Programming the morphology of DNA origami crystals by magnesium ion strength.

Harnessing the programmable nature of DNA origami for controlling structural features in crystalline materials affords opportunities to bring crystal engineering to a remarkable level. However, the challenge of crystallizing a single type of DNA origami unit into varied structural outcomes remains, given the requirement for specific DNA designs for each targeted structure. Here, we show that crystals with distinct equilibrium phases and shapes can be realized using a single DNA origami morphology with an allosteric factor to modulate the binding coordination. As a result, origami crystals undergo phase transitions from a simple cubic lattice to a simple hexagonal (SH) lattice and eventually to a face-centered cubic (FCC) lattice. After selectively removing internal nanoparticles from DNA origami building blocks, the body-centered tetragonal and chalcopyrite lattice are derived from the SH and FCC lattices, respectively, revealing another phase transition involving crystal system conversions. The rich phase space was realized through the de novo synthesis of crystals under varying solution environments, followed by the individual characterizations of the resulting products. Such phase transitions can lead to associated transitions in the shape of the resulting products. Hexagonal prism crystals, crystals characterized by triangular facets, and twinned crystals are observed to form from SH and FCC systems, which have not previously been experimentally realized by DNA origami crystallization. These findings open a promising pathway toward accessing a rich phase space with a single type of building block and wielding other instructions as tools to develop crystalline materials with tunable properties.

Domain-Targeted Membrane Partitioning of Specific Proteins with DNA Nanodevices.

The cell membrane exhibits a remarkable complexity of lipids and proteins that dynamically segregate into distinct domains to coordinate various cellular functions. The ability to manipulate the partitioning of specific membrane proteins without involving genetic modification is essential for decoding various cellular processes but highly challenging. In this work, by conjugating cholesterols or tocopherols at the three bottom vertices of the DNA tetrahedron, we develop two sets of nanodevices for the selective targeting of lipid-order (Lo) and lipid-disorder (Ld) domains on the live cell membrane. By incorporation of protein-recognition ligands, such as aptamers or antibodies, through toehold-mediated strand displacement, these DNA nanodevices enable dynamic translocation of target proteins between these two domains. We first used PTK7 as a protein model and demonstrated, for the first time, that the accumulation of PTK7 to the Lo domains could promote tumor cell migration, while sequestering it in the Ld domains would inhibit the movement of the cells. Next, based on their modular nature, these DNA nanodevices were extended to regulate the process of T cell activation through manipulating the translocation of CD45 between the Lo and the Ld domains. Thus, our work is expected to provide deep insight into the study of membrane structure and molecular interactions within diverse cell signaling processes.

The Endocannabinoid System in Alzheimer's Disease: A Network Meta-Analysis.

The findings concerning the association between endocannabinoid system (ECS) and Alzheimer's disease (AD) exhibited inconsistencies when examining the expression levels of endocannabinoids. This study aimed to provide a comprehensive summary of the studies regarding alterations of the ECS in AD. Six databases were thoroughly searched for literature to select relevant studies investigating the ECS in AD, including changes in cannabinoid receptors (CB1R and CB2R), endocannabinoids (2-AG and AEA), and their associated enzymes (FAAH and MAGL). Traditional meta-analysis evaluated the expression levels of the ECS in AD, and the results showed no significant differences in ECS components between healthy controls and AD patients. However, subgroup analysis revealed significantly lower expression levels of CB1R in AD than in controls, particularly in studies using western blot (SMD = -0.88, p < 0.01) and in studies testing CB1R of frontal cortex (SMD = -1.09, p < 0.01). For studies using HPLC, the subgroup analysis indicated significantly higher 2-AG levels in AD than in controls (SMD = 0.46, p = 0.02). Network meta-analysis examined the rank of ECS alterations in AD compared to controls, and the findings revealed that 2-AG and MAGL exhibited the largest increase and CB1R showed the largest decrease relative to the control group. Based on the findings of traditional meta-analysis and network meta-analysis, we proposed that AD patients may present decreased expression levels of CB1R and increased expression levels of 2-AG and its degrading enzyme MAGL. Our results may contribute to the growing body of research supporting the therapeutic potential of ECS modulation in the management of AD.

HUH Endonuclease: A Sequence-specific Fusion Protein Tag for Precise DNA-Protein Conjugation.

Synthetic DNA-protein conjugates have found widespread applications in diagnostics and therapeutics, prompting a growing interest in developing chemical biology methodologies for the precise and site-specific preparation of covalent DNA-protein conjugates. In this review article, we concentrate on techniques to achieve precise control over the structural and site-specific aspects of DNA-protein conjugates. We summarize conventional methods involving unnatural amino acids and self-labeling proteins, accompanied by a discussion of their potential limitations. Our primary focus is on introducing HUH endonuclease as a novel generation of fusion protein tags for DNA-protein conjugate preparation. The detailed conjugation mechanisms and structures of representative endonucleases are surveyed, showcasing their advantages as fusion protein tag in sequence selectivity, biological orthogonality, and no requirement for DNA modification. Additionally, we present the burgeoning applications of HUH-tag-based DNA-protein conjugates in protein assembly, biosensing, and gene editing. Furthermore, we delve into the future research directions of the HUH-tag, highlighting its significant potential for applications in the biomedical and DNA nanotechnology fields.

Controllable assembly of synthetic constructs with programmable ternary DNA interaction.

Compared with the dual binding components in a binary interaction, the third component of a ternary interaction often serves as modulator or regulator in biochemical processes. Here, we presented a programmable ternary interaction strategy based on the natural DNA triplex structure. With the DNA triplex-based ternary interaction, we have successfully demonstrated controllable hierarchical assemblies from nanometer scale synthetic DNA nanostructure units to micrometer scale live bacteria. A selective signaling system responsive to orthogonal nucleic acid signals via ternary interaction was also demonstrated. This assembly method could further enrich the diversified design schemes of DNA nanotechnology.

Microvascular Perfusion Imaging in Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of dementia worldwide and significantly impacts the essential functions of daily life and social activities. Research on AD has found that its pathogenesis is related to the extracellular accumulation of amyloid-beta (Aß) plaques and intracellular neurofibrillary tangles in the cortical and limbic areas of the human brain, as well as cerebrovascular factors. The detection of Aß or tau can be performed using various probes and methodologies. However, these modalities are expensive to implement and often require invasive procedures, limiting accessibility on a large scale. While magnetic resonance imaging (MRI) and computed tomography (CT) are generally used for morphological and structural brain imaging, they show wide variability in their accuracy for the clinical diagnosis of AD. Several novel imaging modalities have emerged as alternatives that can accurately and vividly display the changes in blood flow and metabolism in each brain area and enable physicians and researchers to gain insights into the generation and progression of the cerebro-microvascular pathologies of AD. In this review, we summarize the current knowledge on microvascular perfusion imaging modalities and their application in AD, including MRI (dynamic susceptibility contrast-MRI, arterial spin labeling-MRI), CT (cerebral CT perfusion imaging), emission computed tomography (positron emission tomography (PET), single-photon emission computed tomography (SPECT)), transcranial doppler ultrasonography (TCD), and retinal microvascular imaging (optical coherence tomography imaging, computer-assisted methods for evaluating retinal vasculature).

Layer-Controllable "2.5D" DNA Origami Crystals Synthesized by a Hierarchical Assembly Strategy.

The finite periodic arrangement of functional nanomaterials on the two-dimensional scale enables the integration and enhancement of individual properties, making them an important research topic in the field of tuneable nanodevices. Although layer-controllable lattices such as graphene have been successfully synthesized, achieving similar control over colloidal nanoparticles remains a challenge. DNA origami technology has achieved remarkable breakthroughs in programmed nanoparticle assembly. Based on this technology, we proposed a hierarchical assembly strategy to construct a universal DNA origami platform with customized layer properties, which we called 2.5-dimensional (2.5D) DNA origami crystals. Methodologically, this strategy divides the assembly procedure into two steps: 1) array synthesis, and 2) lattice synthesis, which means that the layer properties, including layer number, interlayer distance, and surface morphology, can be flexibly customized based on the independent designs in each step. In practice, these synthesized 2.5D crystals not only pioneer the expansion of the DNA origami crystal library to a wider range of dimensions, but also highlight the technological potential for templating 2.5D colloidal nanomaterial lattices.

Mechanochemical "Cage-on-MOF" Strategy for Enhancing Gas Adsorption and Separation through Aperture Matching.

Post-modification of porous materials with molecular modulators has emerged as a well-established strategy for improving gas adsorption and separation. However, a notable challenge lies in maintaining porosity and the limited applicability of the current method. In this study, we employed the mechanochemical "Cage-on-MOF" strategy, utilizing porous coordination cages (PCCs) with intrinsic pores and apertures as surface modulators to improve the gas adsorption and separation properties of the parent MOFs. We demonstrated the fast and facile preparation of 28 distinct MOF@PCC composites by combining 7 MOFs with 4 PCCs with varying aperture sizes and exposed functional groups through a mechanochemical reaction in 5 mins. Only the combinations of PCCs and MOFs with closely matched aperture sizes exhibited enhanced gas adsorption and separation performance. Specifically, MOF-808@PCC-4 exhibited a significantly increased C2H2 uptake (+64%) and a longer CO2/C2H2 separation retention time (+40%). MIL-101@PCC-4 achieved a substantial C2H2 adsorption capacity of 6.11 mmol/g. This work not only highlights the broad applicability of the mechanochemical "Cage-on-MOF" strategy for the functionalization of a wide range of MOFs but also establishes potential design principles for the development of hybrid porous materials with enhanced gas adsorption and separation capabilities, along with promising applications in catalysis and intracellular delivery.

A Membrane-Embedded Macromolecular Catalyst with Substrate Selectivity in Live Cells.

Substrate selectivity is one of the most attractive features of natural enzymes from their "bind-to-catalyze" working flow and is thus a goal for the development of synthetic enzyme mimics that mediate abiotic transformations. However, despite the recent success in the preparation of substrate-selective enzyme mimics based on single-chain nanoparticles, examples extending such selectivity into living systems have been absent. In this article, we report the in cellulo substrate selectivity of an enzyme-mimicking macromolecular catalyst based on a cationic dense-shell nanoparticle (DSNP) scaffold. With a systematic study on DSNP's structure-activity relationship, we demonstrate that the DSNP has excellent membrane affinity that is governed by several contributing factors, namely, charge density, type of charge, and particle size, and the best-performing phosphonium-rich DSNP can be used as a membrane-embedded catalyst (MEC) for efficient on-membrane synthesis. Importantly, the DSNP catalyst retains its selectivity toward lipophilic and anionic substrates when working as an MEC for on-membrane ligation. The usefulness of such substrate selectivity and on-membrane catalysis strategy was exemplified with several molecules of interest with low cell permeability and anionic nature, which were successfully transported into eukaryotic cells by after their formation directly on the cell membrane.

Chemical Biology Approaches toward Precise Structure Control of IgG-Based Antibody-Oligonucleotide Conjugates.

Antibody-oligonucleotide conjugates (AOCs) are important tools for drug development and biochemical analysis. However, the structural heterogeneity of AOCs synthesized through conventional coupling methods raises reproducibility and safety concerns in clinical trials. To address these issues, different covalent coupling approaches have been developed to synthesize AOCs with precise site-specificity and degree of conjugation. This Concept article categorizes these approaches as linker-free or linker-mediated and provides details on their chemistry and potential applications. Several factors, including site-specificity, conjugation control, accessibility, stability, and efficiency, are highlighted when evaluating the pros and cons of these approaches. The article also discusses the future of AOCs, including the development of better conjugation approaches to ensure stimuli-responsive release and the application of high-throughput methods to facilitate their development.

Hybrid Model Teaching in the Postepidemic Period: From Nucleic Acid to Antigen for the Fluorescence Analysis of SARS-CoV-2.

Owing to the global spread of the coronavirus disease 2019 (COVID-19), education has shifted to distance online learning, whereas some face-to-face courses have been resumed with the improvement of the outbreak prevention and management situation, including a laboratory course for senior undergraduate students in chemical biology. Here, we present an innovative chemical biology experiment covering COVID-19 topics, which was created for third-year undergraduates. The basic principles of two nucleic-acid- and antigen-based diagnostic techniques for SARS-CoV-2 are demonstrated in detail. These experiments are designed to provide students with comprehensive knowledge of COVID-19 and related diagnoses in daily life. Crucially, the biosafety of this experimental manipulation was ensured by using artificial nucleic acids and recombinant protein. Furthermore, an interactive hybrid online-facing teaching model was designed to cover the key mechanism regarding PCR and serological tests of COVID-19. Finally, a satisfactory evaluation was obtained through a questionnaire, and simultaneously, reasonable improvements to the course design were suggested. The proposed curriculum provides all the necessary information for other instructors to create new courses supported by research.

Metal-Organic Framework Nanoparticles with Universal Dispersibility through Crown Ether Surface Coordination for Phase-Transfer Catalysis and Separation Membranes.

Dispersing metal-organic framework (MOF) solids in stable colloids is crucial for their availability and processibility. Herein, we report a crown ether surface coordination approach for functionalizing the surface-exposed metal sites of MOF particles with amphiphilic carboxylated crown ether (CEC ). The surface-bound crown ethers significantly improve MOF solvation without compromising the accessible voids. We demonstrate that CEC -coated MOFs exhibit exceptional colloidal dispersibility and stability in 11 distinct solvents and six polymer matrices with a wide range of polarities. The MOF-CEC can be instantaneously suspended in immiscible two-phase solvents as an effective phase-transfer catalyst and can form various uniform membranes with enhanced adsorption and separation performance, which highlights the effectiveness of crown ether coating.

A Biomimetic Approach for Spatially Controlled Cell Membrane Engineering Using Fusogenic Spherical Nucleic Acid.

Engineering of the cell plasma membrane using functional DNA is important for studying and controlling cellular behaviors. However, most efforts to apply artificial DNA interactions on cells are limited to external membrane surface due to the lack of suitable synthetic tools to engineer the intracellular side, which impedes many applications in cell biology. Inspired by the natural extracellular vesicle-cell fusion process, we have developed a fusogenic spherical nucleic acid construct to realize robust DNA functionalization on both external and internal cell surfaces via liposome fusion-based transport (LiFT) strategy, which enables applications including the construction of heterotypic cell assembly for programmed signaling pathway and detection of intracellular metabolites. This approach can engineer cell membranes in a highly efficient and spatially controlled manner, allowing one to build anisotropic membrane structures with two orthogonal DNA functionalities.

Detection of Carotid Atherosclerotic Intraplaque Neovascularization Using Superb Microvascular Imaging: A Meta-Analysis.

OBJECTIVES: Although superb microvascular imaging (SMI) (Toshiba/Canon, Tokyo, Japan) has enabled routine characterization of intraplaque neovascularization (IPN) features in patients with carotid stenosis, no reports have been published on the multicenter and large sample size research in this aspect. The efficacy of SMI in detecting carotid IPN has not been concluded. This study aimed to assess the efficacy of SMI comparing with contrast-enhanced carotid ultrasonography (CEUS) in the detection of carotid IPN or pathologic evaluations of IPN correlated with a history of stroke or transient ischemic attack (TIA). METHODS: Web of Science, Cochrane Library, PubMed, Embase, and Scopus were searched up to August 2020 to identify peer-reviewed human studies on the diagnostic accuracy of SMI in detecting IPN. For the selected study, the correlation coefficient R and Kappa index between SMI and CEUS in detecting IPN were calculated. The correlation coefficient R between SMI in identifying IPN and pathologic evaluations of IPN and the odds ratio of IPN detected by SMI and history of stroke or TIA were also extracted. The subgroup analysis was performed to indicate the source of heterogeneity. RESULTS: Our search identified 11 reports enrolling a total of 605 carotid stenosis patients. Carotid IPN detected by SMI was significantly correlated with which detected by CEUS (R, 0.89; 95% CI, 0.80-0.94; P = .00, and Kappa index, 0.73; 95% CI, 0.67-0.80; P = .00). Notably, a significant correlation was observed in SMI in detecting IPN and pathologic evaluations of IPN (R, 0.52; 95% CI, 0.40-0.62; P = .00). The odds ratio of IPN detected by SMI and history of stroke or TIA was pooled summary with statistical significance (OR, 3.33; 95% CI, 1.78-6.23; P = .00). In subgroup analysis, lower heterogeneity was associated with the degree of carotid stenosis, patients from which country, and types of equipment. CONCLUSIONS: SMI and CEUS display an excellent agreement in detecting carotid IPN. IPN detected by SMI shows high consistency with pathologic evaluations of IPN. Individuals with carotid IPN are more likely to develop stroke or TIA than those without carotid IPN.

Dual-Readout Sandwich Immunoassay for Device-Free and Highly Sensitive Anthrax Biomarker Detection.

We report a dual-readout, AuNP-based sandwich immunoassay for the device-free colorimetric and sensitive scanometric detection of disease biomarkers. An AuNP-antibody conjugate serves as a signal transduction and amplification agent by promoting the reduction and deposition of either platinum or gold onto its surface, generating corresponding colorimetric or light scattering (scanometric) signals, respectively. We apply the Pt-based colorimetric readout of this assay to the discovery of a novel monoclonal antibody (mAb) sandwich pair for the detection of an anthrax protective antigen (PA83). The identified antibody pair detects PA83 down to 1 nM in phosphate-buffered saline and 5 nM in human serum, which are physiologically relevant concentrations. Reducing gold rather than platinum onto the mAb-AuNP sandwich enables scanometric detection of subpicomolar PA83 concentrations, over 3 orders of magnitude more sensitive than the colorimetric readout.

Functional DNA Molecules Enable Selective and Stimuli-Responsive Nanoparticles for Biomedical Applications.

Nanoparticles (NPs) have enormous potential to improve disease diagnosis and treatment due to their intrinsic electronic, optical, magnetic, mechanical, and physiological properties. To realize their full potential for nanomedicine, NPs must be biocompatible and targetable toward specific biomolecules to ensure selective sensing, imaging, and drug delivery in complex environments such as living cells, tissues, animals, and human bodies. In this Account, we summarize our efforts to impart specific biocompatibility and biorecognition functionality to NPs by developing strategies to integrate inorganic and organic NPs with functional DNA (fDNA), including aptamers, DNAzymes, and aptazymes to create fDNA-NPs. These hybrid NPs take advantage of fDNA's ability to either bind targets or catalyze reactions in the presence of targets selectively and utilize their unique physicochemical properties including small size, low immunogenicity, and ease of synthesis and chemical modification in comparison with other molecules such as antibodies. By integrating inorganic NPs such as gold NPs, quantum dots, and iron oxide nanoparticles with fDNA, we designed stimuli-responsive fDNA-NPs that exhibit target induced assembly and disassembly of NPs, resulting in a variety of colorimetric, fluorescent, and magnetic resonance imaging (MRI)-based sensors for diagnostic of a broad range of analytes. To impart both biocompatibility and selectivity on inorganic NPs for targeted bioimaging, we have demonstrated DNA-mediated surface functionalization, shape-controlled synthesis, and coordinative assembly of such NPs as specific bioprobes. A highlight is provided on the construction of fDNA-based nanoprobes with light-activatable sensing and imaging functions, which provides precise control of recognition properties of fDNA with high spatiotemporal resolution. To explore the potential of organic NPs for biosensing applications, we have developed an enzyme-responsive fDNA-liposome as a universal sensing platform compatible with diverse biological targets as well as different detection methods including fluorescence, MRI, or temperature, making possible point-of-care diagnostics. To expand the application regime of organic NPs, we collaborated with the Zimmerman group to prepare single-chain block copolymer-based NPs and incorporated it with a variety of functions, including monovalent DNA for assembly, tunable surface chemistry for cellular imaging, and coordinative Cu(II) sites for catalyzing intracellular click reactions, demonstrating the potential of using organic NPs to create promising fDNA-NP systems with programmable functionalities. Furthermore, we survey our recent endeavor in integration of cell-specific aptamers with different NPs for targeted drug delivery, showing that introducing stimuli-responsive properties into NPs that target tumor microenvironments would enable safer and more effective therapy for cancers. Finally, current challenges and future perspectives in fDNA-mediated engineering of NPs for biomedical applications are discussed.

Levetiracetam induction of theta frequency oscillations in rodent hippocampus in vitro.

Levetiracetam (LEV) has been demonstrated to improve cognitive function. Hippocampal theta rhythm (4-12 Hz) is associated with a variety of cognitively related behaviors, such as exploration in both humans and animal models. We investigated the effects of LEV on the theta rhythm in the rat hippocampal CA3 in hippocampal slices in vitro. We found that LEV increased the theta power in a dose-dependent manner. The increase in theta power can be blocked by GABAA receptor (GABAAR) or NMDA receptor (NMDAR) antagonists but not by AMPA receptor antagonist, indicating the involvement of GABAAR and NMDAR in the induction of theta activity. Interestingly, LEV enhancement of theta power can be also blocked by taurine or GABA-A agonist THIP, indicating that LEV induction of theta may be related to the indirect boosting of GABA action via reduction of extrasynaptic GABAAR activation. Furthermore, the increased theta power can be partially reduced by the mACh receptor (mAChR) antagonist atropine but not by nACh receptor antagonists, suggesting that mAChR activation provides excitatory input into local network responsible for LEV-induced theta. Our study demonstrated that LEV induced a novel theta oscillation in vitro, which may have implications in the treatment of the neuronal disorders with impaired theta oscillation and cognitive function.

[Pre-formulation physicochemical properties of component-based Chinese medicine of Qinqi Fengshi Fang].

Pre-formulation physicochemical properties of the component-based Chinese medicine of Qinqi Fengshi Fang were investigated to provide a research basis for the design of the dosage form for component-based Chinese medicine of Qinqi Fengshi Fang. The macroporous resin adsorption and refining technology was used to prepare the total glycosides extract of Gentianae Macrophyllae Radix, Panacis Majoris Rhizome and Corni Fructus respectively in the prescription of Qinqi Fengshi Fang. Their physicochemical properties were investigated, including solubility, wettability, hygroscopicity, equilibrium solubility, oil-water partition coefficient, and stability. The results showed that the total glycosides of Gentianae Macrophyllae Radix, Panacis Majoris Rhizome and Corni Fructus all had good solubility and wettability. The solubility index of each total glycoside component was greater than 85%, and the water absorption index was greater than 50%. In the range of pH 2.0-7.4, the equilibrium solubility of three kinds of total glycosides all increased with the increase of pH, showing a consistent change trend of solubility. The hydrophilicity was also suitable and similar. Overall, three kinds of total glycosides showed good stability, but strong hygroscopicity. The degree of hygroscopicity was as follows: total glycosides of Gen-tianae Macrophyllae Radix > total glycosides of Corni Fructus > total glycosides of Panacis Majoris Rhizome. Therefore, the hygroscopi-city needed to be considered in the preparation of the component-based Chinese medicine of Qinqi Fengshi Fang. The excipients and packaging materials can be properly selected to reduce the hygroscopicity of the preparation. This study provides a reference for the dosage form design of the component-based Chinese medicine of Qinqi Fengshi Fang.

Programmable Assembly of Nano-architectures through Designing Anisotropic DNA Origami Patches.

Programmable assembly of nanoparticles (NPs) into well-defined architectures has attracted attention because of tailored properties resulting from coupling effects. However, general and precise approaches to control binding modes between NPs remain a challenge owing to the difficulty in manipulating the accurate positions of the functional patches on the surface of NPs. Here, a strategy is developed to encage spherical NPs into pre-designed octahedral DNA origami frames (DOFs) through DNA base-pairings. The DOFs logically define the arrangements of functional patches in three dimensions, owing to the programmability of DNA hybridization, and thus control the binding modes of the caged nanoparticle with designed anisotropy. Applying the node-and-spacer approach that was widely used in crystal engineering to design coordination polymers, patchy NPs could be rationally designed with lower symmetry encoded to assemble a series of nano-architectures with high-order geometries.

Fluorescent Graphitic Carbon Nitride-Based Nanozymes with Peroxidase-Like Activities for Ratiometric Biosensing.

While breakthroughs in peroxidase-like nanozymes for bioanalysis have been made, most of current nanozyme biosensing systems are based on a single signal output. Such sensing systems could be easily influenced by environmental and personal factors. We envision that nanozyme sensing systems with ratiometric signal outputs would provide more reliable and robust sensing performance. Herein, to construct such ratiometric sensing systems, three fluorescent graphitic carbon nitride (C3N4)-based nanozymes (i.e., C3N4-Ru, C3N4-Cu, and C3N4-hemin) with excellent peroxidase-like activities were prepared. These fluorescent nanozymes emitted a fluorescence at 438 nm when excited at 385 nm. Interestingly, when o-phenylenediamine (OPD) was catalytically oxidized to oxidized OPD (OPDox) in the presence of H2O2 and nanozymes, the OPDox not only emitted an emerging fluorescence at 564 nm but also quenched the fluorescence at 438 nm of the nanozymes. We therefore employed the ratio of the fluorescent intensity at 564 and 438 nm (i.e., F564/F438) as the signal output to construct the ratiometric biosensing systems. First, we used the C3N4-Ru nanozyme to construct the ratiometric H2O2 sensing system, which showed not only the enhanced robustness but also wider linear range and better sensitivity than most reported H2O2 sensors based on nanozymes. Second, with the assistance of glucose oxidase, glucose can be detected by such ratiometric sensing systems. Third, we used three different C3N4-based nanozymes to construct ratiometric sensor arrays for the detection and discrimination of five phosphates. This study provides new insights for constructing robust nanozyme biosensing systems.