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Membrane protein engineering exhibits great potential for cell functionalization. Although genetic strategies are sophisticated for membrane protein engineering, there still exist some issues, including transgene insertional mutagenesis, laborious, complicated procedures, and low tunability. Herein, we report a DNA-templated anchoring of exogenous proteins on living cell membranes to realize programmable functionalization of living cells. Using DNA as a scaffold, the model cell membranes are readily modified with proteins, on which the density and ratio of proteins as well as their interactions can be precisely controlled through predictable DNA hybridization. Then, the natural killer (NK) cells were engineered to gain the ability to eliminate the immune checkpoint signaling at the NK-tumor synapse, which remarkably promoted NK cell activation in immunotherapy. Given the versatile functions of exogenous proteins and flexible designs of programmable DNA, this method has the potential to facilitate membrane-protein-based cell engineering and therapy.

Genetically encoded fluorescent proteins have been used for metal ion detections by combining fluorescent proteins with metal-binding proteins or peptides. However, their applications are largely restricted to a limited number of metal ions, such as Ca(2+) and Zn(2+), due to the lack of available metal-binding proteins or peptides that can be fused to fluorescent proteins and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. To overcome these limitations, we report herein the use of Mg(2+)-specific 10-23 or Zn(2+)-specific 8-17 RNA-cleaving DNAzymes to regulate the expression of fluorescent proteins as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green fluorescent protein, by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red fluorescent protein, is not affected. The Mg(2+) or Zn(2+) in Hela cells can be detected using both fluorescent confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes, such as for Mg(2+), Na(+), Cu(2+), Zn(2+), Pb(2+), Hg(2+), Ag(+), and UO(2)(2+), can be obtained through in vitro selection, and the resulting DNAzymes often share a similar secondary structure and reaction mechanism, the method described in this work can likely be applied to imaging many other metal ions and thus significantly expand beyond the range of the current genetically-encoded fluorescent proteins, allowing this class of sensors to be even more powerful in providing deeper understanding of the roles of metal ions in biology.

Nonspecific adhesivity of nanoparticles to cells is regarded as a significant issue of nanomedicine, which brings about many serious drawbacks in applications, including low detection sensitivity, non-targeted biotoxicity and poor diagnostic accuracy. Here, we propose for the first time, DNA-decorated semiconductor polymer nanoparticles (SPN-DNAs), whose adhesivity can be significantly alleviated by controlling the density and thickness of DNA layers. This property is demonstrated to be independent of external conditions such as temperature, concentration, incubation time, ionic strength and cell lines. The mechanism of this phenomenon is also discussed. Finally, based on minimized nonspecific adhesivity to cells, a triggered nanoswitch can be constructed to control cellular internalization and drug delivery.

Filamentous fungi are abundant resources of bioactive natural products. Here, 151 marine-derived fungi were collected from the north Yellow Sea and identified by an internal transcribed spacer (ITS) sequence. The crude extracts of all strains were evaluated for their antimicrobial activities and analyzed by HPLC fingerprint. Based on these, strain

Artificial enzyme cascade systems with confinement effect are highly important in synthetic biology and biomedicine. Herein, a framework nucleic acid-based confined enzyme cascade (FNA-CEC) for synergistic cancer therapy in vivo was developed. The FNA-CEC consisted of glucose oxidase and horseradish peroxidase precisely assembled on an addressable DNA tetrahedron scaffold within few nanometers. Glucose oxidase (GOx) can trigger efficient glucose depletion for tumor starvation therapy, and increase the local concentration of H2O2 in situ for enhanced downstream horseradish peroxidase (HRP)-activated prodrug therapy. Due to the spatial-confinement on DNA tetrahedron scaffold, the efficiency of intermediate metabolites transportation between the enzyme cascades was improved. Moreover, FNA-CEC was applied for efficient synergistic cancer therapy in vitro and in vivo . As a simple and efficient approach, the FNA-CEC is expected to expand the toolbox of technologies in synthetic biology and biomedicine.

Natural living systems are driven by delicate protein networks whose functions are precisely controlled by many parameters, such as number, distance, orientation, and position. Focusing on regulation rather than just imitation, the construction of artificial protein networks is important in many research areas, including biomedicine, synthetic biology and chemical biology. DNA origami, sophisticated nanostructures with rational design, can offer predictable, programmable, and addressable scaffolds for protein assembly with nanometer precision. Recently, many interdisciplinary efforts have achieved the precise construction of DNA origami-based protein networks, and their emerging application in many areas. To inspire more fantastic research and applications, herein we highlight the applicability and potentiality of DNA origami-based protein networks. After a brief introduction to the development and features of DNA origami, some important factors for the precise construction of DNA origami-based protein networks are discussed, including protein-DNA conjugation methods, networks with different patterns and the controllable parameters in the networks. The discussion then focuses on the emerging application of DNA origami-based protein networks in several areas, including enzymatic reaction regulation, sensing, bionics, biophysics, and biomedicine. Finally, current challenges and opportunities in this research field are discussed.

The quality and application of super-resolution fluorescence imaging greatly lie in the dyes' properties, including photostability, brightness, and Stokes shift. Here we report a synergistic strategy to simultaneously improve such properties of regular fluorophores. Introduction of quinoxaline motif with fine-tuned electron density to conventional rhodamines generates new dyes with vibration structure and inhibited twisted-intramolecular-charge-transfer (TICT) formation synchronously, thus increasing the brightness and photostability while enlarging Stokes shift. The new fluorophore YL578 exhibits around twofold greater brightness and Stokes shift than its parental fluorophore, Rhodamine B. Importantly, in Stimulated Emission Depletion (STED) microscopy, YL578 derived probe possesses a superior photostability and thus renders threefold more frames than carbopyronine based probes (CPY-Halo and 580CP-Halo), known as photostable fluorophores for STED imaging. Furthermore, the strategy is well generalized to offer a new class of bright and photostable fluorescent probes with long Stokes shift (up to 136 nm) for bioimaging and biosensing., Super-resolution microscopy is a powerful tool for cellular studies but requires bright and stable fluorescent probes. Here, the authors report on a strategy to introduce quinoxaline motifs to conventional probes to make them brighter, more photostable, larger Stokes shift, and demonstrate the probes for biosensing applications.

Plasma membranes are the fundamental mediators through which cells communicate with their surrounding environment. The techniques to monitor or synthetically manipulate the cell membranes are attractive tools to engineer the functions of cells as well as their local microenvironment. Current advances of biomolecular science enable the insertion of functional compounds onto cell-surface via external integration or genetic engineering to manipulate cell membrane function. Recently, the DNA nanotechnology made it possible to use synthetic DNA as an emerging and promising molecular toolkit for anchoring and exploring cell-surface. In this review, the latest advances of DNA nanotechnology on cell-surface are summarized. We first give an overview of commonly used strategies for installing DNA nanodevices onto cell-surface including amphiphilic interaction, covalent modification, and affinity labeling. Then the biological applications of DNA nanodevices on cell membranes are reviewed. By integrating functional nucleic acids as recognition elements, DNA sensors are fabricated to monitor the cellular microenvironment and membrane activities. In addition, the programmable behaviors of DNA on cell-surface are also discussed, which include biomimicry and the regulation of membrane functions. Finally, we analyze the current challenges in the development of DNA nanotechnology on cell-surface as well as their prospects in bioimaging and cancer therapy.

Ligand-functionalized plasmonic nanoparticles have been widely used for targeted imaging in living systems. However, ligand presentation and encoding on the nanoparticle's surface in a stoichiometrically controllable manner remains a great challenge. Herein, we propose a method to construct ligand-engineered plasmonic nanoprobes by using nanoparticle encapsulation with topological DNA tetrahedrons, which enables the programmed ligand loading for precise regulation of targeting efficiency of nanoprobes in biorelated applications. With this method, we demonstrated the preparation of functionalized plasmonic nanoprobes by programmed loading of RGD peptides and aptamers onto the DNA tetrahedron encapsulated gold nanoparticles with controllable stoichiometric ratios. The cell imaging and particle counting assays suggested that the targeting efficiency of the nanoprobes could be readily modulated by tailoring the number and stoichiometric ratios of the loaded ligands, respectively. It can be anticipated that this robust strategy could provide new opportunities for the construction of efficacious nanoprobes and delivery systems for versatile bioapplications.

The quality and application of super-resolution fluorescence imaging greatly lie in the properties of fluorescent probes. However, conventional fluorophores in a cellular environment often suffer from low brightness, poor photostability, and short Stokes shift (BDQF-6 exhibits around twofold greater brightness (ε × Φ = 6.6 × 104 L·mol− 1·cm− 1) and Stokes shift (56 nm) than its parental fluorophore, Rhodamine B. Importantly, in Stimulated Emission Depletion (STED) microscopy, BDQF-6 derived probe possesses a superior photostability and thus renders threefold more frames than carbopyronine- and JF608-based probes, known as photostable fluorophores for STED imaging. More BDQF-6 derivatives were developed next, allowing us to perform wash-free organelles (mitochondria and lysosome) staining and protein labeling with ultrahigh signal-to-noise ratios (up to 106 folds) in confocal and STED microscopy of live cells, or two-photon and 3D STED microscopy of fixed cells. Furthermore, the strategy was well generalized to different types of dyes (pyronin, rhodol, coumarin, and Boranil), offering a new class of bright and photostable fluorescent probes with long Stokes shift (up to 136 nm) for bioimaging and biosensing.

The enzymatic-assisted signal amplification of DNA sensors is rarely applied in living cells due to the difficulties in protein delivery. In this study, we have proposed a biomineralization-based DNA nanoprobe to transport nucleases and DNA sensors for enzyme-assisted imaging of microRNA in living cells.

Monitoring the distribution and concentration fluctuation of cellular compositions at the molecular level is of great significance in biological studies and medical diagnostics. Towards this goal, lots of fluorescent probes for cellular imaging have been developed based on different kinds of materials. Among them, newly emerged DNA nanostructures possess the advantages of precisely programmable interactions and predictable thermodynamics, which provide powerful toolkits for the design of fluorescent probes. In this review, the recent advances of DNA nanostructure-based fluorescent probes for cellular imaging are summarized. The preparation and properties of DNA nanostructures were first introduced. Then we focused on the applications of DNA nanostructures for fluorescence biosensing of various kinds of cellular targets, including nucleic acids, proteins, small molecules and physical features. Finally, the current challenges and future directions in the research field are discussed.

Spherical nucleic acids (SNAs) play critical roles in many fields, such as molecular diagnostics, disease therapeutics, and materials application. Due to the important role of DNA density on the properties of SNAs, the controlled synthesis of monodisperse SNAs with precise DNA density is an important approach for the structure-function relationship study and finite functions regulation of SNAs. In particular, the construction of monodisperse SNAs in a valency-tunable and site-specific manner is highly important; however, it is still challenging. Herein, on the basis of the high controllability, nanometer precision, and addressable modification ability of framework nucleic acid (FNA), we develop the concept of valency-controlled framework nucleic acid core-based molecular spherical nucleic acids (FNA-mSNAs) with tunable biosensing performances. The FNA-mSNAs consist of a valency-tunable FNA-based DNA nanocube as the core and a controlled, precise number of DNA strands per core. By simply alternating the binding site number for shell DNA strands on the DNA nanocube, homogeneous FNA-mSNAs with different valencies were easily designed, which enabled the molecular level study of the effect of valency on their properties, such as nuclease stability and cellular uptake. Furthermore, taking advantage of the addressable modification ability of FNA, the first heterogeneous molecular SNAs with tunable valency were demonstrated. Importantly, the valency of heterogeneous FNA-mSNAs was able to tune their biosensing performance, such as response dynamics, detection sensitivity, and response range. With these remarkable features, FNA-mSNAs provide new research methods for the development of functional SNAs at the molecular level for a wide range of biological applications.

Many different metal ions are involved in various biological functions including metallomics and trafficking, and yet there are currently effective sensors for only a few metal ions, despite the first report of metal sensors for calcium more than 40 years ago. To expand upon the number of metal ions that can be probed in biological systems, we and other laboratories employ the in vitro selection method to obtain metal-specific DNAzymes with high specificity for a metal ion and then convert these DNAzymes into fluorescent sensors for these metal ions using a catalytic beacon approach. In this Forum Article, we summarize recent progress made in developing these DNAzyme sensors to probe metal ions in living cells and in vivo, including several challenges that we were able to overcome for this application, such as DNAzyme delivery, spatiotemporal control, and signal amplification. Furthermore, we have identified a key remaining challenge for the quantitative detection of metal ions in living cells and present a new design and the results of a Forster resonance energy transfer (FRET)-based DNAzyme sensor for the ratiometric quantification of Zn2+ in HeLa cells. By converting existing DNAzyme sensors into a ratiometric readout without compromising the fundamental catalytic function of the DNAzymes, this FRET-based ratiometric DNAzyme design can readily be applied to other DNAzyme sensors as a major advance in the field to develop much more quantitative metal-ion probes for biological systems.

Filamentous fungi are abundant resources of bioactive natural products. Here, 151 marine-derived fungi were collected from the north Yellow Sea and identified by an internal transcribed spacer (ITS) sequence. The crude extracts of all strains were evaluated for their antimicrobial activities and analyzed by HPLC fingerprint. Based on these, strain Penicillium oxalicum MEFC104 was selected for further investigation. Two new polyketide–amino acid hybrid compounds with feature structures of tetramic acid, oxopyrrolidine A and B, were isolated. Their planner structures were assigned by HRESIMS and 1D/2D NMR experiments. The absolute configurations were determined by modified Mosher’s method, J-based configuration analysis, and ECD calculations. Furthermore, the biosynthetic pathway was identified by bioinformatic analysis and gene-deletion experiments. This study established a link between oxopyrrolidines and the corresponding biosynthesis genes in P. oxalicum.

Cell membrane-targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl-4H-chromen-4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.

Aptamer-based sensors have emerged as a major platform for detecting small-molecular targets, because aptamers can be selected to bind these small molecules with higher affinity and selectivity than other receptors such as antibodies. However, portable, accurate, sensitive, and affordable detection of these targets remains a challenge. In this work, we developed an aptasensing platform incorporating magnetic beads and a DNAzyme for signal amplification, resulting in high sensitivity. The biosensing platform was constructed by conjugating a biotin-labeled aptamer probe of small-molecular targets such as toxins and a biotin-labeled substrate strand on magnetic beads, and the DNAzyme strand hybridized with the aptamer probe to block the substrate cleavage activity. The specific binding of the small-molecular target by the aptamer probe can replace the DNAzyme strand and then induce the hybridization between the DNAzyme strand and substrate strand, and the iterative signal amplification reaction of hydrolysis and cleavage of the substrate chain occurs in the presence of a metal ion cofactor. Using invertase to label the substrate strand, the detection of small molecules of the toxin is successfully transformed into the measurement of glucose, and the sensitive analysis of small molecules such as toxins can be realized by using the household portable glucose meter as a readout. This platform is shown to detect ochratoxin, a common toxin in food, with a linear detection range of 5 orders of magnitude, a low detection limit of 0.88 pg/mL, and good selectivity. The platform is easy to operate and can be used as a potential choice for quantitative analysis of small molecules, at home or under point-of-care settings. Moreover, by changing and designing the aptamer probe and the arm of DNAzyme strand, it can be used for the analysis of other analytes.

A series of physiological barriers have impeded nanoparticle-based drug formulations (NDFs) from reaching their targeted sites and achieving therapeutic outcomes. In this study, we develop size-controllable stealth doxorubicin-loaded nanodrug coated with CD47 peptides (DOX/sNDF-CD47) based on supramolecular chemistry to overcome multiple biological barriers. The smart DOX/sNDF-CD47 can efficiently decrease sequestration by macrophages and disassemble into poly(amidoamine) dendrimers with nuclear localization sequences (DOX/PAMAM-NLS) in the presence of matrix metalloproteinase-2 (MMP-2). Such structure transformation endows DOX/sNDF-CD47 with the ability of deep penetration in multicellular tumor spheroid, lysosomal escape, and nucleus localization, resulting in excellent cytotoxicity and drug resistance combating. In vivo experiments further confirmed that DOX/sNDF-CD47 has good tumor-targeting ability and can significantly improve therapeutic efficacy of DOX on xenograft tumor model. The ability to overcome multiple biological barriers makes sNDF-CD47 a promising NDFs to treat cancer expressing MMP-2 and combating drug resistance.

Precision diagnosis and effective treatment are the cores of early cancer therapy. Here, for the first time, we report a hybridization chain reaction-based nanoprobe for selective and sensitive cancer cell recognition and amplified photodynamic therapy.

Spatial and temporal distributions of metal ions in vitro and in vivo are crucial in our understanding of the roles of metal ions in biological systems, and yet there is a very limited number of methods to probe metal ions with high space and time resolution, especially in vivo. To overcome this limitation, we report a Zn(2+)-specific near infrared (NIR) DNAzyme nanoprobe for real-time metal ion tracking with spatiotemporal control in early embryos and larvae of zebrafish. By conjugating photocaged DNAzymes onto lanthanide-doped upconversion nanoparticles (UCNPs), we have achieved upconversion of a deep tissue penetrating NIR 980 nm light into 365 nm emission. The UV photon then efficiently photo-decages a substrate strand containing a nitrobenzyl group at the 2’-OH of adenosine ribonucleotide, allowing enzymatic cleavage by a complementary DNA strand containing a Zn(2+)-selective DNAzyme. The product containing a visible FAM fluorophore that is initially quenched by BHQ1 and Dabcyl quenchers, is released after cleavage, resulting in higher fluorescent signals. The DNAzyme-UCNP probe enables Zn(2+) sensing by exciting in the NIR biological imaging window in both living cells and zebrafish embryos, and detecting in the visible region. This report introduces a platform that can be used to understand the Zn(2+) distribution with spatiotemporal control, thereby giving insights into the dynamical Zn(2+) ion distribution in intracellular and in vivo models.

The monitoring of K+ is very important and emergency because of their unique relationship in various disease diagnosis and treatment. G-quadruplex analogue is a classical recognition unit for K+ detection and has been widely applied in K+ relevant research. Common fluorescent dyes were employed for design of G-quadruplex structure-based K+ probes which suffered from the aggregation-caused quenching effect, and possibly limited the biological applications in living systems. Herein, we report an aggregation-induced emission (AIE) effect-based fluorescent probe for cellular K+ analysis and imaging. Benefitting from the K+ triggered AIE phenomenon, the designed TPE derivative modified guanine (G)-rich oligonucleotide fluorescent probe (TPE-oligonucleotide probe) exhibits high sensitivity (∼10-fold higher than most reported G-quadruplex-based probes) with extended photostability which facilitates the prolonged fluorescence observations of K+ in living cells. On the basis of these advantages, the TPE-oligonucleotide probe serves as a promising candidate for the functional study and analysis of K+.

DNA nanostructures hold great promise for various applications due to their remarkable properties, including programmable assembly, nanometric positional precision, and dynamic structural control. The past few decades have seen the development of various kinds of DNA nanostructures that can be employed as useful tools in fields such as chemistry, materials, biology, and medicine. Aptamers are short single-stranded nucleic acids that bind to specific targets with excellent selectivity and high affinity and play critical roles in molecular recognition. Recently, many attempts have been made to integrate aptamers with DNA nanostructures for a range of biological applications. This review starts with an introduction to the features of aptamer-functionalized DNA nanostructures. The discussion then focuses on recent progress (particularly during the last five years) in the applications of these nanostructures in areas such as biosensing, bioimaging, cancer therapy, and biophysics. Finally, challenges involved in the practical application of aptamer-functionalized DNA nanostructures are discussed, and perspectives on future directions for research into and applications of aptamer-functionalized DNA nanostructures are provided.

RNA-cleaving DNAzymes have been demonstrated as a promising platform for sensing metal ions. However, the poor biological imaging performance of RNA-cleaving DNAzyme-based fluorescent probes has limited their intracellular applications. Compared with traditional one-photon fluorescence imaging, two-photon (TP) fluorescent probes have shown advantages such as increased penetration depth, lower tissue autofluorescence, and reduced photodamage. Herein, for the first time, we developed an RNA-cleaving DNAzyme-based TP imaging probe (TP-8–17ES–AuNP) for Zn2+ detection in living cells by modifying a Zn2+-specific DNAzyme (8–17) with a TP fluorophore (TP-8–17ES) and using gold nanoparticles (AuNPs) for intracellular delivery. The modified TP-8–17ES exhibits good two-photon properties and excellent photostability. For the TP-8–17ES–AuNP, in the absence of Zn2+, the TP fluorophore is quenched by both AuNPs and the molecular quencher. Only in the presence of Zn2+ does the DNAzyme cleave the TP fluorophore-labeled s...

A two photon-excited fluorescent supramolecular nanoplatform is first designed for quantitative analysis with host molecules, sensing probes and an internal reference., Fluorescence quantitative analyses for vital biomolecules are in great demand in biomedical science owing to their unique detection advantages with rapid, sensitive, non-damaging and specific identification. However, available fluorescence strategies for quantitative detection are usually hard to design and achieve. Inspired by supramolecular chemistry, a two-photon-excited fluorescent supramolecular nanoplatform (TPSNP) was designed for quantitative analysis with three parts: host molecules (β-CD polymers), a guest fluorophore of sensing probes (Np–Ad) and a guest internal reference (NpRh–Ad). In this strategy, the TPSNP possesses the merits of (i) improved water-solubility and biocompatibility; (ii) increased tissue penetration depth for bioimaging by two-photon excitation; (iii) quantitative and tunable assembly of functional guest molecules to obtain optimized detection conditions; (iv) a common approach to avoid the limitation of complicated design by adjustment of sensing probes; and (v) accurate quantitative analysis by virtue of reference molecules. As a proof-of-concept, we utilized the two-photon fluorescent probe NHS–Ad-based TPSNP-1 to realize accurate quantitative analysis of hydrogen sulfide (H2S), with high sensitivity and good selectivity in live cells, deep tissues and ex vivo-dissected organs, suggesting that the TPSNP is an ideal quantitative indicator for clinical samples. What’s more, TPSNP will pave the way for designing and preparing advanced supramolecular sensors for biosensing and biomedicine.

Cell-surface fluorescent probes are effective tools in cell biology and engineering. Here, we for the first time report a diacyllipid–aptamer conjugate-based fluorescent probe which could anchor on cell membrane for real-time tracking of potassium ions in the cell microenvironment.

Two-dimensional graphitic carbon nitride nanosheets (CNNSs) with planar graphene-like structure have stimulated increasingly research interest in recent years due to their unique physicochemical properties. CNNSs possess superior stability, high fluorescence quantum yield, low-toxicity, excellent biocompatibility, unique electroluminescent and photoelectrochemical properties, which make them appropriate candidates for biosensing. In this review, we first introduce the preparation and unique properties of CNNSs, with emphasis on their superior properties for biosensing. Then, recent advances of CNNSs in photoelectrochemical biosensing, electrochemiluminescence biosensing and fluorescence biosensing are highlighted. An additional attention is paid to the marriage of CNNSs and nucleic acids, which exhibits great potentials in both biosensing and intracellular imaging. Finally, current challenges and opportunities of this 2D material are outlined. Inspired by the unique properties of CNNSs and their advantages in biological applications, we expect that more attention will be drawn to this promising 2D material and extensive applications can be found in bioanalysis and diseases diagnosis.

A FRET-based two-photon fluorescent probe, , which exhibited a fast and high selective ratiometric response to nitroxyl, was first proposed. was successfully applied to two-photon dual-channel imaging of nitroxyl in living cells and tissues with less cross-talk between channels and satisfactory deep-tissue imaging depth.