key: cord-0068920-ieymuksv authors: Li, Xiaodan; Guo, Mengyu; Chen, Chunying title: Graphdiyne: from Preparation to Biomedical Applications date: 2021-10-23 journal: Chem Res Chin Univ DOI: 10.1007/s40242-021-1343-8 sha: bb6a09594125a766aa999e1716580cd35ee431d0 doc_id: 68920 cord_uid: ieymuksv Graphdiyne(GDY) is a kind of two-dimensional carbon nanomaterial with specific configurations of sp and sp(2) carbon atoms. The key progress in the preparation and application of GDY is bringing carbon materials to a brand-new level. Here, the various properties and structures of GDY are introduced, including the existing strategies for the preparation and modification of GDY. In particular, GDY has gradually emerged in the field of life sciences with its unique properties and performance, therefore, the development of biomedical applications of GDY is further summarized. Finally, the challenges of GDY toward future biomedical applications are discussed. Carbon has four electrons in the outer layer of nucleus, so it can form three common hybrid structures(sp, sp 2 , sp 3 ) [1, 2] . Furthermore, abundant carbon allotropes can be formed through covalent bonds (Fig.1 ). After the diamond(sp 3 ) and graphite(sp 2 ) have been known, fullerene(C60, sp 2.28 ), a spherical carbon molecule composed of 12 five-membered rings and 20 six-membered rings, was developed in 1985 by Kroto et al. [3] Driven by fullerene research, in 1991, a more peculiar carbon structure, carbon nanotube, was discovered by Lijima of NEC [4] ， which further deepened peopleʹs understanding of low dimensional carbon materials. In 2004, Geim et al. [5] firstly prepared a two-dimensional(2D) material, graphene, which was composed of sp 2 hybrid carbon atoms arranged in a hexagon honeycomb structure periodically and connected with each other in the form of covalent bond. Graphene is a zero-band gap semiconductor material with excellent electrical properties [6] . However, the energy band and performance regulation of graphene is still an unsolved difficulty [7, 8] . Therefore, we are looking for a new type of carbon-based 2D structure with both excellent band gap and higher carrier mobility. As early as 1987, Baughman et al. [9] proposed a kind of two-dimensional allotrope material of carbon, which is formed by the arrangement of carbon atoms in sp and sp 2 hybrid states according to a certain periodic rule, which was called graphynes. This new carbon allotrope, especially graphdiyne (GDY), has different active sp hybrid carbon and special conjugated structure from graphene, which has aroused researchers' exploration of its properties. In 1997, GDY was first proposed by Haley et al. [10] , then it was predicted to be a semiconductor material with direct band gap(0.46 eV) and high room temperature carrier mobility(10 4 -10 5 cm 2 ·V -1 ·s -1 ) [11] . However, due to the instability of its monomers and the existence of carbon-carbon triple bonds in the structure, the experimental synthesis of graphynes presents a huge challenge. It was not until 2010 that GDY was first synthesized by Li et al. [12] using the in situ Glaser coupling reaction of hexaethynylbenzene(HEB) monomer on the copper substrate. This new type of carbon-based 2D atomic crystal material was synthesized on the copper substrate by solution chemistry. After that, scientists are committed to the exploration of high-quality preparation of GDY and broadening the applications of its special properties. In biomedical field, taking special pore structure, π-π conjugated systems, special photoresponse properties and geometrical properties as advantages, GDY has new understanding and applications in Graphynes have well-distributed pore structures and large πconjugated systems, endowing them with potential applications in biosensors, biological imaging, cancer treatment, antibacterial and radiation protection [13] . In these biomedical applications, graphyne-based materials show better performance and higher stability than other carbon based materials. Huang et al. [18] calculated the Gibbs free energy of various carbon allotropes with graphyne and hydrogen as reactants, and found that the Gibbs free energy of graphynes was 0.803 eV, which was larger than those of diamond(-0.022 eV), C60(0.364 eV) and carbon nanotube (6, 6)(0.014 eV). These results indicated that graphynes were the most unstable kind of carbon allotropes. Properties determine applications. Graphynes benefit from the unique structure, such as GDY, which can be applied in many fields. Zhang et al. [19] compared the fracture stress, strain and Youngʹs modulus of several kinds of graphynes and graphene. They found that the introduction of -C≡Creduced the rigidity and enhanced the flexibility of graphynes, so they could produce greater deformation under stress. In addition, Shao et al. [20] found that the mechanical properties of graphynes were related to temperature. With the increase of temperature, the mechanical properties of graphynes decreased obviously. Electrical property is one of the most attractive properties Review of graphyne allotropes. Long et al. [11] calculated the band gaps of graphene and graphynes were 0.46-1.22 eV and 0.44-1.18 eV. At room temperature, the electron mobility of graphynes could reach 2.1×10 5 cm 2 ·V -1 ·s -1 . Similar to other carbon materials, physical properties of graphynes can be changed through chemical doping, such as the band gap adjusting [21] . Since GDY is a semiconductor with a direct band-gap, the optical performance can be studied by adjusting the inherent microstructure(such as sizes, layer number, stack configuration, etc.), chemical doping, external strain and electric field, etc. GDY possesses high hole mobility and narrow band gap, so it can be a new kind of carbon-based photoluminescence(PL) material, and can have many potential applications, such as UV photodetectors and bioimaging system [22] . Chen et al. [23] and Luo et al. [24] revealed the effect of graphyne allotropes stacking structure on the optical properties by density functional theory, which provided a theoretical basis for the structural identification of graphynebased photocatalyst. According to the results of many studies, the light absorption of GDY was related to its layer number and stacking arrangement. GDY also showed a high fluorescence quenching ability, which could be used as a platform for fluorescence sensing, and the quenching ability of oxidized GDY was higher than that of GDY [25] [26] [27] [28] . It is critical to understand the intrinsic structure and physical properties of GDY for its basic research and practical application. However, due to the difficulty of obtaining high quality GDY up to now, the current theoretical research is far beyond experimental research. So far, in order to further study the intrinsic structure and properties of GDY, researchers have devoted themselves to the controllable synthesis of highquality GDY with single or few layers. In the past decade, various approaches have been developed to synthesize GDY with controllable layer and different morphologies, such as GDY films [12] , nanowalls [14] , nanoribbons [11, 18] , nanosheets [10, 11] , nanotubes [29] and free standing three-dimensional(3D) GDY [26] . Kong et al. [30] summarized the gaps between reality and ideality of GDY, and revealed that the synthesis of GDY needed a long way to go (Fig.4) . Generally, there are two traditional strategies for the preparation of 2D nanomaterials: one is the bottom-up category starting from existing precursors/monomers, the other is the top-down preparation method starting from block GDY, which has not been realized at present. So, the existing synthesis technology of GDY can be divided into dry chemistry and wet chemistry (Fig.5) . Moreover, the Fig.4 Schematic illustration of the gap between reality and ideality of GDY [30] Black ball and green ball, carbon atom; red ball, oxygen atom; blue ball, hydrogen atom. Reprinted with permission from ref. [30] . Copyright 2020, Elsevier Ltd. [14] Reprinted with permission from ref. [14] . Copyright 2019, Royal Society of Chemistry. functionalized GDY a nd other GDY-based materials are synthesized with different novel strategies, especially for its deep use in biomedical area. In order to obtain high-quality GDY with high crystallinity and low defects, the preparation methods have emerged one after another [10, 31, 32] . Dry chemical synthesis is based on methods of controlled synthesis under strict, non-solution conditions. At present, the dry synthesis methods of GDY include surface preparation under high vacuum of STM, chemical vapor deposition synthesis and explosion method [33] [34] [35] [36] [37] [38] . The above synthesis method of GDY is precise, but it is not suitable for mass production and commercial application. Due to the problems of monomer stability, rotation of carbon-carbon single bond and low specific surface area of substrate, the quality control and preparation efficiency of GDY are limited. Synthesizing GDY is via the wet chemical routes possesses: Cu-surfacemediated synthesis, interface-assisted synthesis, and solutionphase van der Waals epitaxy. According to different functional groups, the methods for preparing GDY based on coupling reaction can be roughly divided into two categories: the first is the homogeneous coupling reaction between monomers and terminal alkynyl groups. Among them, the most commonly https://doi.org/10.1007/s40242-021-1343-8 Review used are Glaser, Glaser Hay and Eglinton coupling reactions [30, [38] [39] [40] . However, due to the high reaction activity, these monomers face the risk of oxidative decomposition in the whole process and usually require additional heating. The other type is homocoupling of terminal alkynyl-silane groups, which can enhance the reaction efficiency without deprotection(such as Hiyama coupling) [41] . Due to the low efficiency and various by-products, it is still aimed at developing new precursors with high stability and high reactivity to improve the preparation of GDY [42] . The coupling reaction mechanism is shown in Fig.6 . It is also necessary to develop a method for directly growing GDY with specific structure and properties to a specific target substrate. This method can keep the intrinsic properties and aggregation morphology of GDY unchanged, and GDY combined with different functional substrates can realize the construction of a variety of functional materials, greatly expanding the application field of GDY [38, 43] . Confined synthesis strategy uses strategies, such as concentration diffusion, template effect, interface restriction or temperature gradient to limit the target substrate or react in a certain space, which can prepare 2D materials. These confined strategies can deal with the poor stability of the monomer, the lower reaction efficiency and side reactions, and also the single bond between the diacetylenefree rotation [37, 44, 45] . [30] (A) Concluded synthetic route for GDY based on HEB or HEB-TMS precursor; (B) proposed mechanism for classical Glaser coupling reaction; (C) proposed mechanism for Glaser-Hay coupling reaction; (D) proposed mechanism for Eglinton coupling reaction; (E) proposed mechanism for alkynyl-silane coupling reaction. Reprinted with permission from ref. [30] . Copyright 2020, Elsevier Ltd. Although bulk GDY can be exfoliated into single-or few-layered GDY with high exfoliation efficiency(75%, mass fraction) [46] , it is still a great challenge to synthesize single/ few layer GDY single crystal films that can keep their twodimensional material characteristics. The main problems are as follows. 1) The single bond connecting benzene ring and alkyne bond in the monomer can rotate freely, which will lead to the formation of highly branched or cross connected threedimensional skeleton structure in the reaction process, so we cannot get the expected high-quality layered crystal samples. 2) The traditional epitaxial substrate surface has dangling bonds, which makes the epitaxial layer and substrate have strong interaction, so the lattice of epitaxial layer and substrate should be matched. 3) Due to the existence of ES barrier, the 'step-down' diffusion of monomers adsorbed on the epitaxial layer is inhibited, which will lead to the aggregation and nucleation of monomers on the surface of epitaxial layer, resulting in the formation of thick layer GDY samples(layer by layer stacking out of plane growth mode). The poor solubility and processing properties of GDY are the main obstacles that limit its applications, especially in biological systems. The high activity of the acetylene bond unit in GDY provides a good platform for its chemical modification and doping. At present, progress has been made in nonmetallic heteroatom doping, metal atom modification, and surface modification. The photoelectric properties, energy storage, conversion efficiency, and catalytic performance of GDY are improved a lot. In particular, many important results have been achieved in theoretical research, and experimental work has gradually increased in recent years, but the research on GDY is still in the preliminary stage of development. Herein, we made a more comprehensive summary and discussion of the current modification strategies of GDY, which will also provide gist for the wider application of graphyne allotropes in the future. At present, the main bottleneck hindering researchersʹ further study is the poor solubility and processability of GDY. But there is still a long way to explore the preparation of higher quality GDY, and researchers prefer to use the alkyne bond/sub nanopore and the semiconductor energy band structure of GDY for chemical functionalization, and the fact also proves the importance of chemical modification. Hydrogen modified GDY is often called hydrogenated GDY. The sp and sp 2 hybridization and large π-π conjugated system of GDY and its derivatives can provide more adsorption space for H2 adsorption. GDY can covalently bind with one or more hydrogen atoms to transform sp 2 and sp carbon atoms into sp 3 or sp 2 carbon atoms, thus laying a foundation for the preparation of new carbon allotropes. At present, the study of γ-GDY-H system is mainly focused on two aspects: (I) the effect of hydrogen adsorption on the regulation of the properties of γ-GDY itself, and (II) the use of γ-GDY for hydrogen storage. Regarding the influence of hydrogenation on the intrinsic properties of GDY, the researchers found that the increase in the degree of hydrogenation has a negative impact on the mechanical properties of GDY from the perspective of mechanical properties [47] . Some researchers also simulated the combination of hydrogen atom and GDY with orbital acetylene bond. In the research, it was found that the priority of hydrogen atom binding to sp and sp 2 acetylenic bonds was different under different circumstances [48, 49] . Li et al. [50] used first-principles systems to study the dynamic stability and electronic structure changes of hydrogenated GDY. By analyzing the enthalpy of formation, they pointed out that the hydrogenation configuration(eHH) containing only sp 3 hybrid carbon atoms was more stable than the hydrogenation configuration(eH) of each carbon atom passivated by a single hydrogen atom. However, the eHH of each carbon atom passivated by a single hydrogen atom is more stable. The temperature dependence of the Mholtz free energy indicates that eH is more favorable than eHH below Oxygen modification is a promising method for functionalizing GDY to achieve specified properties. Most studies have focused on the mechanism of oxidation on the regulation of band structure and photoelectric properties of GDY. In the biological field, the oxidation treatment of GDY is often used to improve the hydrophobicity and dispersion. Zhang et al. [51] further studied the effect of the coverage of oxygen-containing functional groups on the structure and properties of GDY band, which provided a basis for the oxidative modification of GDY. Mohajeri et al. [52, 53] The specific calculation showed that the functionalization of GDY sheet containing C=O group made HOMO move towards high energy, but LUMO change little. In contrast, the -COOH-group was more effective for LUMO and transferred LUMO to a lower energy. These shifts reduced the threshold of optical transition between HOMO region and LUMO region, and the peak appeared in a low energy region. Above are the research results on the modification of GDY by oxidation found in the current research. In recent years, many researchers are also committed to exploring the application of GDY in the field of biology [13] . Li et al. [54] [55] [56] applied GDY and GDYO to many fields including the biological research. In order to increase the compatibility and dispersion of GDY in the biological aqueous environment, the researchers obtained GDYO through oxidation treatment to ensure better effect. More recently, Yan et al. [57] made further study of GDYO, and they gave new comments for the unique 2D GDYO. Wang et al. [58] Currently, GDY applied in the field of biology has been commonly modified by oxidation to increase the dispersion and hydrophilicity of its basic aqueous solution system. Other studies have also shown that GDYO has more excellent biocompatibility and safety, and lower biotoxicity compared to graphene oxide(GO) and other carbon materials [57, 59] Although GDY has many different allotropes, they are all composed of sp and sp 2 hybrid carbon atoms. In γ-GDY, for example, the sp hybrid carbon atoms form an alkyne bond, which is very beneficial to the addition reaction with halogen. The halogenation reaction of γ-GDY was studied by Leeʹs group [60] . The study showed that the halogen atom(M) was preferentially combined with the sp hybrid carbon atom when the halogen atom was added to the GDY, with one halogen atom above the ring plane and the other halogen atom below the ring plane. According to the theoretical calculation, the halogen atoms in the halogenation reaction can only be outside the original γ-GDY plane, but not inside the plane, which is mainly because of the larger atomic radius of the halogen atoms, resulting in a larger steric resistance. Compared with the chlorination, bromination and iodization of GDY, the fluorination reaction of GDY is very different. It is found that γ-GDY can fully open the triple bond and get the form of sp 3 hybridization when the fluorination reaction occurs [21] . Li et al. [61] prepared two-dimensional At present, the prepared GDY still has defects, which limits its further development. However, the exploration of the preparation process is slow, and the functional modification (like doping) of GDY on the existing basis is one of the best ways to improving the application limitations of GDY. The effect of defects(such as vacancy defects) on materials may cause various changes in properties. Various studies on preventing vacancy defects can change the electronic properties of carbon nanomaterials, such as the electronic properties of GDY and spin technology. Kim et al. [62] revealed using density function theory The doping of heteroatoms makes it an effective method to adjust and improve the electronic structure and surface chemical activity of GDY itself through the electronegativity difference between carbon and heteroatoms. It is also considered to be a quick and effective way to preparing new graphyne derivatives. The most common heteroatoms used for doping modified graphyne allotropes include N, B, P, F, S, etc. [50] . Single atom doping: doping of a single element can not only effectively improve the catalytic performance of oxygen reduction of GDY, but also has good storage functions of electric energy and hydrogen energy. Prof. Chen et al. [63] reported B, N single-doped GDY and its catalytic effect on oxygen reduction reaction. The results showed that when pyridine was used as the probe, the Bsubstituted GDY was a good substrate for the Raman enhanced spectra, and was beneficial to the catalytic oxygen reduction reaction. The doping of B atom will introduce the hole, resulting in the local center of positive charge, and change the electronic structure of the GDY plane. The N atom in pyridine, which is negatively charged, will be attracted to these positive centers by electrostatic attraction. The strong bond between pyridine and GDY affects the mechanism of some reactions. For example, electron transport between the two contributes to Raman enhancement under incident light. In addition to the common doping of nitrogen, boron, chlorine and other elements mentioned above, the introduction of oxygen-containing functional groups into GDY can also be considered as oxygen doping from another perspective, which also has a great regulatory influence on the adsorption capacity and energy storage properties of GDY [63] [64] [65] [66] [67] [68] [69] . Multi-element doping: single atom doping can give different properties of GDY, and multiple heteroatom codoping can complement the limitation of single atom doping performance regulation, and more effectively regulate the electrical properties and optical properties of GDY [66] . Kangʹs group [70] showed that the co- Bhattacharya et al. [71] and HsGDY [72] , Ghazzal and many scientists [73] continued to study the chemical synthesis doping of GDY. Li et al. [72] also gave full play to the fact that GDY could be prepared by [74] [75] [76] [77] . Based on the structural influence of the interaction between metal atoms and GDY, many researches have analyzed the N-type doping of metal atoms in the structure of GDY based on theoretical calculation. The widely studied metal atoms doped with GDY include Au, Cu, Fe, Pt, Ni, Mn, Mo, Ru, etc. [78] [79] [80] . Transition metal doping can further improve the catalytic performance of graphite catalysts, in which Fe is the most effective metal element. Li et al. [81] used Fe atoms to incorporate with GDY, endowing the magnetite/GDY heterojunctions with highly photocatalytic performances. Sun et al. [79] revealed that GDY anchored with single 3d transition-metal atom showed different structures and properties, which opened the way for the deeper cognation of GDY and its wider application. At present, there are few applications in the field of biology. In reported in the literature [82] , the [3+2] Huisgen cycloaddition reaction catalyzed by copper(I) has been used for the preparation of side chain functional linear polymers. Therefore, it is an ideal method to modify GDY and improve its water solubility by quick click chemical synthesis using GDY endacetylene and azide compounds. https://doi.org/10.1007/s40242-021-1343-8 According to this, Li et al. [82] functionalized GDY through Huisgen cycloaddition based on click chemistry (Fig.7) . 1,2,3-Triazole was produced by Huisgen 1,3-dipole cycloaddition at the end of GDY and alkyl azide to obtain soluble GDY. Modified GDY had improved solubility and processability with remaining their intrinsic properties. The reaction could be carried out at a high yield under mild conditions without destroying the acetylene group. In addition, products with triazole bonds were extremely stable for hydrolysis, oxidation, or reduction. The modified GDY not only significantly improves its solubility and processing performance, but also maintains its inherent properties, resulting in a high-quality film. Such soluble GDYs can provide promising applications for the development of GDY-based materials. This method is suitable for introducing a wide range of chemical functional structures. Reprinted with permission from ref. [82] . Copyright 2019, Elsevier Ltd. GDY holds great promise for applications in batteries, solar cells, catalysis, and energy storage. As the increasing applications of GDY were reported, the biocompatibility of GDY attracted more attention. Zheng et al. [59] compared the physicochemical, biological and mutagenic effects of GO and GDYO. The results showed that GO was only soluble in H2O, but accumulated dynamically in 0.9% NaCl, phosphate buffered saline and cell culture medium, while GDYO could be well dissolved in various media. GO nanoparticles adhered and aggregated on the membrane of human hepatocyte cells, leading to cell stress and the production of reactive oxygen species, thus resulting in cell membrane wrinkle, methionine poisoning and apoptosis. In contrast, as shown in Fig.8 , GDYO did not adhere to the cell membrane, confirming the biocompatibility of the GDY. Table 1 summarizes the main applications of GDY in the biological field, which shows GDY has great application potential. Fig.8 Comparisons between graphene oxide and graphdiyne oxide in physiochemistrybiology and cytotoxicity [59] Reprinted with permission from ref. [59] . Copyright 2018, Amierican Chemical Society. Alkynyl groups in GDY play important roles in enhancing sensing properties (Fig.9 ). Due to the planar structure and large surface area, GDY can easily capture biomolecules and maintain the maximum quenching efficiency of dyes. GDY also exhibits excellent sensing performances due to the light absorption covering from the UV-Vis region to the IR region. In addition, GDY is used as a support material for immobilizing enzymes to target biomolecules, thereby improving the selectivity of biosensors [83, 84] . Fig.9 Characteristics of semiconductive GDY for biochemical sensing device [85] Reprinted with permission from ref. [85] . Copyright 2020, Springer Nature. Benefiting from unique structure and properties, graphenebased devices have been applied for fast and cheap DNA sequencing technology. The density functional theory calculation indicated that the interaction between GDY and 6-carboxyfluorescein(FAM) was stronger than that of other 2D nanomaterials, such as graphene [85] , confirming that the presence of acetylene in GDY facilitated the single-stranded DNA(ssDNA) adsorption, which led to a further increase of fluorescence quenching [54, 83] . In addition, GDY was used as a support material for immobilizing enzymes to target biomolecules, thereby improving the selectivity of electrochemical biosensors [84] . Based on the mechanisms, such as fluorescence resonance energy transfer(FRET) or photoinduced electron transfer(PET), dye-labelled DNA was self-assembled onto the surface of GDY-based nano-quenchers by π-π stacking and/or hydrophobic interaction. Wang et al. [54] used oxidized GDY to establish a new platform for effective fluorescence sensing of DNA and thrombin (Fig.10 ). This study extends GDY to the field of fluorescence sensing for the first time, stimulating scientistsʹ interest in 2D nanostructure fluorescence sensing. Until 2019, Chang et al. [87] reported a few layers of GDY NS (0.9 nm) as a fluorescence probe with ultra-high fluorescence quenching ability. It was found that the thinner the GDY, the stronger the fluorescence quenching effect. With the affinity difference for ssDNA and dsDNA, a method for M. Tuberculosis(Mtb) and its drug resistance genes was developed. This sensing platform can be further applied for the Mtb detection fromclinical samples with a low background and a high signal-to-noise ratio. Reprinted with permission from ref. [54] . Copyright 2016, Royal Society of Chemistry. Enzymes and proteins play important roles in electron transfer, https://doi.org/10.1007/s40242-021-1343-8 Review dioxygen binding, activation, and reduction [88] . Bisphenol [84] fabricated GDY-based tyrosinase biosensor, which showed remarkable analytical performances for BPA detection with fast response, high sensitivity, good operation repeatability and low detection limit. MicroRNA plays a key role in regulating human gene expression and widely exists in human tissues and body fluids. Therefore, ultrasensitive and feasible microRNA detection can achieve predictive diagnosis of a variety of human diseases, especially cancer [89, 90] . Photoelectrochemical(PEC) methods featuring high sensitivity and feasibility might be a potential way to detecting MicroRNAs [12, 92] . Compared with graphene, GDY has a naturally band gap of 1.12 eV, and possesses a high electron conductivity, manifesting that GDY has a promising application in PEC [93] [94] [95] . Li et al. [132] fabricated a photoactive material, which loaded GDY with AuNPs(AuNPs-GDY). AuNPs-GDY took advantage of the band-gap of GDY to produce hole-electron pairs, and the plasmon resonance effect of AuNPs to achieve a high PEC response. When the mass ratio of GDY to tetra-chloroauric acid was 1:2.5, AuNPs-GDY exhibited the best PEC performance. MicroRNA let-7a, a cancer marker, was chosen as a detection model. With a detection limit of 3.3×10 -19 mol/L and a good linearity with microRNA concentration ranging from 1.0×10 -18 mol/L to 1.0×10 -10 mol/L, this PEC biosensor provided a promising platform based on GDY to detect MicroRNA at ultralow levels for diagnoses. Small biomolecules, such as carbohydrates and hydrogen peroxide are important biomarkers and mediators in many biological processes. Compared with graphene, GDY has larger pores composed of π-conjugated acetylenic bonds, which may have strong adsorption to biomolecules. Amino acids detector: Chen et al. [91] reported the interactions between single-layer GDY/Graphene sheet and [133, 134] . Due to the high adsorption capacity and active sites on the GDY surface, Liu et al. [94] prepared a GDY based glucose detection platform with dual enzyme activity by immobilizing ferrous ions and glucose oxidase on GDY tablets, providing a new perspective for the immobilization of ions and enzymes by 2D materials (Fig.11 ). H2O2 detector: Hydrogen peroxide(H2O2) is an important mediator in many biological processes. The conventional H2O2 detection methods include electrochemical method [98, 99] , and coloring method [135] , etc. Zhuang et al. [93] reported an in situ synthesis of Prussian blue nanoparticles(PB) on GDYO. The PB/GDYO hybrid was used as an electrode with high electrochemical catalytic activity towards hydrogen peroxide. GDYO was able to anchor PB in the process of nanoparticle formation and stabilization. The PB/GDYO hybrid showed high electrochemical catalytic activity and stability for the detection of hydrogen peroxide. Transition metal atoms can often be used as adsorption centers for small molecules. The π bond on the surface of graphene is saturated and chemically inert, so the adsorption energy of metal atoms on graphene surface is less than 2 eV and the migration barrier is less than 0.8 eV [96, 97] . Lower adsorption energy and migration barrier make metal atoms aggregate on GDY at room temperature. Therefore, GDY is easier to adsorb metal atoms than graphene, making it a potential molecular detection material. Reprinted with permission from ref. [94] . Copyright 2019, Amierican Chemical Society. The unique acetylene nanostructure and strong hydrophobic skeleton make GDY a kind of new potential materials for H2O sensing. The necessary properties for humidity sensor include stable hydrophobic skeleton and extended hydrophilic Review functional groups with water absorption ability. Yan et al. [57] showed that sensor based on GDYO had ultrafast humidity response, which was better than the sensor based on GO. The Reprinted with permission from ref. [100] . Copyright 2021, Royal Society of Chemistry. Owing to the excellent extinction coefficient of GDY in the near-infrared region, GDY NSs have been explored as good imaging agents for cancer diagnose. The chemical modification of GDY using light elements is a possible route to regulate its unique structure and optoelectronic properties. Directly heating the mixture of xenon difluoride and GDY produces partially fluorinated GDY, whose covalent C-F bond is hybridized with local sp 2 carbon because the alkyne bond is broken. It can be seen that the fluorescence of GDY is The fluorescence enhancement is considered to be caused by defect states, which shows the potential properties of GDY as light-emitting devices, such as biosensors [137] . As a 0-dimensional nano material, GDY quantum dots (GDYQDs) have some unique advantages because of size effect, such as band gap generated by quantum confinement, good dispersion, richer active sites, biocompatibility, etc. Therefore, they are expected to break through the problem of GDY dispersion and dissolution and be applied to biological imaging and other fields. Experiments have proved that GDYQD has excellent light stability, can stimulate pH dependent fluorescence emission, has effective cell uptake and cell imaging ability, and will not induce detectable cytotoxic effects in vitro [138] . Zhang et al. [139] and Guo et al. [140] combined GDY with functional chromophores with high chemical stability and quantum yield, such as pyrene, and synthesized GDY-Py QDs with strong fluorescence and good dispersion through the Sonogashira cross coupling reaction. It can be used for cell imaging and effectively overcomes the shortcomings of photobleaching with traditional fluorescent organic dyes. GDYQDs are expected to be used to construct high-quality fluorescent probes for biomedical localization and cell monitoring. Because of the overuse of antibiotics in biomedicine and [101] [102] [103] , oxidative stress [103] , cell/membrane component rupture [104, 105] and inhibition of bacterial metabolism [106] . Liu et al. [111] developed an efficient nanozyme constructed by MoS2/rGO with great capturing ability, as alternative antibiotics used in antibacterial field. Liu et al. [112] have also Zhu et al. [107] studied the bacterial toxicity of GDY and GDYO, and further explored the antibacterial mechanism of GDY. The investigation of the antibacterial behaviors of GDY based nanomaterials may provide useful guidelines for the future design and application of this novel molecular allotrope of carbon [107] . Similar to the destructive effect of graphene on bacterial pathogen membrane [141, 142] , GDY can also induce the collapse of bacterial cell membrane by capturing or wrapping bacterial cell membrane. Zhu et al. [107] and abundant phospholipids in bacterial membrane [143, 144] . Theoretical simulation shows that there is a strong van der Waals and hydrophobic interaction between GDY and bacterial membrane, which can damage the bacterial by wrapping or trapping bacterial membrane. The antibacterial effect of GDY is not very strong through the chemical effect of oxidative stress [145, 146] . Therefore, GDY composite antibacterial system is usually further constructed based on a unique large π-conjugated system and a highly active hybrid structure, such as modifying TiO2 nanofibers, using alkyne bond to bind photosensitive materials, such as zinc phthalocyanine and so on. Although TiO2 has photocatalytic activity for producing ROS, the recombination of electrons and holes limits its antibacterial ability. Wang et al. [147] modified TiO2 nanofibers through GDY. The system had excellent biocompatibility and bone induction ability to induce cell adhesion and differentiation. The system not only improved the anti-drug-resistant-bacterial ability, but also helped promote the process of bone tissue regeneration. In order to understand the potential mechanism of antibacterial effect of GDY, as mentioned above, Zhu et al. [107] explored the effect of GDY on the bacterial metabolism through differential gene expression analysis. The analysis of gene expression showed that the presence of GDY would disturb multiple genes responsible for bacterial metabolism. GDY could downregulate the metabolism-related genes and also compromise the bacterial membrane. A combination of 'physical' and 'chemical' effects is accounted for the antimicrobial activity of GDY based nanomaterials. As shown in Fig.13 , when bacteria directly contacted with GDY, GDY wrapped the bacterial membrane due to physical adsorption. At the same time, GDY NS has the function of blade similar to graphene nanosheet, whose sharp edge can be inserted into bacterial membrane, resulting in the leakage of substances in cells. On the other hand, GDY may chemically induce the production of ROS, thereby interfering with specific microbial Review Fig.13 Possible antibacterial mechanisms of GDY based nanomaterials [107] Reprinted with permission from ref. [107] . Copyright 2020, John Wiley & Sons, Inc. processes. However, experiments showed that the 'physical' effect of GDY played a major role in the antibacterial process, and the 'chemical' effect of GDY had little effect on bacterial metabolism, which indicated that GDY induced oxidative stress was a secondary bactericidal factor. Unlike graphene, due to the hybridization of sp and sp 2 in carbon atoms, GDY shows a unique semiconductor electron transport structure and a larger conjugate system. This phenomenon enhances the electron transport, which is not only conducive to the adsorption of reactants and intermediates, but also conducive to the fixation of single atoms on GDY, especially after doping modification. The formation of high active centers improves the catalytic performance of metals and their oxides. Therefore, GDY is expected to show better effects in the fields of catalysis, including photocatalysis, electrocatalysis and biocatalysis. With the rapid development of nanoscience, some inorganic nanomaterials have been found to have enzyme-like catalytic activity. These nanomaterials can catalyze the substrate reaction of natural enzymes and have a catalytic mechanism similar to that of natural enzymes, so they are defined as nanozymes [108] [109] [110] . However, due to their low catalytic activity, poor bacterial capturing capacity, and complicated material design, the feasibility of nanozymes is still far from satisfactory [111, 112] . GDY with both sp 2 -and sp-hybridized carbon atoms is introduced to anchor ultrasmall nanoparticles with persistent enzyme-like activities. GDY is expected to become a nanozyme because of the high specific surface area, rich surface chemistry and easy functionalized structure. Ma et al. [148] developed the oxidized form of GDY, which could serve as a new kind of carbon nanozyme mimicking peroxidase. For the peroxidase-like activity of carbon-based nanomaterials, from the experimental and theoretical results, the ketone functional group unit was considered to be the catalytic center for H2O2 decomposition. Therefore, compared with GDY, GDYO with the oxygencontaining groups plays an important role in simulating the activity of peroxidase. Oxygen-containing groups are mainly used as active sites to absorb H2O2 and destroy O-H bonds with HO2 • and H + . In addition, the high specific surface area, unique pore structure and conjugated electronic structure of GDYO also provide rich transport channels and adsorption sites for the substrate [149] [150] [151] . Zhou et al. [113] developed a novel GDY, which can firmly The 2D semiconductor properties of GDY provide good photogenerated electron-hole transmission ability [11, 152] . With rich electron and good hole-transfer ability, GDY has great application potential in the field of photocatalysis. Using the adsorption capacity, which benefitted by its active defect sites, we can more effectively compared with traditional catalysts to prepare efficient semiconductor heterojunctions, which can be used not only for catalytic energy fields [153] [154] [155] , but also as a biological nanozyme with high catalytic activity for catalytic antibacterial and tumor treatment. The presence of high π-conjugation, numerous open cavities, and superior specific surface area make GDY an ideal delivery system for anticancer drugs. Based on these properties, GDY is used in a variety of tumor treatment strategies. For solid tumors, the abnormally rapid value-added of cancer https://doi.org/10.1007/s40242-021-1343-8 Review cells causes the core position of the tumor to become a low-oxygen environment. In the hypoxia environment, tumor cells are resistant to killing by ionizing radiation [115] . Therefore, increasing molecular oxygen content and lowering hypoxiainducing factor-1(HIF-1) levels can effectively inhibit tumor growth [115] . As shown in the Fig.14 , Liu et al. [116] Reprinted with permission from ref. [116] . Copyright 2020, Elsevier Ltd. Photothermal therapy(PTT) is an advanced type of localized tumor treatment [117] . GDY has high photothermal conversion efficiency and was recently used as a photothermal agent in PTT. Li et al. [118] reported GDY NSs with PEGylation(GDY-PEG) as photothermal-acoustic wave nano-transducers for PTT in 2017 for the first time. To improve its biocompatibility, GDY was accompanied by PEGylation. Similar to other 2D materials, GDY-PEG had a broad optical absorption from the UV to the NIR regions. GDY-PEG also showed superior photostability with high photothermal conversion efficiency of 42%, which was higher than that of other classic PTT agents [119, 120] . Because Photodynamic therapy is now well established and is used in the treatment of cancer and some superficial tumors, such as esophageal and bladder cancers [122] [123] [124] . As mentioned above, Jiang et al. [58] invented an iRGD Reprinted with permission from ref. [58] . Copyright 2019, American Chemical Society. Immunotherapy eliminates tumor cells by strengthening the bodyʹs own immune system, such as activating macrophages. https://doi.org/10.1007/s40242-021-1343-8 Researches have shown that nanomaterials themselves can promote the release of inflammatory factors and induce M1 polarization of marophages [125, 126, 156] . Guo et al. [126] 16 Schematic overview of the mechanism whereby GDYO nanosheets re-educate immunosuppressive macrophages through the intracellular corona and facilitate cancer immunotherapy [126] Reprinted with permission from ref. [126] . Copyright 2021, American Chemical Society. GDY possesses great fascination for radioprotection mainly due to its high free radical scavenging ability, unique optical properties, good chemical stability and good biocompatibility. Radiotherapy has been widely used in cancer treatment because of its advantages of noninvasive and good effect. However, because the high-energy ionizing radiation inevitably damages normal cells, it is increasingly important to take radiation protection to avoid radiation damage. Therefore, in recent years, many therapeutic systems for radiation protection have been developed by combining the design of nanomaterials with biosafety research. Many scholars have developed GDY-based radiation protective agents. For the first time, Xie et al. [127] synthesized bovine serum albumin(BSA)-modified GDY nanoparticles(GDY-BSA NPs) to study the application of GDY for the radioprotection both in cell and in animal models. The results indicated that the GDY-BSA NPs could effectively scavenge the DPPH, ABTS, O 2− · , and Ps. The research group further studied the effects of GDY-BSA NPs on gastrointestinal radiation protection [128] . The results further confirmed that GDY held the unique advantages of strong free radical scavenging ability, good chemical stability in gastric acid environment, long residence time in gastrointestinal tract and good biosafety through oral administration, which provided favorable preconditions for their application as gastrointestinal radiation protective agents. In addition to the radiation of radiotherapy, ultraviolet is also a major killer of human cells. In recent years, some scholars [129] have found that GDY had ultrafast photoelectric characteristics better than graphene, and possessed unique optical nonlinear adsorption abilities because of the unique sp hybrid structure and large π-conjugated system. In particular, GDY has excellent UV nonlinear characteristics, which can just perfectly absorb UV. Tissue engineering refers to the use of artificial biological tissue to replace human tissue, exercising the structure and function. However, even in the field of more mature skin tissue engineering, the functional repair of the nervous system is still a difficult problem. Nervous system requires synapses to respond for receiving and transmitting electric signals. GDYbased artificial synapse(GAS), invented by Wei et al. [130] in 2020, exhibited intrinsic short-term plasticity. GAS has been proposed to mimic the biological signal transmission behaviors. The relatively low diffusion barrier for ions in GDY benefited from its surface adsorption and interlayer insertion. The rapid diffusion and storage of ions profited from the existence of triangular pores in GDY structure and sp hybrid carbon. At the same time, because of the good biocompatibility, GDY could bind to pre-synaptic neurons to form a hybrid system to achieve synaptic plasticity and complete the transmission of biological signal. Based on the previous work, Wei et al. [131] connected gas with GDY based artificial muscle, completed the integration and output of artificial efferent nerve information, and opened up an interesting way for the later development and construction of GDY nerve. https://doi.org/10.1007/s40242-021-1343-8 Nucleic acids usually including DNA, mRNA, siRNA, miRNA and immunostimulatory nucleic acids, etc. are used to treat major diseases such as cancer [157] . Realizing the designed delivery of nucleic acids can refine the treatment to the gene level, but many levels of barriers in the biological environment must also be overcome in the process to deliver them to the cells. Vaughan et al. [157] summarized the development of nanomaterials for targeted nucleic acid delivery for the treatment of cancer. With the continuous development of nanotechnology, the research of nanomaterials as nucleic acid delivery vehicles is also constantly updated. GDY, with sp hybridized carbon atoms, a π-conjugate structure, a large specific surface area and an active alkyne bond, is a good type of nucleic acid delivery vector. As mentioned above, Zhou et al. [113] used GDY to uniformly anchor CeO2 nanoparticles to form GDY-CeO2, as an expedient MicroRNA vehicle (Fig.17 ), which could keep higher stability and efficiency to deliver miR181a to tumor, exhibiting low Fig.17 Multifunctional GDY-CeO 2 nanozymes facilitating microRNA delivery and attenuating tumor hypoxia for highly efficient radiotherapy of esophageal cancer [113] Reprinted with permission from ref. [113] . At present, the New Coronary Pneumonia Epidemic is hitting the world, and the field of biomedicine is committed to the research and development of COVID-19 vaccines. As one of the most effective strategies to prevent and control infectious diseases and some non-communicable diseases, especially cancer, the design and development of vaccines are particularly critical. Appropriate addition of adjuvants and carriers to vaccine preparations can not only improve the immunogenicity of the antigen, but also induce long-term immunity. Chenʹs group [158] are also working on developing vaccine adjuvants, such as manganese adjuvants, using nanobiotechnology to improve vaccine efficacy. In recent years, researchers have also made research based on carbon nanomaterials [159, 160] . Especially erewhile, researchers have tried to develop various functionalized GO vaccines and adjuvants based on the unique 2D properties of GO. Through adhesion and immune response experiments, Zhan et al. [161] fully confirmed that GO nanosheets could be used as DC Due to the hybrid structure and intrinsic hydrophobic properties of GDY, modification methods, such as oxidation and doping are still needed to develop to improve its hydrophilic, so as to increase its utilization efficiency. In recent years, because of its unique structure and excellent performance, GDY has shown great potential in biological applications. First, due to its surface adsorption Review characteristics and conjugated structure, GDY can be used as a carrier in the fields of biocatalysis, drug delivery and so on. Then, the excellent photoelectric characteristics make GDY have unique performance in the design of biosensors and fluorescent probes. Third, after oxidative modification, the distribution of oxygen-containing functional groups on its surface was also found to be closely bound to the structure of tumor immune microenvironment related protein STAT3, which promoted the research on the mechanism and application of GDY in tumor immunotherapy. In addition, benefiting by the UV nonlinear absorption characteristics and free radical scavenging ability of GDY, it can be used in the field of UV and radiation protection. GDY possesses excellent semiconductor and photoelectric properties, which can also establish contact with neural synapses in tissue engineering. In conclusion, as a new 2D carbon nanomaterial, GDY has great application potential with its unique hybrid structure and advantages. At present, the main factors limiting its application in the biological field are as follows: (1) Material intrinsic limitations: as a new 2D carbon allotrope, GDY with high crystallinity has not been perfectly prepared. Researchers have also been limited to large-scale preparation of GDY. Besides, the experimental part is difficult to catch up with theoretical calculation. At present, the intrinsic problems of materials that limit their application, especially in the biological field, can be summarized as follows. (a) The hydrophobicity of GDY leads to its poor solubility in the biological liquid environment. Therefore, most studies based on biological liquid environment are carried out by obtaining GDYO rich in hydroxyl and carboxyl groups after oxidation treatment. (b) From the perspective of material preparation, the crystallinity of existing GDY is not high, and the quality needs to be improved. The defects of GDY may interfere with the cognition of practical application and mechanism exploration. Therefore, the research results so far cannot explain the specific action mechanism and effect of GDY, which also requires more efforts. (2) Unclear Nano-Bio interaction mechanisms: nanomaterial-biological(Nano-Bio) interaction occurs at the interface between the surface of nanomaterials and surrounding biological liquids or biomolecules(such as proteins, DNA and lipids) [162] . Because of its unique hybrid structure, porous structure, large specific surface area and excellent surface adhesion, GDY can specifically interact with biological molecules in the biological environment, but its nano-bio interaction mechanisms still remain unclear enough. In 2021, Guo et al. [126] explored the intracellular protein corona(ICPC) of GDY. The researchers used the isotope 13 C to label GDYO, quantitatively analyzed the interaction ratio between GDYO and intracellular proteins. At the same time, the distribution and metabolism of GDYO in peritoneal macrophages and tumors after administration were analyzed, which provided a direct, accurate and quantitative analysis method for the assessment of the biological behavior of carbon nanomaterials in vivo. Several typical studies based on theoretical simulation explored the functional mechanisms of the interaction between GDY and biological macromolecules in vivo. Through largescale all atom molecular dynamics simulation, Luan et al. [142] revealed that GDY could indeed destroy the potential toxicity mechanism of protein-protein interaction(PPI) by cutting proteins, which provided a strong basis for reducing the toxic effect of GDY in biological cells. Zhang et al. [163] The review of the physical properties, modification and the relative applications should be valuable to further studies. Synthesis decides the destiny. We believe that GDY and its derivatives have greater application potential in many fields, especially in the biological systems. MRS Online Proceedings Library Advanced Functional Materials Superlattices and Microstructures Proceedings of the National Academy of Sciences of the united states of America Proceedings of the National Academy of Sciences of the United States of America Advanced Healthcare Materials Proceedings of the National Academy of Sciences Handbook of Carbon-Based Nanomaterials The authors declare no conflicts of interest.