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.

Switchable Site-Selective Benzanilide C(sp2)-H Bromination via Promoter Regulation.

Regioselective benzanilide bromination that generates either regioisomer from the same starting material is desirable. Herein, we develop switchable site-selective C(sp2)-H bromination by promoter regulation. This protocol leads to regiodivergent brominated benzanilide starting from the single substrate via selection of promoters. The protocol demonstrates excellent regioselectivity and good tolerance of functional groups with high yields. The utility effectiveness of this method has been well exemplified in the late-stage modification of biologically important molecules.

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.

CHEMICAL REACTION MONITORING BY AMBIENT MASS SPECTROMETRY.

Chemical reactions conducted in different media (liquid phase, gas phase, or surface) drive developments of versatile techniques for the detection of intermediates and prediction of reasonable reaction pathways. Without sample pretreatment, ambient mass spectrometry (AMS) has been applied to obtain structural information of reactive molecules that differ in polarity and molecular weight. Commercial ion sources (e.g., electrospray ionization, atmospheric pressure chemical ionization, and direct analysis in real-time) have been reported to monitor substrates and products by offline reaction examination. While the interception or characterization of reactive intermediates with short lifetime are still limited by the offline modes. Notably, online ionization technologies, with high tolerance to salt, buffer, and pH, can achieve direct sampling and ionization of on-going reactions conducted in different media (e.g., liquid phase, gas phase, or surface). Therefore, short-lived intermediates could be captured at unprecedented timescales, and the reaction dynamics could be studied for mechanism examinations without sample pretreatments. In this review, via various AMS methods, chemical reaction monitoring and mechanism elucidation for different classifications of reactions have been reviewed. The developments and advances of common ionization methods for offline reaction monitoring will also be highlighted.

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.

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.

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.

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.

An Acetone Sensor Based on Plasma-Assisted Cataluminescence and Mechanism Studies by Online Ionizations.

The new development of noninvasive diagnosis in breath analysis requires the fabrication of inexpensive and rapid sensing technologies, whose sensing mechanisms are further encouraged to be studied. Here based on the dramatically enhanced plasma-assisted cataluminescence (PA-CTL) signals, a new sensor was constructed for the detection of acetone, an important biomarker of diabetes mellitus in breath. As demonstrated, the PA-CTL-based sensor showed good sensitivity, repeatability, and selectivity in acetone detection, which also displayed good recovery and stability in exhaled breath. An online ionization system of low-temperature plasma mass spectrometry was designed to couple with the PA-CTL sensor for examining changes of molecules during the sensing. For the first time, some important ions were recorded in real time, which helped to clear the reaction mechanism of radical-based catalytic oxidation combined with the CTL data. By the fabrication and relative comprehensive understanding of the PA-CTL sensor for acetone detection, this work would promote the development of CTL sensing techniques and inspire a potential pathway for rapid and noninvasive diagnosis.

A "Soft" and "Hard" Ionization Method for Comprehensive Studies of Molecules.

Ambient mass spectrometry can be rapidly and directly effective for molecular studies, while there still seems to be a gap between two major groups of electrospray ionization (ESI)- and atmospheric pressure chemical ionization (APCI)-related techniques, for detection of moderately polar to polar and low polar to nonpolar molecules in a relatively low mass range, respectively. Here, an extensively applicable "soft" and "hard" ionization method, spray-dependent plasma mass spectrometry (SDP MS), was established for detecting various molecules with diverse polarities or molecular weights. By SDP MS, both fragment ions and intact molecular ions can be obtained. Significantly, cluster ions of aggregates in high mass range formed by weak molecular interactions can also be well recorded, much softer than traditional ESI MS. By filling the gap between ESI-based and APCI-based ionization techniques, SDP MS would enhance MS performance for comprehensive molecular studies and be extensively applicable in fields of organic synthesis, biological chemistry, medical chemistry, and clinical diagnosis.

A dynamic cascade DNA nanocomplex to synergistically disrupt the pyroptosis checkpoint and relieve tumor hypoxia for efficient pyroptosis cancer therapy.

Pyroptosis has attracted widespread concerns in cancer therapy, while the therapeutic efficiency could be significantly restricted by using the crucial pyroptosis checkpoint of autophagy and tumor hypoxia. Herein, a DNA nanocomplex (DNFs@ZnMn), containing cascade DNAzymes, promoter-like ZnO2-Mn nanozymes and photosensitizers, was constructed in one pot through rolling circle amplification reactions to induce pyroptosis through disrupting autophagy. After targeting cancer cells with a high expression of H+ and glutathione, DNFs@ZnMn decomposed to expose DNAzymes and promoter-like ZnO2-Mn nanozymes. Then, sufficient metal ions and O2 were released to promote cascade DNA/RNA cleavage and relieving of tumor hypoxia. The released DNAzyme-1 self-cleaved long DNA strands with Zn2+ as the cofactor and simultaneously exposed DNAzyme-2 to cleave ATG-5 mRNA (with Mn2+ as the cofactor). This cascade DNAzyme-mediated gene regulation process induced downregulation of ATG-5 proteins to disrupt autophagy. Simultaneously, the released ZnO2 donated sufficient H2O2 to generate adequate O2 to relieve tumor hypoxia, obtaining highly cytotoxic 1O2 to trigger pyroptosis. By using dynamic cascade gene silencing to disrupt the pyroptosis checkpoint and synergistic relieving of hypoxia, this DNA nanocomplex significantly weakened cellular resistance to achieve efficient pyroptosis therapy both in vitro and in vivo.

Competitive photooxidation of small colorless organics controlled by oxygen vacancies under visible light.

Visible-light photooxidation sensitized by surface attachment of small colorless organics on semiconductor photocatalysts has emerged as an economical method for photocatalytic synthesis or degradation. In particular, heteroatom (X = N and Cl)-containing substrates could undergo either C-N coupling or dechlorination degradation via sensitizing TiO2, but the mechanism in conducting the competitive visible-light sensitized photooxidations is still vague. Herein, the visible-light photooxidation of colorless 4-chlorobenzene-1,2-diamine (o-CAN) on TiO2 was revealed, contributing to selective C-N coupling rather than dechlorination. Oxygen vacancies (OVs) were in situ generated on the TiO2 surface, which could be dominant in weakening the Cl-Ti adsorption of o-CAN and regulating the activation of O2 for selective C-N coupling. The C-N coupling product, functionalized as the sensitizer, further promoted the visible-light photooxidation upon N-Ti and Cl-Ti coordination. This process was then confirmed by on-line mass spectrometric analysis, and the intermediates as well as their kinetics were determined. Thereby, theoretical calculations were employed to verify the roles of OVs in competitive photooxidation and lowering the energy barriers as well. Based on the comprehensive characterizations of both the catalysts and intermediates, this work has provided insights into competitive photooxidations under visible light.

OH radical-initiated single-electron transfer for accelerated degradation via carbocation intermediates.

Single electron transfer (SET) has made great contributions to a broad range of chemical processes, whose radical cation and carbocation intermediates are important for mechanism studies. Herein, hydroxyl radical (˙OH)-initiated SET was revealed in accelerated degradations, via the online examination of radical cations and carbocations by electrosonic spray ionization mass spectrometry (ESSI-MS). In the green and efficient non-thermal plasma catalysis system (MnO2-plasma), hydroxychloroquine was efficiently degraded upon SET via carbocations. In the plasma field full of active oxygen species, ˙OH was generated on the MnO2 surface to initiate SET-based degradations. Furthermore, theoretical calculations revealed that ˙OH preferred to withdraw the electron from the N atom that was conjugated to the benzene ring. This facilitated the generation of radical cations through SET, which was followed by the sequential formation of two carbocations for accelerated degradations. Transition states and energy barriers were calculated to study the formation of radical cations and subsequent carbocation intermediates. This work demonstrates an ˙OH-initiated SET for accelerated degradation via carbocations, providing a deeper understanding and the potential for the wider application of SET in green degradations.

Water-resistant organic-inorganic hybrid perovskite quantum dots activated by the electron-deficient d-orbital of platinum atoms for nitrogen fixation.

Due to their special physicochemical properties, organic-inorganic hybrid perovskite quantum dots (OIP QDs) are ideal and potential catalysts for the nitrogen reduction reaction (NRR). However, the OIP QD-based NRR is limited by poor water resistance, competitive suppression by the hydrogen evolution reaction, and inefficient active sites on the catalyst surfaces. Herein, to ensure an efficient NRR in aqueous solution, a water-resistant polycarbonate-part-encapsulated heterojunction of Zn,PtIV co-doped PbO-MAPbBr3 (PtIV/Zn/PbO/PC-Zn/MAPbBr3) is prepared through one-step electrospray-based microdroplet synthesis. Confirmed by both experimental and theoretical examinations, PbO is exposed on the PC-part-encapsulated surface to construct a Type I heterojunction. This heterojunction is further improved by synergistic co-doping with PtIV to facilitate efficient electron transfer for efficient photocatalysis of the NRR. Due to the active sites of the d-orbital electron-deficient Pt atoms (exhibiting a lower reaction energy barrier and highly selective N2 adsorption), the ammonia yield rate is 40 times higher than that without doping. This work initiates and develops on the application of OIP QDs in the NRR.

SiRNA-templated 3D framework nucleic acids for chemotactic recognition, and programmable and visualized precise delivery for synergistic cancer therapy.

Developments in framework nucleic acids (FNAs) are limited by complicated synthesis, by-product interference, and low framework utilization. Herein, simple core-shell spherical 3D FNAs (ST-SFNAs) preparation is presented based on siRNA-templated linear polymerization followed by hybridization chain reaction branched polymerization. Without by-products, all components exhibited their special functions to obtain high space utilization of ST-SFNAs. ST-SFNAs were covered by catalase and folic acid-functionalized liposome membranes. The catalase endowed ST-SFNAs with chemotactic activities in the H2O2 reaction catalyzed by catalase. Furthermore, combined with functionalized folic acids' targeting folate receptors, the synergistic chemotactic recognition of cancer cells was obtained. This dramatically promoted targeted cellular uptakes compared with traditional active or passive targeting pathways. Subsequently, the cascaded-logical programmable release of drugs was precisely controlled by targeting glutathione and ATP (via S-S bond and ATP aptamer on the inner g-DNA cover). This was visualized by "turn on" fluorescent signals generated by special hybridization of released hairpin DNAs with survivin mRNA biomarkers. Simultaneously, biocompatible synergistic therapy was achieved by simultaneously releasing doxorubicin and siRNA. With its high utilization for synergistic chemotactic recognition, programmable and visualized delivery, as well as synergistic therapy, an efficient platform for maximizing the therapeutic efficacy has been developed. This would initiate further FNA-based material development for a variety of biological applications.

Understanding of TEMPO-electrocatalyzed acceptorless dehydrogenation of tetrahydroquinoline by in situ extractive electrospray ionization mass spectrometry.

The TEMPO-electrocatalyzed acceptorless dehydrogenation of tetrahydroquinoline, a transformation in green synthesis and hydrogen storage, has been investigated by coupling of an electrocatalytic system with in situ extraction electrospray ionization mass spectrometry. Dynamic changes in important species and intermediates were monitored, which evoked an updated AD understanding.

Observation of intermediates by online mass spectrometry to demonstrate the multiple mechanisms of dye-sensitized photocatalysis.

Online mass spectrometry was applied to reveal multiple mechanisms of visible-light irradiated dye-sensitized photocatalysis for o-phenylenediamine oxidation. The reactants, products and short-lived intermediates were recorded and dynamically tracked. Dimer and unexpected trimer intermediates were observed to deduce the stepwise aerobic photooxidation mechanism with multiple routes, which was supported by theoretical calculations.

Accelerating ambient soft-landing for the separation of aggregation-induced emission luminogens with unique properties.

The separating and obtaining of AIEgens with narrow-sized distribution is significant for aggregation-induced emission (AIE) examinations and applications, so far no effective pathway has been reported for separation of AIEgens with weak interactions. Here, with the aid of deflecting electric fields and accelerating electric fields, we fabricated an accelerating ambient soft-landing (AASL) as a soft separation method for accelerating and separating charged particles. By AASL, AIEgens of Cu nanoclusters (NCs) were sprayed and converted into charged particles of vapor-phase by electrospray, thereby to be selected by entering defecting electric field, then accelerated in the following accelerating electric field for the separation based on sizes, and finally landed on the receptor surface for collections. Without breaking the weak interactions between AIEgens of CuNCs, this separation can maintain the original fluorescence (FL) properties and sizes, which have been confirmed by transmission electron microscope (TEM), FL microscope, and atomic force microscope (AFM). In addition, the accumulation of uniform-sized AIEgens on the surface showed the positive correlation between AIEgens size and the landing distance, which helped to make a better collection of different size of AIEgens. This work provides a promising pathway to obtain particular monodispersed AIEgens with uniform sizes, which would be significant for further examinations and applications of AIE materials.

Biodegradable nanosyringes for intracellular amplification-based dual-diagnosis and gene therapy in single living cells.

The efficient delivery of biomolecules into living cells as well as their easy biodegradation have been challenges for the application of intracellular amplification for sensitive multiple-diagnosis and gene therapy for cancer. Herein, new strategies of amplification-based dual-detection of cancer biomarkers (Let-7a miRNA and VEGF) and gene therapy for cancers are put forward. These are achieved through biodegradable nanosyringes (NSs), rigid and sharp in vitro but degradable in vivo, which are applied for the efficient loading, delivery and release of biomolecules (enzymes, nucleic acids, and even silencing RNA) into living cells. After penetrating cell membranes and escaping from endosomes through their rigid and sharp tips, NSs release biomolecules for fast and easy "one-step" rolling circle amplification (ring formation and amplification) in single living cells. Therefore, based on signals from two probes, FAM-Probe and Cy5-Probe, that selectively bind to amplification products, 100 aM of Let-7a and 100 fM of VEGF could be detected, which are much lower than reported values. Furthermore, siRNAs can also be delivered by NSs for gene therapy, and their therapeutic effect was evaluated by their in vivo antitumor efficacy in CCRF-CEM subcutaneous xenograft nude mice. Rigid in vitro and degradable in vivo, NSs show potential for achieving fast, sensitive and safe cancer diagnosis and efficient therapy.