Rearranging Spin Electrons by Axial-Ligand-Induced Orbital Splitting to Regulate Enzymatic Activity of Single-Atom Nanozyme with Destructive d-π Conjugation.

Most of the nanozymes have been obtained based on trial and error, for which the application is usually compromised by enzymatic activity regulation due to a vague catalytic mechanism. Herein, a hollow axial Mo-Pt single-atom nanozyme (H-MoN5@PtN4/C) is constructed by a two-tier template capture strategy. The axial ligand can induce Mo 4d orbital splitting, leading to a rearrangement of spin electrons (↑ ↑ → ↑↓) to regulate enzymatic activity. This creates catalase-like activity and enhances oxidase-like activity to catalyze cascade enzymatic reactions (H2O2 → O2 → O2•-), which can overcome tumor hypoxia and accumulate cytotoxic superoxide radicals (O2•-). Significantly, H-MoN5@PtN4/C displays destructive d-π conjugation between the metal and substrate to attenuate the restriction of orbitals and electrons. This markedly improves enzymatic performance (catalase-like and oxidase-like activity) of a Mo single atom and peroxidase-like properties of a Pt single atom. Furthermore, the H-MoN5@PtN4/C can deplete overexpressed glutathione (GSH) through a redox reaction, which can avoid consumption of ROS (O2•- and •OH). As a result, H-MoN5@PtN4/C can overcome limitations of a complex tumor microenvironment (TME) for tumor-specific therapy based on TME-activated catalytic activity.

Multienzyme-Like Polyoxometalate-Based Single-Atom Enzymes for Cancer-Specific Therapy Through Acid-Triggered Nontoxicity-to-Toxicity Transition.

Single-atom enzymes (SAzymes) exhibit great potential for chemodynamic therapy (CDT); while, general application is still challenged by their instability and unavoidable side effects during delivery. Herein, a manganese-based polyoxometalate single-atom enzyme (Mn-POM SAE) is first introduced into tumor-specific CDT, which exhibits tumor microenvironment (TME)-activated transition of nontoxicity-to-toxicity. Different from traditional POM materials, the aggregates of low-toxic Mn-POM SAE nanospheres are obtained at neutral conditions, facilitating efficient delivery and avoiding toxicity problems in normal tissues. Under acid TME conditions, these nanospheres are degraded into smaller units of toxic Mn(II)-PW11; thus, initiating cancer cell-specific therapy. The released active units of Mn(II)-PW11 exhibit excellent multienzyme-like activities (including peroxidase (POD)-like, oxidase (OXD)-like, catalase (CAT)-like, and glutathione peroxidase (Gpx)-like activities) for the synergistic cancer therapy due to the stabilized high valence Mn species (MnIII/MnIV). As demonstrated by both intracellular evaluations and in vivo experiments, ROS is generated to cause damage to lysosome membranes, further facilitating acidification and impaired autophagy to enhance cancer therapy. This study provides a detailed investigation on the acid-triggered releasing of active units and the electron transfer in multienzyme-mimic-like therapy, further enlarging the application of POMs from catalytical engineering into cancer therapy.

Dual Active Centers Linked by a Reversible Electron Station as a Multifunctional Nanozyme to Induce Synergetically Enhanced Cascade Catalysis for Tumor-Specific Therapy.

Nanozymes have shown great promise in reactive oxygen species (ROS)-mediated tumor therapy with mitigated side effects but are normally limited by the complex tumor microenvironment (TME). Herein, to overcome the adverse effects of TME, such as tumor hypoxia and high endogenous glutathione (GSH), an aptamer-functionalized Pd@MoO3-x nano-hydrangea (A-Pd@MoO3-x NH) is constructed for high-efficiency cancer therapy. Utilizing the irregular shape characteristics of nano Pd, the A-Pd@MoO3-x NH nanozyme simultaneously exposes catalase-like Pd(111) and oxidase-like Pd(100) surface facets as dual active centers. This can catalyze cascade enzymatic reactions to overcome the negative effects of tumor hypoxia caused by the accumulation of cytotoxic superoxide (O2•-) radicals in TME without any external stimuli. In addition, the nanozyme can effectively degrade the overexpressed glutathione (GSH) through the redox reaction to avoid nontherapeutic consumption of O2•- radicals. More significantly, as a reversible electron station, MoO3-x can extract electrons from H2O2 decomposing on Pd(111) or GSH degradation and transfer them back to Pd(100) through oxygen bridges or few Mo-Pd bonds. This can synergistically enhance enzyme-like activities of dual active centers and the GSH-degrading ability to enrich O2•- radicals. In this way, the A-Pd@MoO3-x NH nanozyme can selectively and remarkably kill tumor cells while keeping the normal cell line unharmed.

Single-Molecule Evaluation of the SARS-CoV-2 Nucleocapsid Protein Using Gold Particle-in-a-Frame Nanostructures Enhanced Fluorescent Assay.

Ultrasensitive evaluation of low-abundance analytes, particularly with limits approaching a single molecule, is a key challenge in the design of an assay for profiling severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen. Herein, we report an aptamer claw strategy for directly evaluating the SARS-CoV-2 antigen based on gold particle-in-a-frame nanostructures (Au PIAFs). Au PIAF was used as a metal-enhanced fluorescence material. The assay integrated with a microplate reader achieved a sensitivity of 44 fg·mL-1 in under 3 min and accurately detected the SARS-CoV-2 nucleocapsid protein (N protein) in human saliva samples. When our assay is combined with a single-molecule counting platform, the limit of detection can be as low as 0.84 ag·mL-1. This rapid and ultrasensitive assay holds promise as a tool for screening SARS-CoV-2 and other contagious viruses.

Computation-Assisted Design of ssDNA Framework Nanorobots for Cancer Logical Recognition, Toehold Disintegration, Visual Dual-Diagnosis, and Synergistic Therapy.

Single-stranded DNA (ssDNA) allows flexible and directional modifications for multiple biological applications, while being greatly limited by their poor stability, increased folding errors, and complicated sequence optimizations. This greatly challenges the design and optimization of ssDNA sequences to fold stable 3D structures for diversified bioapplications. Herein, the stable pentahedral ssDNA framework nanorobots (ssDNA nanorobots) were intelligently designed, assisted by examining dynamic folding of ssDNA in self-assemblies via all-atom molecular dynamics simulations. Assisted by two functional siRNAs (S1 and S2), two ssDNA strands were successfully assembled into ssDNA nanorobots, which include five functional modules (skeleton fixation, logical dual recognition of tumor cell membrane proteins, enzyme loading, dual-miRNA detection and synergy siRNA loading) for multiple applications. By both theoretical calculations and experiments, ssDNA nanorobots were demonstrated to be stable, flexible, highly utilized with low folding errors. Thereafter, ssDNA nanorobots were successfully applied to logical dual-recognition targeting, efficient and cancer-selective internalization, visual dual-detection of miRNAs, selective siRNA delivery and synergistic gene silencing. This work has provided a computational pathway for constructing flexible and multifunctional ssDNA frameworks, enlarging biological application of nucleic acid nanostructures.

Examination of electronically mismatched Diels-Alder reaction by on-line mass spectrometry.

RATIONALE: Electronically mismatched Diels-Alder reactions have gained much attention as an alternative pathway for C-C bond formation. To facilitate the development of facile organic transformations, mechanistic investigations are required. Spectroscopic methods (NMR, electron paramagnetic resonance and UV-visible) are normally adopted for mechanistic examinations, but further improvements in directly obtaining structural information of short-lived intermediates are encouraged. Herein, an electronically mismatched Diels-Alder reaction between indole and 1,3-cyclohexadiene was studied using in situ electrospray ionization mass spectrometry (in situ ESI-MS). Based on direct sampling and detection of the in situ ESI-MS without sample pretreatment, the structures and dynamics of important intermediates were examined on-line. METHODS: A syringe-based photocatalytic reactor and in situ ambient MS (AMS) evaluation system was constructed for mechanism studies. The role of oxygen was confirmed via control reaction employed in the N2 -bubbled system. The stepwise cation radical-based pathway and the [2 + 2] cycloaddition process were determined through a series of experiments, including solvent evaluation, MS/MS experiments and dynamic monitoring. RESULTS: The dependence of the reaction on solvent polarity demonstrated that the reaction occurs via the formation of cation radicals, which were captured, identified and dynamically monitored via in situ ESI-MS. Without pre-separation, the intermediate of [2 + 2] cycloaddition was identified and the cycloaddition process was thereby determined to be the combination of [4 + 2] cycloaddition and [2 + 2] cycloaddition. In addition, oxygen was proved to act as an electron mediator for both catalyst Ru(bpz)3 (PF6 )2 and radical cations. CONCLUSIONS: The mechanism of an electronically mismatched Diels-Alder reaction was successfully deduced by in situ MS associated with a syringe-based photocatalytic reactor. The structures and dynamics of cation radicals, the effect of O2 for the reaction and the detailed process of [2 + 2] cycloaddition have been well demonstrated. This work could not only promote the understanding and development of facile photocatalytic transformations, but also enlarge the application range of AMS in on-line monitoring.

Confined surface-enhanced indole cation-radical cyclization studied by mass spectrometry.

Reactions in confined spaces exhibit unique reactivity, while how the confinement effect enhances reactions remains unclear. Herein, the reaction in the confined space of a nanopipette reactor was examined by in situ nano-electrospray mass spectrometry (nanoESI-MS). The indole cation-radical cyclization was selected as the model reaction, catalyzed by a common visible-light-harvesting complex Ru(bpz)3(PF6)2 (1% eq.) rather than traditional harsh reaction conditions (high temperature or pressure, etc.). As demonstrated by in situ nanoESI-MS, this reaction was readily promoted in the nanopipette under mild conditions, while it was inefficient in both normal flasks and microdroplets. Both experimental and theoretical evidence demonstrated the formation of concentrated Ru(II)-complexes on the inner surface of the nanopipette, which facilitated the accelerated reactions. As a result, dissociative reactive cation radicals with lower HOMO-LUMO gap were generated from the Ru(II)-complexes by ligand-to-metal charge transfer (LMCT). Furthermore, the crucial cation radical intermediates were captured and dynamically monitored via in situ nanoESI-MS, responsible for the electronically matched [4 + 2] cycloaddition and subsequent intramolecular dehydrogenation. This work inspires a deeper understanding of the unique reactions in confined spaces.

Germacrane Sesquiterpene Dilactones from Mikania micrantha and Their Antibacterial and Cytotoxic Activity.

Four new germacrane sesquiterpene dilactones, 2ß-hydroxyl-11ß,13-dihydrodeoxymikanolide (1), 3ß-hydroxyl-11ß,13-dihydrodeoxymikanolide (2), 1α,3ß-dihydroxy-4,9-germacradiene-12,8:15,6-diolide (3), and (11ß,13-dihydrodeoxymikanolide-13-yl)-adenine (4), together with five known ones (5-9) were isolated from the aerial parts of Mikania micrantha. Their structures were elucidated on the basis of extensive spectroscopic analysis. Compound 4 is featured with an adenine moiety in the molecule, which is the first nitrogen-containing sesquiterpenoid so far isolated from this plant species. These compounds were evaluated for their in vitro antibacterial activity against four Gram-(+) bacteria of Staphyloccocus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus (BC) and Curtobacterium. flaccumfaciens (CF), and three Gram-(-) bacteria of Escherichia coli (EC), Salmonella. typhimurium (SA), and Pseudomonas Solanacearum (PS). Compounds 4 and 7-9 were found to show strong in vitro antibacterial activity toward all the tested bacteria with the MIC values ranging from 1.56 to 12.5 µg/mL. Notably, compounds 4 and 9 showed significant antibacterial activity against the drug-resistant bacterium of MRSA with MIC value 6.25 µg/mL, which was close to reference compound vancomycin (MIC 3.125 µg/mL). Compounds 4 and 7-9 were further revealed to show in vitro cytotoxic activity toward human tumor A549, HepG2, MCF-7, and HeLa cell lines, with IC50 values ranging from 8.97 to 27.39 µM. No antibacterial and cytotoxic activity were displayed for the other compounds. The present research provided new data to support that M. micrantha is rich in structurally diverse bioactive compounds worthy of further development for pharmaceutical applications and for crop protection in agricultural fields.

Biomineralization of DNA Nanoframeworks for Intracellular Delivery, On-Demand Diagnosis, and Synergistic Cancer Treatments.

DNA nanoframeworks, with great biological information and controlled framework structures, exhibit great potentials in biological applications. Their applications are normally limited by unstable structures susceptible to hydrolysis, depurination, depyrimidination, oxidation, alkylation, or nuclease degradations. Herein, to ensure the mechanical and chemical stabilities of DNA nanoframeworks for intracellular applications, biomineralization of multifunctional DNA nanoframeworks with a tetrahedral skeleton is employed. Via silicification, the S-S bond is simultaneously introduced to obtain the silica-armored DNA nanoframeworks (Si-DNA nanoframeworks), mechanically and chemically stabilized for efficient intracellular deliveries. This successfully prevents degradations and leakages of reagents loaded on Si-DNA nanoframeworks, including biomolecular siRNA and small DOX drugs. Furthermore, the nucleic acid strands of the nanoframeworks are labeled with FAM and the quencher, facilitating miRNA detection upon "turn-on" signals from hybridizations. Therefore, the nanoframeworks collapse via double responses of the silica coating (silica acidic dissolution and S-S reduction by GSH) in cancer cells, realizing on-demand reagent release for miRNA detection and synergistic treatments (by siRNA and DOX). Demonstrated by both in vivo and in vitro experiments, the biomineralization has stabilized DNA nanomaterials for biological applications.

Synthesis and Characteristics of Self-Assembled Multifunctional Ln3+ -DNA Hybrid Coordination Polymers.

Owing to the unique catalytic, optical and magnetic properties, lanthanides (Ln) as multicomponent biomarkers, are widely used in the field of optical sensing, mass spectrometry and magnetic resonance imaging. As ligands, DNA molecules have good biocompatibility, high stability, cost efficiency, programmability and biodegradability. Based on the coordination-driven self-assembly between Ln ions (Ln3+ ) and DNA molecules, a multifunctional Ln3+ -DNA hybrid coordination polymers (CPs) were synthesized. Not only a series of different Ln3+ (single Ln3+ ) and DNA hybrid CPs were synthesized, but one hybrid CP contains two kinds of Ln3+ was obtained. Besides, the synthetic CPs in cell fluorescence imaging and miRNA sensing also exhibited high performance. This work provides a novel idea for the synthesis of DNA based nanomaterials, which is promising for biologically-related applications.

Detection of glutathione, cysteine, and homocysteine by online derivatization-based electrospray mass spectrometry.

RATIONALE: Electrospray ionization mass spectrometry (ESI-MS) is one of the most popular techniques for obtaining structural information, which is commonly used in bioanalysis and clinical diagnostics. However, for the detection of complicated samples with high reactivities (such as reactive sulfur species, RSS), traditional ESI-MS usually suffers from overlapped and inaccurate signals. In this study, based on the multiphase flow of extractive electrospray ionization (MF-EESI), an ambient MS technique of online derivatization was proposed to detect thiols without any other sample pretreatment. METHODS: RSS molecules and the derivatization reagent of 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) were introduced into the internal and innermost capillary of the MF-EESI system, respectively. By a high-velocity nebulizing stream of N2 gas through an external capillary, both flows of innermost biothiols and internal NBD-Cl were electrosprayed and mixed for online reactions. Therefore, the fast derivatization of thiols was used to generate stable ionized derivatives for MS detection. RESULTS: By evaluating the changes in MS signals before and after the derivatization, the ions of RSS were identified simply and correctly. Without any sample pretreatment, the fast detection of cysteine, homocysteine, and glutathione has been achieved in the complicated samples. CONCLUSIONS: The present online derivatization-based MF-EESI was successfully used for fast, simple, and accurate detection of biothiols. This presented a potential pathway for the fast identification of thiols in complicated samples.

Label- and enzyme-free plasmon-enhanced single molecule fluorescence detection of HIV DNA fragments based on a catalytic hairpin assembly.

We developed a label- and enzyme-free single molecule fluorescence counting strategy for HIV DNA fragments detection. The nucleic acid biosensor consists of a 5' terminal connected with a triangular gold nanoplate, 3' terminal rich in guanine hairpin probe (HP1) and a hairpin probe HP2 complementary to the partial sequence of HP1. Without the existence of the target DNA, the DNA fragment rich in the guanine region is locked in a hairpin structure and cannot form a G-quadruplex, hence NMM exhibits a low fluorescence signal. When the target DNA exists, the hairpin assembly will trigger a strand displacement amplification reaction that produces a great number of G-quadruplexes, and the fluorescence brightness of NMM will be enhanced. The plasmon resonance effect of the triangular gold nanoplates will further amplify the fluorescence signal. This method can analyze the target DNA with high sensitivity and selectivity, and the detection limit is 0.83 fM. The analysis of the HIV DNA fragments in diluted human serum samples was successfully achieved, and the recovery rate was 92%-104%. Because of its easy operation and low cost, it has broad development potential in biochemical analysis and clinical applications.

Integrating Near-Infrared Visual Fluorescence with a Photoelectrochemical Sensing System for Dual Readout Detection of Biomolecules.

Compared with traditional visible light-driven fluorescence visualization (FV), near-infrared (NIR)-induced FV is an interesting and promising method, while photoelectrochemical (PEC) immunoassay sensing possesses the advantages of high sensitivity, low cost, and simple instrumentation. We combined PEC sensing with NIR-induced FV together and developed a dual readout sensing platform. In this protocol, based on the antibody-analyte (i.e., antigen, DNA, and RNA) reaction and the sandwich-type structure, CuInS2 microflowers as the matrix provided the original background photocurrent; chlorin e6 (Ce6) was conjugated to antibody-modified upconversion nanoparticles and formed a signal label for the PEC sensing and naked-eye readout. Different from traditional PEC immunosensors, under NIR illumination, the developed dual mode sensing platform could achieve quick qualitative analysis and quantitative analysis. Preliminary application performance of the proposed biosensor in prostate-specific antigen analysis is acceptable, indicating its promising potential in clinical/biological studies.

Spatiotemporally Controlled DNA Nanoclamps: Single-Molecule Imaging of Receptor Protein Oligomerization.

Cell membrane surface receptor proteins play an important role in cellular biological processes. There are numerous methods to detect receptors, yet developing an artificially controlled and specific detection and treatment strategy remains a challenge. Herein, we develop such a strategy based on upconversion nanoparticles (UCNPs) loaded DNA probes that enable two-color ratiometric imaging excitated by a 980 nm laser. The light response controllable signal opening strategy avoids waste during probe transportation and improves sensitivity. Thereby the number of receptors on individual DU145 cell membranes is counted by single-molecule detection. Due to the different expression of specific receptor proteins, the number of single fluorescent dots counted can be used as a basis for distinguishing DU145 from other cells. This work is highly controllable to increase sensitivity, providing a platform for cancer diagnosis and treatment.

One-Step Prepared Water-Resistant Organic-Inorganic-Hybrid Perovskite Quantum Dots with Zn-Oxygen Vacancies for Attempts at Nitrogen Fixation.

Applying organic-inorganic hybrid perovskite quantum dots (PQDs) to photocatalytic nitrogen fixation is hindered long-term by the inherent instability in water and tedious preparations. Here, to realize PQD-catalyzed photocatalytic N2 reduction reaction (NRR), water-resistant PQDs are simply prepared through one-step electrospray synthesis in microseconds. During the fast electrospray, PQDs of Zn/PbO-doped methylammonium lead bromide (Zn/PbO/PC-Zn/MAPbBr3 , MA: CH3 NH3 ) are prepared and part-encapsulated by polycarbonate. The synthesis maintains good water resistance, whose restriction on charge transport is overcome skillfully. Simultaneously, substitution of Zn with Pb on water-resistant surface is also achieved, which fabricates new Zn-oxygen vacancies (Zn-OVs) with Zn/PbO-Zn/MAPbBr3 type I heterojunction. This facilitates efficient electron transfer from internal heterojunction interface of Zn/MAPbBr3 PQDs to the surface of Zn/PbO. Demonstrated by theoretical calculations, Zn-OVs promote chemisorption and polarization of N2 . In addition, s-electrons in exposed Zn become active due to changes of electron filling of Zn orbitals under OVs' co-doping. Thus, photocatalytic N2 reduction reaction catalyzed by organic-inorganic hybrid PQDs is first achieved in aqueous phase without sacrificial agents being added. This initiates possibilities for photocatalytic applications of organic-inorganic hybrid PQDs in aqueous phase.

Multi-Dimensionally Extended Functionalization Innovates to an Entropy-Driven Detection of Multi-miRNAs for One-Step Cancer Screening and Diagnosis in Living Cells.

Compared with tedious multi-step detections, multi-functional nanoprobes are effective for one-step screening and diagnosis of cancers by multi-detection of microRNAs (miRNAs). However, limited probe density, spatial mutual interference, and low target-triggered hybridization efficiency of nanoprobes will hinder intracellular applications. Here, for obtaining high loading density but low spatial mutual interference between functional biomolecules on nanoprobes, an extended biofunctionalization in three dimensions (the two-dimensional surface and a special "height" direction) is designed. Therefore, a multi-functional probe is constructed for one-step detection of multi-miRNAs for cancer screening and diagnosis. With linker-bridged multiple single-stranded DNAs swung out rigidly, multi-dimensionally extended upconversion nanorods (ME-UCNRs) covered by chitosan are constructed to load and deliver multiple biomolecules into living cells. Escaping from endolysosomes, ME-UCNRs maintain good biological activities of functionalized DNAs for effective detection of multi-miRNAs in living cells. Thereby, with multiple targets of miRNAs, toehold-mediated entropy-driven strand displacements are employed to give respectively changed fluorescent signals via fluorescence resonance energy transfer. Thus, a universal cancer biomarker of miR-21 and two specific liver-cancer biomarkers (miR-199a and miR-224) are efficiently detected through a one-step detection. By discriminating cancer cells from normal ones and determining liver-cancer cells simultaneously, this work innovates an efficient and definite one-step strategy for fast screening and early cancer diagnosis.

Mannose Promotes Metabolic Discrimination of Osteosarcoma Cells at Single-Cell Level by Mass Spectrometry.

Discrimination of cancer cells at the single-cell metabolic level is crucial for early diagnosis. However, some cancer cells share similar metabolic information with normal cells, making them difficult to be distinguished using mass spectrometry. Herein, we propose a method by treating osteosarcoma cells and normal human osteoblasts with mannose as a stimulant, which greatly promotes the metabolic discrimination of osteosarcoma cells at the single-cell level. The low PMI (mannose 6-phosphate isomerase) level of both osteosarcoma cell lines compared to normal human osteoblasts is the reason for the abnormal metabolic pathway of two osteosarcoma cell lines with mannose treatment. We also found that the level of hexoses-6P in osteosarcoma cells significantly increased after mannose treatment, while no such increase was found in normal human osteoblasts. The proposed method is very meaningful for early diagnosis of cancer.

Sequencing of Small DNA Fragments with Aggregated-Induced-Emission Molecule-Labeled Nucleotides.

Sequencing by synthesis is a significant method for high-throughput DNA sequencing. Herein, we synthesized terminal aggregated-induced-emission luminogen (AIEgen) labeled nucleotides (dNTPs-HCAP) that could serve as substrates for some polymerases and applied them into the sequencing of small DNA fragments. In the process of DNA amplification, ratiometric AIEgens are released from dNTPs-HCAP and aggregate through the effects of phosphatase, which results in changes in the ratiometric fluorescent signals. With the AIEgen-labeled nucleotides, we accomplished the sequencing of small DNA fragments through double changes in fluorescence. In addition, we achieved the differentiation of single nucleotide polymorphisms through rolling circle amplification reactions without the addition of signal probes, which is fast and cost-effective. The introduction of ratiometric AIEgens into DNA synthesis makes the detection of DNA sequences more efficient and accurate. Therefore, the development of AIEgen-labeled nucleotides is meaningful for the study of DNA sequencing methods.

Target-Triggered Assembly of Nanogap Antennas to Enhance the Fluorescence of Single Molecules and Their Application in MicroRNA Detection.

Nanogap antennas are plasmonic nanostructures with a strong electromagnetic field generated at the gap region of two neighboring particles owing to the coupling of the collective surface plasmon resonance. They have great potential for improving the optical properties of fluorophores. Herein, nanogap antennas are constructed using an aqueous solution-based method to overcome the defects of weak fluorescence and photobleaching associated with traditional organic dyes, and a highly sensitive nanogap antenna-based sensing strategy is presented for the detection of low-abundance nucleic acid biomarkers via a target-triggered strand displacement amplification (SDA) reaction between two DNA hairpins that are tagged to the tips of gold nanorods (Au NRs). In the presence of targets, end-to-end Au NR dimers gradually form, and the fluorophores quenched by the Au NRs exhibit a dramatic fluorescence enhancement due to the plasmon-enhanced fluorescence effect of nanogap antennas. Meanwhile, the SDA reaction results in secondary amplification of fluorescence signals. Combined with single-molecule counting, this method applied in miRNA-21 detection can achieve a low detection limit of 97.2 × 10-18 m. Moreover, accurate discrimination between different cells through miRNA-21 imaging demonstrates the potential of this method in monitoring the expression level of low-abundance nucleic acid biomarkers.

Visualizations of Mercury Methylation and Dynamic Transformations by In Vivo Imaging.

Visualization of Hg(II) and MeHg in their native contexts is significant for examining mercury poisoning, while it is challenging because of indistinguishable fluorescent (FL) signals during FL imaging. Herein, visualizations of mercury methylation and dynamic transformations of Hg(II) and MeHg are achieved in living biological systems. Well distinguishable FL responses (blue emission for Hg(II), yellow emission for MeHg) are obtained by a double-response FL probe (DPAHB) without any interference. As demonstrated by experimental and computational studies, the distinguishable signals are attributed to selective binding with DPAHB and different inhibition of excited-state proton transfer. Through control tests for live-dead markers, mercury methylation is demonstrated to be employed in living biological systems. Therefore, the methylation and dynamic transformations of both ions are monitored in zebrafish by imaging, and these results are confirmed by traditional high-performance liquid chromatography-based methods. The methylation of Hg(II) to MeHg, dynamic transformations and final accumulations of both species in zebrafish tissues are visualized successfully. This method is also convenient for fast evaluation of detoxification reagents. This is the first visualization of in vivo mercury methylation and dynamic transformation of both species and is effective for studying pathological processes in their native contexts.