酶振荡"探针"研究生物活性氧的新进展

评述了研究生物活性氧的意义和方法，简要概述了时空振荡反应中酶振荡的某些研究进展，提出了以酶振荡“探针”研究生物活性氧的新方法。     

Electrochemical sensors for oxidative stress monitoring

Electrochemical sensors are ideally suited for the detection of reactive oxygen and nitrogen species (ROS and RNS) generated during biological processes. This review discusses the latest work in the development of electrochemical microsensors for ROS/RNS and their applications for monitoring oxidative stress in biological systems. The performance of recent designs of microelectrodes and electrode materials is discussed along with their functionality in preclinical models of drug efficacy, mitochondrial distress, and endothelial dysfunction. Challenges and opportunities in translating this methodology to study the pathophysiology associated with various diseases are discussed.     

Turn-on DNA damage sensors for the direct detection of 8-oxoguanine and photoproducts in native DNA

The integrity of the genetic information in all living organisms is constantly threatened by a variety of endogenous and environmental insults. To counter this risk, the DNA-damage response is employed for repairing lesions and maintaining genomic integrity. However, an aberrant DNA-damage response can potentially lead to genetic instability and mutagenesis, carcinogenesis, or cell death. To directly monitor DNA damage events in the context of native DNA, we have designed two new sensors utilizing genetically fragmented firefly luciferase (split luciferase). The sensors are comprised of a methyl-CpG binding domain (MBD) attached to one fragment of split luciferase for localizing the sensor to DNA (50-80% of the CpG dinucleotide sites in the genome are symmetrically methylated at cytosines), while a damage-recognition domain is attached to the complementary fragment of luciferase to probe adjacent nucleotides for lesions. Specifically, we utilized oxoguanine glycosylase 1 (OGG1) to detect 8-oxoguanine caused by exposure to reactive oxygen species and employed the damaged-DNA binding protein 2 (DDB2) for detection of pyrimidine dimer photoproducts induced by UVC light. These two sensors were optimized and validated using oligonucleotides, plasmids, and mammalian genomic DNA, as well as HeLa cells that were systematically exposed to a variety of environmental insults, demonstrating that this methodology utilizing MBD-directed DNA localization provides a simple, sensitive, and potentially general approach for the rapid profiling of specific chemical modifications associated with DNA damage and repair.     

Patterning, integration and characterisation of polymer optical oxygen sensors for microfluidic devices

This paper describes a process for the layer-by-layer fabrication and integration of luminescent dye-based optical oxygen sensors into microfluidic devices. Application of oxygen-sensitive platinum(ii) octaethylporphyrin ketone fluorescent dye dissolved in polystyrene onto glass substrates by spin-coating was studied. Soft lithography with polydimethylsiloxane (PDMS) stamps and reactive ion etching in oxygen plasma were used to produce sensor patterns with a minimum feature size of 25 microm. Sensors patterns were integrated into a PDMS microfluidic device by plasma bonding. No degradation of the sensor response as a result of the lithography and pattern-transfer processes was detected. Gaseous and dissolved oxygen (DO) detection was characterised using fluorescence microscopy. The intensity signal ratio of the sensor films was found to increase almost two-fold from 3.6 to 6.8 by reducing film thickness from 1.3 microm to 0.6 microm. Calibration of DO measurement showed linear Stern-Volmer behaviour that was constant for flow rates from 0.5 to 2 mL min(-1). The calibrated sensors were subsequently used to demonstrate laterally resolved detection of oxygen inside a microfluidic channel. The fabrication process provides a novel, easy to use method for the repeatable integration of optical oxygen sensors into cell-culture and lab-on-a-chip devices.     

Biochemistry of Oxidative Stress

As a normal attribute of aerobic life, structural damage to organic compounds of a wide variety (DNA, proteins, carbohydrates and lipids) may occur as a consequence of oxidative reactions. Oxidative damage inflicted by reactive oxygen species has been called “oxidative stress”. Biological systems contain powerful enzymatic and nonenzymatic antioxidant systems, and oxidative stress denotes a shift in the prooxidant/antioxidant balance in favor of the former. Diverse biological processes such as inflammation, carcinogenesis, ageing, radiation damage and photobiological effects appear to involve reactive oxygen species. This field of research provides new perspectives in biochemical pharmacology, toxicology, radiation biochemistry as well as pathophysiology.     

Highly selective two-photon fluorescent probe for imaging of nitric oxide in living cells简

A new two-photon fluorescent probe, ADNO, for nitric oxide （NO） based on intramolecular photoinduced electron transfer （PET） mechanism d/splays a rapid response to NO with a remarkable fluorescent enhancement in PBS buffer. The excellent chemoselectivity of ADNO for NO over other ROS/RNS （reactive oxygen species or nitrogen species） and common metal ions was observed. Moreover, ADNO has been successfully applied in fluorescence imaging of NO of living cells using both one-photon microscopy （OPM） and two-~hoton microscopy （TPM）,     

Cancerous cell death from sensitizer free photoactivation of singlet oxygen

Singlet oxygen ((1)O(2)) is an electronic state of molecular oxygen which plays a major role in many chemical and biological photo-oxidation processes. It has a high chemical reactivity which is commonly harnessed for therapeutic issues. Indeed, (1)O(2) is believed to be the major cytotoxic agent in photodynamic therapy. In this treatment of cancer, (1)O(2) is created, among other reactive species, by an indirect transfer of energy from light to molecular oxygen via excitation of a photosensitizer (PS). This PS is believed to be necessary to obtain an efficient (1)O(2) production. In this paper, we demonstrate that production of (1)O(2) is achieved in living cells from PS-free 1270 nm laser excitation of molecular oxygen. The quantity of (1)O(2) produced in this way is sufficient to induce an oxidative stress leading to cell death. Other effects such as thermal stress are discriminated and we conclude that cell death is only due to (1)O(2) creation. This new simplified scheme of (1)O(2) activation can be seen as a breakthrough for phototherapies of malignant diseases and/or as a noninvasive possibility to generate reactive oxygen species in a tightly controlled manner.     

新型钙荧光探针在H2O2对HL-60胞浆钙影响研究中的应用

采用H2O2、黄嘌呤和黄嘌呤氧化酶、H2O2/抗坏血酸/Fe^2＋（或H2O2/Fe^＋）、H2O2和辣根过氧化酶（HRP）等不同活性氧产生体系,对自行合成的胞浆特异性钙荧光探针STDIn的活性氧耐受性进行了考察.以STDIn-AM为探针标记HL-60细胞,激光共聚焦技术检测单细胞内Ca^2＋-STDIn荧光强度的变化;探讨了不同浓度的H2O2对胞内Ca^2＋浓度变化的影响,从膜结构和生物信号传导的角度研究了氧自由基对培养细胞的生物效应.     

Singlet oxygen in plants--its significance and possible detection with double (fluorescent and spin) indicator reagents

Direct detection of reactive oxygen species (ROS), especially singlet oxygen, in plants under stress conditions is of special importance, not only to identify primary events of oxidative damage, but also in studies exploring the potential role of ROS as signal molecules. Due to short life-times and diffusion distances of ROS, these tasks require highly reactive and selective indicator reagents, localized at the presumed site of production. In the present study, we compared four double sensors: ROS indicator reagents in which partial fluorescence quenching of a dansyl moiety occurs as a result of nitroxide radical formation from a sterically hindered amine constituent. Our experiments support the idea that shorter donor-acceptor distances within these molecules result in higher reactivity to ROS. The presence of a diethylaminoethyl side chain resulted in better selectivity to singlet oxygen: reagents lacking such substituent had an additional reactivity to superoxide anions, probably as a result of the formation of zwitterionic structures. Fluorescence localization studies of the indicator reagents in tobacco leaves and in Chlamydomonas cells show promising perspectives of their applications to plant stress studies.     

ROS-responsive cyclodextrin nanoplatform for combined photodynamic therapy and chemotherapy of cancer

Cyclodextrin(CD) has special spatial structure and well biological safety,so it has been widely used for constructing CD-based na noplatforms.Through functionalization,cyclodextrin can form various stimulusresponse nanoplatforms,such as pH,temperature,redox,light and magnetic fields.In this study,we designed a highly sensitive reactive oxygen species(ROS)-responsive polymer PCP which encapsulated doxorubicin(DOX) and purpurin 18(P18) to achieve the syne rgy of photodynamic and chemotherapy.The high content of reactive oxygen species(ROS) in the tumor microenvironment(TME) triggers the cleavage of the borate bond of MPEG-CD-PHB(PCP),thereby promoting the re lease of drugs.When irradiated with nea rinfrared laser,the photosensitizer P18 released by polymer micelles can produce reactive oxygen species to promote cell apoptosis.Compared with monotherapy,a series of experiments confirmed that our micelles had enhanced anti-cancer activity.This work was beneficial to the design of ROS-responsive materials and provides an effective strategy for the application of collaborative anti-tumor therapy.     

A protein-targeted photosensitizer for highly efficient cancer therapy

Photodynamic therapy typically employs photo-triggered photosensitizers to generate reactive oxygen species to destroy cancer cells. However, the therapeutic effect of photodynamic therapy is often limited owing to the ultrashort diffusion distance of reactive oxygen species and easy efflux of photosensitizers. Herein, we design and synthesize a protein-targeted molecular photosensitizer for highly efficient photodynamic therapy. The designed photosensitizer can covalently bind with the sulfhydryl groups of intracellular proteins to achieve the protein targeting. Under irradiated with near infrared laser, the photosensitizer was locally activated, and the produced reactive oxygen species directly destroy intracellular bioactive proteins, causing cell dysfunction and ultimately inducing cell apoptosis. Significantly, the leakage of molecular photosensitizer is effectually avoided due to the protein targeting. In vivo experimental results indicated that the effect of treatment was efficiently enhanced with the protein-targeted strategy. This work can offer new insights for designing protein-based therapeutic drugs.     

Interplay between O2 and SnO2: oxygen ionosorption and spectroscopic evidence for adsorbed oxygen.

Tin dioxide is the most commonly used material in commercial gas sensors based on semiconducting metal oxides. Despite intensive efforts, the mechanism responsible for gas-sensing effects on SnO(2) is not fully understood. The key step is the understanding of the electronic response of SnO(2) in the presence of background oxygen. For a long time, oxygen interaction with SnO(2) has been treated within the framework of the "ionosorption theory". The adsorbed oxygen species have been regarded as free oxygen ions electrostatically stabilized on the surface (with no local chemical bond formation). A contradiction, however, arises when connecting this scenario to spectroscopic findings. Despite trying for a long time, there has not been any convincing spectroscopic evidence for "ionosorbed" oxygen species. Neither superoxide ions O(2)(-), nor charged atomic oxygen O,(-) nor peroxide ions O(2)(2-) have been observed on SnO(2) under the real working conditions of sensors. Moreover, several findings show that the superoxide ion does not undergo transformations into charged atomic oxygen at the surface, and represents a dead-end form of low-temperature oxygen adsorption on reduced metal oxide.     

氧气常压介质阻挡放电的发射光谱及能量传递机理

为研究氧气常压介质阻挡放电中的物理化学行为, 以纯氧作为放电体系, 用发射光谱(optical emission spectroscopy)诊断技术分析了等离子体中可能存在的化学活性物种. 利用在500-950 nm范围的氧原子发射光谱计算出等离子体中的电子温度为(1.02±0.03) eV; 观测了760 nm处的具有清晰转动结构的氧气A带(atmospheric band)O2(b1∑+g-X3∑-g), 并用其转动结构计算了转动温度(气体温度)为(650±20) K; 在500-700 nm范围观测了氧气的第一负带系(first negative system) O+2(b4∑-g-a4∏u), 在190-240 nm范围观测了微弱但特征清晰的氧气的Hopfield带系O+2(c4∑+u-b4∑-g). 研究发现, 在氧气常压介质阻挡放电等离子体中存在多种激发态氧原子、激发态氧气分子、基态和激发态氧气分子离子等反应活性物种, 这些活性物种的形成涉及氧气分子的激发、解离和电离等多种过程, 每个过程都包含多个能量传递步骤, 氧分子解离产生的氧原子是导致一系列高激发态氧原子生成和氧气电离激发的主要因素.     

Skin photosensitizing agents and the role of reactive oxygen species in photoaging.

In this paper, the role of reactive oxygen species in photoaging is presented. Many photosensitizing agents are known to generate reactive oxygen species (singlet oxygen (1O2), superoxide anion (O2.-) and .OH radicals). Although photoaging (dermatoheliosis) of human skin is caused by UVB and UVA radiation, the hypothesis tested here in the pathogenesis of photoaging of human skin is the free radical theory involving the generation of reactive oxygen species by UVA (320-400 nm) radiation and their damaging oxidative effects on cutaneous collagen and other model proteins. The UVA-generated reactive oxygen species cause cross-linking of proteins (e.g. collagen), oxidation of sulfydryl groups causing disulfide cross-links, oxidative inactivation of certain enzymes causing functional impairment of cells (fibroblasts, keratinocytes, melanocytes, Langerhans cells) and liberation of proteases, collagenase and elastase. The skin-damaging effects of UVA appear to result from type II, oxygen-mediated photodynamic reactions in which UVA or near-UV radiation in the presence of certain photosensitizing chromophores (e.g., riboflavin, porphyrins, nicotinamide adenine dinucleotide phosphate (NADPH), etc.) leads to the formation of reactive oxygen species (1O2, O2.-, .OH). Four specific observations are presented to illustrate the concept: (1) the production of 1O2 and O2.- by UVB, UVA and UVA plus photosensitizing agents (such as riboflavin, porphyrin and 3-carbethoxypsoralens) as a function of UV exposure dose, the sensitizer concentration and the pH of the irradiated solution; (2) the formation of protein cross-links in collagen, catalase and superoxide dismutase by 1O2 and O2.- (.OH) and the resulting denaturation of proteins and enzyme activities as a function of UVA exposure dose; (3) the protective role of selective quenchers of 1O2 and O2.- (e.g. alpha-tocopherol acetate, beta-carotene, sodium azide, ascorbic acid, etc.) against the photoinactivation of enzymes and the prevention of the protein cross-linking reaction; (4) the possible usefulness of certain antioxidants or quenchers that interact with the UVA-induced generation of reactive oxygen species in the amelioration of the process of photoaging.     

光动力抗菌光敏剂的研究进展

光动力抗菌化学疗法是一种结合光敏剂分子和可见光产生的活性氧物种杀灭病原微生物的抗感染治疗方法.活性氧物种能够与致病菌中的多种生物活性分子反应,这一特性使得微生物不易对该方法产生耐药性,这也是该方法近年来备受关注的主要原因.本文重点介绍了近年来光动力抗菌化学疗法领域新型光敏剂药物的研究进展,包括卟啉类衍生物、BODIPY化合物、共轭聚合物和钌多吡啶配合物.     

Synthesis and characterization of a new fluorescent probe for reactive oxygen species

We report the development of a new fluorescence sensor for reactive oxygen species (ROS) based on a benzofurazan structure. The sensor, NBFhd, is initially non-fluorescent and reacts with peroxyl radicals by hydrogen transfer in an aqueous medium under neutral conditions to release the fluorescent N-methyl-4-amino-7-nitrobenzofurazan (NBF) and 1,4-benzoquinone. NBFhd shows excellent contrast and no interference in the region of cell autofluorescence and is a new tool to detect ROS in homogeneous and biological systems.     

A highly selective fluorescent probe for the detection and imaging of peroxynitrite in living cells

We have found a specific reaction between ketone 1 and peroxynitrite (ONOO-), rather than other reactive oxygen species and reactive nitrogen species generated in the biological system. On the basis of this reaction, we have successfully developed a new fluorescent probe HKGreen-1, which is highly selective for the detection of peroxynitrite in living cells. Before the oxidation with peroxynitrite, the dichlorofluorescein part is masked and the probe is nonfluorescent. However, upon reaction with peroxynitrite, the fluorophore is released, resulting in strong enhancement in fluorescence intensity.     

A Novel Phenoxazine-based Fluorescent Probe for the Detection of HOCl in Living Cells

Hypochlorous acid (HOCl), one of the reactive oxygen species (ROS), is a key microbicidal agent which is used for natural defense. However, it is also linked to varieties of human diseases owing to the overproduction of HOCl. Much effort has been made to exploit selective fluorescent sensors for the detection of HOCl, but most of them have some disadvantages such as short excitation wavelength, low selectivity, and slow response and so on. These restrict the biological application of the probes. In this work, BR-O was designed and synthesized on the base of phenoxazine for the detection of HOCl. BR-O exhibited a violent fluorescence enhancement in the presence of HOCl, showing excellent selectivity and high sensitivity. More importantly, the probe BR-O was capable of detecting exogenous and endogenous HOCl in living cells.     

Initiator‐Loaded Gold Nanocages as a Light‐Induced Free‐Radical Generator for Cancer Therapy

Tumor hypoxia greatly suppresses the therapeutic efficacy of photodynamic therapy (PDT), mainly because the generation of toxic reactive oxygen species (ROS) in PDT is highly oxygen‐dependent. In contrast to ROS, the generation of oxygen‐irrelevant free radicals is oxygen‐independent. A new therapeutic strategy based on the light‐induced generation of free radicals for cancer therapy is reported. Initiator‐loaded gold nanocages (AuNCs) as the free‐radical generator were synthesized. Under near‐infrared light (NIR) irradiation, the plasmonic heating effect of AuNCs can induce the decomposition of the initiator to generate alkyl radicals (R^.), which can elevate oxidative‐stress (OS) and cause DNA damages in cancer cells, and finally lead to apoptotic cell death under different oxygen tensions. As a proof of concept, this research opens up a new field to use various free radicals for cancer therapy.     

氧对n-BuLi/THF引发丁二烯苯乙烯共聚合的影响

研究了氧分子与活性种快速失活的定量关系 ,二代杂质对活性种及聚合物微观结构的影响 .从聚合动力学及聚合物相对分子质量等方面来测定氧分子快速杀死活性种数 ,并考察了体系中氧产生的偶联反应及其对丁苯共聚物物理机械性能、动态力学性能及耐磨性能的影响 .结果表明 :氧对n BuLi THF体系丁苯共聚合有不利影响 .