natural proteins and peptides, the feature of designable ability at molecular level makes peptoids seem to be suitable for fabricating highly tunable 2D nanostructures by self-assembly.
5 Summary and outlook
In this review, the recent progress in strategies for fabricating 2D bio-nanostructures through self-assembly of proteins, peptides, or peptoids have been summarized. We focused on the elucidation of the self-assembly mechanisms, hierarchical structure, and practical or potential applications of a series of 2D bio-nanostructures.
In our opinion, there are some areas that should be further investigated. One is fundamental knowledge of molecular dynamics or self-assembly kinetics of building blocks by using computer simulation techniques or other advanced instrument and equipment. The development in fundamental research will supply continuing inspiration and guidance for controllable growth and formation of 2D or 3D bio-nanostructures. Second, more efforts should be made on the discovery, selection, and development of proteins, peptides, or peptoids with specific sequence that are more suitable for nanofabrication. Last but not least, the new and valuable application of 2D bio-nanostructures should be exploited. The use of phase-transitioned lysozyme (PTL) nanofilm as an effective template to prepare artificial bone and tooth is an excellent example. As research continues, it is to be hoped that such extraordinary work in the field of biotechnologies and biomaterials will increase gradually.
P.Y. thanks the funding from the National Natural Science Foundation of China (Grant Nos. 51673112 and 21374057), the 111 Project (Grant No. B14041), and Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT_14R33) as well as Open Project of State Key Laboratory of Supramolecular Structure and Materials (Grant No. sklssm201727). W. Z. thanks the support of Natural Science Basic Research Plan in Shaanxi Province (No. 2016JM5024), China Postdoctoral Science Foundation (No. 2014M560747), and Scientific Research Project of Xianyang Normal University (No. 13XSYK017).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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