Versatile Fluorescent Probes for Imaging the Superoxide Anion in Living Cells and In Vivo

The superoxide anion (O₂^.?) is widely engaged in the regulation of cell functions and is thereby intimately associated with the onset and progression of many diseases. To ascertain the pathological roles of O₂^.? in related diseases, developing effective methods for monitoring O₂^.? in biological systems is essential. Fluorescence imaging is a powerful tool for monitoring bioactive molecules in cells and in vivo owing to its high sensitivity and high temporal‐spatial resolution. Therefore, increasing numbers of fluorescent imaging probes have been constructed to monitor O₂^.? inside live cells and small animals. In this minireview, we summarize the methods for design and application of O₂^.?‐responsive fluorescent probes. Moreover, we present the challenges for detecting O₂^.? and suggestions for constructing new fluorescent probes that can indicate the production sites and concentration changes in O₂^.? as well as O₂^.?‐associated active molecules in living cells and in vivo.     

A Novel Fluorescent Probe for Hydrogen Peroxide and Its Application in Bio-Imaging

Hydrogen peroxide (H₂O₂) plays an important role in the human body and monitoring its level is meaningful due to the relationship between its level and diseases. A fluorescent sensor (CMB) based on coumarin was designed and its ability for detecting hydrogen peroxide by fluorescence signals was also studied. The CMB showed an approximate 25-fold fluorescence enhancement after adding H₂O₂ due to the interaction between the CMB and H₂O₂ and had the potential for detecting physiological H₂O₂. It also showed good biocompatibility and permeability, allowing it to penetrate cell membranes and zebrafish tissues, thus it can perform fluorescence imaging of H₂O₂ in living cells and zebrafish. This probe is a promising tool for monitoring the level of H₂O₂ in related physiological and pathological research.     

A High Sensitivity Detection Method of Singlet Oxygen and Superoxide Anion

This paper, for the first time, reports a method that can be used as a highly sensitive probe for singlet oxygen (^1O2) and superoxide anion (O2^-) in vitro or in vivo. FCLA(3,7-dihydro-6-{4-[2-(N‘-(5-fluoresceinyl)thioureido)ethoxy]phenyl}-2-methylimidazo{1,2-a}pyrazin-3-one sodium salt), a chemiluminescence (CL) analysis reagent, has been reported to sensitively react with ^1O2 and O2^- to emit photons with a spectral peak of 525nm. In this work,when human serum albumin (HSA) was added into FCLA solution to enhance the CL intensity,approximately 20 times, compared to that without HSA. The enhanced CL had the same 525 nm spectral peak, identical to that without HSA. By gradually reducing the molecular oxygen content in the solution, we find that the auto-oxidation of oxygen molecules dissolved in the solution plays an important role in the CL process. Based on these experimental evidences, we propose a novel and highly sensitive detection method of ^1O2 and O2^- which may have a great potential in chemical and medical applications.     

若干锰(Ⅱ)配合物的合成及其清除活性氧效能的研究

合成了三种锰(Ⅱ)配合物,通过多种物理方法进行了表征,推断了它们的结构式。用核黄素光照法测定了它们的超氧化物歧化酶活性,并对其清除多形核白细胞(PMNL)呼吸爆发产生的活性氧及黄嘌呤与黄嘌呤氧化酶体系产生的·O₂^-及H₂O₂-Fe²⁺产生的·OH的效能进行了研究,结果表明能降低活性氧导致的鲁米诺化学发光值;降低·O₂^-与DMPO加合物的ESR波谱信号,减少·O₂^-损伤膜提取物产生的脂类过氧化物产量;对·OH与DMPO加合物的ESR波谱无影响,说明这三种配合物确具有SOD样活性。     

Redox Reactions of Biologically Active Molecules upon Cold Atmospheric Pressure Plasma Treatment of Aqueous Solutions

Cold atmospheric pressure plasma (CAPP) is widely used in medicine for the treatment of diseases and disinfection of bio-tissues due to its antibacterial, antiviral, and antifungal properties. In agriculture, CAPP accelerates the imbibition and germination of seeds and significantly increases plant productivity. Plasma is also used to fix molecular nitrogen. CAPP can produce reactive oxygen and nitrogen species (RONS). Plasma treatment of bio-tissue can lead to numerous side effects such as lipid peroxidation, genotoxic problems, and DNA damage. The mechanisms of occurring side effects when treating various organisms with cold plasma are unknown since RONS, UV-Vis light, and multicomponent biological tissues are simultaneously involved in a heterogeneous environment. Here, we found that CAPP can induce in vitro oxidation of the most common water-soluble redox compounds in living cells such as NADH, NADPH, and vitamin C at interfaces between air, CAPP, and water. CAPP is not capable of reducing NAD⁺ and 1,4-benzoquinone, despite the presence of free electrons in CAPP. Prolonged plasma treatment of aqueous solutions of vitamin C, 1,4-hydroquinone, and 1,4-benzoquinone respectively, leads to their decomposition. Studies of the mechanisms in plasma-induced processes can help to prevent side effects in medicine, agriculture, and food disinfection.     

超氧负离子基与槲皮素的反应

氧的浓度超过正常值会引起毒性效应。超氧负离子基(O_2~(?))作为生物体内代谢产生的一种活性中间体,在发现超氧化物歧化酶(SOD)是其专一清除剂后,引起人们广泛的兴趣,并对 O_2~(?)与人类的某些疾病和癌症的关系进行了研究。由于·OH、~1O_2和 HO_2等也是高活性的物质,有人认为在研究 O_2~(?)的毒害效应时,不应忽视这些由 O_2~(?)可衍生出来的活性氧。为阐明 O_2~(?)在生物体内的作用,还需对 O_2~(?)化学作更多更深入的基础工作。黄酮类化合物是一大类重要的有生化意义的化合物,已有许多关于槲皮素氧化反应的报     

嗜中性粒细胞中过氧化氢及其释放过程的电化学检测

活性氧（ROS）是细胞代谢的重要产物之一,其浓度与多种生理、病理现象密切相关;H2O2是ROS的重要代表,对细胞内H2O2及其释放过程的测定有助于理解相关疾病的形成机制,因而具有重要的意义.本文报道一种检测细胞内H2O2浓度、监测其生成和释放过程的电化学方法,该方法利用固定在天然矿物凹土（attapulgite,全称凹凸棒石黏土）表面的血红蛋白（Hb）对H2O2还原的电催化作用实现H2O2的定量测定.将Hb固定到凹土表面制备了Hb-凹土（Hb-Atta）纳米复合材料,用透射电子显微镜（TEM）、原子力显微镜（AFM）、红外光谱（FTIR）、紫外-可见光谱（UV-vis）等对其进行了表征;将该复合物修饰到玻碳（GC）电极表面制成了Hb-Atta/GC电极,用伏安法考察了Hb在凹土表面的直接电子转移性能,结果表明,Hb能进行有效的直接电子转移反应,其伏安曲线上呈现出一对良好的氧化还原峰,式量电位（E0′）约为-350mV（vs.SCE,50mV/s）;进一步的结果表明,Hb-Atta对H2O2的电化学还原具有良好的催化性能,并能用于H2O2的定量测定,具有响应速率快、线性范围宽、检测限低等优点.将Hb-Atta/GC电极应用于嗜中性粒细胞内H2O2的测定,结果表明,该电极不仅能定量地检测细胞内的H2O2含量,还能监测细胞内H2O2生成和释放的过程,而且细胞内其他ROS和电活性物质如OCl-、NO.、ONOO-和抗坏血酸等不产生干扰.本文的结果为测量细胞内H2O2浓度、研究其生成及释放过程动力学以及进一步阐明由H2O2导致的相关疾病机制等提供了一个新的平台,可望在生物电化学、细胞生物学和病理生理学等领域得到应用.     

Biological Risk Assessment of Three Dental Composite Materials following Gas Plasma Exposure

Gas plasma is an approved technology that generates a plethora of reactive oxygen species, which are actively applied for chronic wound healing. Its particular antimicrobial action has spurred interest in other medical fields, such as periodontitis in dentistry. Recent work has indicated the possibility of performing gas plasma-mediated biofilm removal on teeth. Teeth frequently contain restoration materials for filling cavities, e.g., resin-based composites. However, it is unknown if such materials are altered upon gas plasma exposure. To this end, we generated a new in-house workflow for three commonly used resin-based composites following gas plasma treatment and incubated the material with human HaCaT keratinocytes in vitro. Cytotoxicity was investigated by metabolic activity analysis, flow cytometry, and quantitative high-content fluorescence imaging. The inflammatory consequences were assessed using quantitative analysis of 13 different chemokines and cytokines in the culture supernatants. Hydrogen peroxide served as the control condition. A modest but significant cytotoxic effect was observed in the metabolic activity and viability after plasma treatment for all three composites. This was only partially treatment time-dependent and the composites alone affected the cells to some extent, as evident by differential secretion profiles of VEGF, for example. Gas plasma composite modification markedly elevated the secretion of IL6, IL8, IL18, and CCL2, with the latter showing the highest correlation with treatment time (Pearson’s r > 0.95). Cell culture media incubated with gas plasma-treated composite chips and added to cells thereafter could not replicate the effects, pointing to the potential that surface modifications elicited the findings. In conclusion, our data suggest that gas plasma treatment modifies composite material surfaces to a certain extent, leading to measurable but overall modest biological effects.     

电催化过氧化氢还原的纳米材料作为潜在的辐射防护剂

纳米材料由于独特的物理化学性质,在生物医学领域显示出许多潜在的应用前景,诸如医学成像、药物输运和生物传感等. 这篇综述总结了对过氧化氢和氧还原表现出好的电催化活性的一些纳米材料显示了辐射防护性能. 作者讨论了这些纳米材料的辐射防护性能来源于它们的类酶活性,因为它们的催化性质表现为和活性氧的快速反应,为清除体内的自由基提供了一条有效通道. 作者也提出了纳米材料的电催化活性和作为临床转化关键的辐射防护性能之间关系的见解. 最后,作者指出了这些纳米材料作为新的辐射防护剂用于辐射防护治疗辅助成份所面临的挑战和将来的研究方向.     

Tandem Payne/Dakin Reaction: A New Strategy for Hydrogen Peroxide Detection and Molecular Imaging

Hydrogen peroxide (H₂O₂) has been recognized as one of the most significant ROS (reactive oxygen species) in human health and disease. Because of the intrinsic attributes of H₂O₂—such as its low reactivity under physiological pH—it is exceedingly challenging to develop small‐molecule fluorescent probes with high selectivity and sensitivity for visualization of H₂O₂ in an intricate biological milieu. To address this gap, a rationally designed tandem Payne/Dakin reaction is reported that is specific to molecular recognition of H₂O₂. New H₂O₂ probes based on this unique chemical strategy can be easily synthesized by a general coupling reaction, and the practical applicability of those probes has been confirmed by the visualization of endogenously produced H₂O₂ in living cells. In particular, starvation‐induced H₂O₂ production in mouse macrophages has been detected by the novel probe in both confocal imaging and flow cytometry. This tandem Payne/Dakin reaction provides a basis for developing more sophisticated molecular tools to interrogate H₂O₂ functions in biological phenomena.     

Highly Selective Nanostructured Electrochemical Sensor Utilizing Densely Packed Ultrathin Gold Nanowires Film

The number of studies conducted about nonenzymatic electrochemical sensors has increased in recent years due to the development of more stable and robust electrodes using noble metals. One of the key aspects for achieving high sensing performance including detection limit and sensitivity is the design of electrode architecture. Herein, we report a new electrochemical sensing platform featuring ultrathin standing gold nanowires (AuNWs) for nonenzymatic detection of hydrogen peroxide (H₂O₂). The use of AuNWs resulted in an increased electron transfer efficiency due to the higher active surface area compared to traditional gold film electrodes. This sensor demonstrates good selectivity, reproducibility, a linear range up to 49.5 mM of H₂O₂ with a sensitivity of 0.185±0.003 mAmM^?1cm^?2 and a limit of detection of 111 μM. The biological relevance of this sensor was tested in cell culture media to illustrate the performance of the proposed sensing electrode in complex biological media.     

Direct Detection of the Superoxide Anion as a Stable Intermediate in the Electroreduction of Oxygen in a Non‐Aqueous Electrolyte Containing Phenol as a Proton Source

The non‐aqueous Li–air (O₂) battery has attracted intensive interest because it can potentially store far more energy than today′s batteries. Presently Li–O₂ batteries suffer from parasitic reactions owing to impurities, found in almost all non‐aqueous electrolytes. Impurities include residual protons and protic compounds that can react with oxygen species, such as the superoxide (O₂^?), a reactive, one‐electron reduction product of oxygen. To avoid the parasitic reactions, it is crucial to have a fundamental understanding of the conditions under which reactive oxygen species are generated in non‐aqueous electrolytes. Herein we report an in situ spectroscopic study of oxygen reduction on gold in a dimethyl sulfoxide electrolyte containing phenol as a proton source. It is shown directly that O₂^?, not HO₂, is the first stable intermediate during the oxygen reduction process to hydrogen peroxide. The unusual stability of O₂^? is explained using density functional theory (DFT) calculations.     

Singlet-Oxygen-Induced Phospholipase A2 Inhibition: A Major Role for Interfacial Tryptophan Dioxidation

Several studies have revealed that various diseases such as cancer have been associated with elevated phospholipase A₂ (PLA₂) activity. Therefore, the regulation of PLA₂ catalytic activity is undoubtedly vital. In this study, effective inactivation of PLA₂ due to reactive species produced from cold physical plasma as a source to model oxidative stress is reported. We found singlet oxygen to be the most relevant active agent in PLA₂ inhibition. A more detailed analysis of the plasma-treated PLA₂ identified tryptophan 128 as a hot spot, rich in double oxidation. The significant dioxidation of this interfacial tryptophan resulted in an N-formylkynurenine product via the oxidative opening of the tryptophan indole ring. Molecular dynamics simulation indicated that the efficient interactions between the tryptophan residue and phospholipids are eliminated following tryptophan dioxidation. As interfacial tryptophan residues are predominantly involved in the attaching of membrane enzymes to the bilayers, tryptophan dioxidation and indole ring opening leads to the loss of essential interactions for enzyme binding and, consequently, enzyme inactivation.     

Effect of Cation Size on Solvation and Association with Superoxide Anion in Aprotic Solvents

Influence of cation size on solvation strength, diffusion, and kinetics of the association reaction with anions O₂⁻ in aprotic solvents, such as acetonitrile and dimethyl sulfoxide, has been investigated by means of molecular dynamics simulations. The work is motivated by the need to understand the molecular nature of the solvent-induced changes in capacity of Li-air batteries. We have shown that the dependence of the solvation shell stability on the cation size has a maximum at a particular ion radius that corresponds to a solvent coordination number of 4. The shell stability maximum coincides with the diffusion coefficient minimum. The variation of the cation shell stability has a crucial impact on the kinetics of the cation-O₂⁻ association. We have demonstrated that profound inhibition of the association reaction for Li⁺ in dimethyl sulfoxide is a result of the lock-and-key effect that cannot be described in the framework of Hard Soft Acid Base theory.     

The Reaction of Muonium with Hydrogen Peroxide in Aqueous Solution

Rate constants for the reactions of muonium (Mu) (the ultralight isotope of the hydrogen atom) with H₂O₂ in H₂O and D₂O₂ in D₂O have been determined at various temperatures and pH (pD) values. The data are consistent with the three reactions: , , and the equivalent for the deuterated entities. A significant positive H/D isotope effect was found for the undissociated peroxide, while for the anions the effect was negligible or slightly in the opposite direction. In addition, for concentrated solutions of peroxide a study of the muon spin polarization as a function of applied transverse magnetic field yielded results consistent with the rate constants determined from the direct decay measurements, and indicated that the reaction products are diamagnetic, most likely MuH and MuOH, i. e., no muoniated radical products are formed. These results are potentially relevant for management of the radiolysis products in nuclear industry.     

光泽精在肾上腺素自氧化体系中的电化学研究

光泽精与在肾上腺素自氧化体系中（ｐＨ＝１０．２０）产生的超氧阴离子自由基反应,其氧化产物在 －１．１６ Ｖ（ｖｓ．ＳＥＣ）和－１．６０Ｖ（ｖｓ． ＳＥＣ）处有两个还原峰,其中－１．１６ Ｖ处的还原峰是光泽精被氧化的中 间产物的双电子还原峰,－１．６０Ｖ处的还原峰是最终产物 Ｎ－甲基吖啶酮（ＮＭＡ）的双电子还原峰。形成 反应中间体和 ＮＭＡ的反应是一级反应,其反应速度常数分别为 ２．０７× １０－４ｓ－１和 ２．２３ × １０－５ｓ－１。     

Apigenin Isolated from Carduus crispus Protects against H2O2-Induced Oxidative Damage and Spermatogenic Expression Changes in GC-2spd Sperm Cells

Testicular oxidative stress is one of the most common factors underlying male infertility. Welted thistle, Carduus crispus Linn., and its bioactive principles are attracting scientific interest in treating male reproductive dysfunctions. Here, the protective effects of apigenin isolated from C. crispus against oxidative damage induced by hydrogen peroxide (H₂O₂) and dysregulation in spermatogenesis associated parameters in testicular sperm cells was investigated. Cell viabilities, ROS scavenging effects, and spermatogenic associated molecular expressions were measured by MTT, DCF-DA, Western blotting and real-time RT-PCR, respectively. A single peak with 100% purity of apigenin was obtained in HPLC conditions. Apigenin treated alone (2.5, 5, 10 and 20 µM) did not exhibit cytotoxicity, but inhibited the H₂O₂-induced cellular damage and elevated ROS levels significantly (p < 0.05 at 5, 10 and 20 µM) and dose-dependently. Further, H₂O₂-induced down-regulation of antioxidant (glutathione S-transferases m5, glutathione peroxidase 4, and peroxiredoxin 3) and spermatogenesis-associated (nectin-2 and phosphorylated-cAMP response element-binding protein) molecular expression in GC-2spd cells were attenuated by apigenin at both protein and mRNA levels (p < 0.05). In conclusion, our study showed that apigenin isolated from C. crispus might be an effective agent that can protect ROS-induced testicular dysfunctions.     

Gas Plasma-Augmented Wound Healing in Animal Models and Veterinary Medicine

The loss of skin integrity is inevitable in life. Wound healing is a necessary sequence of events to reconstitute the body’s integrity against potentially harmful environmental agents and restore homeostasis. Attempts to improve cutaneous wound healing are therefore as old as humanity itself. Furthermore, nowadays, targeting defective wound healing is of utmost importance in an aging society with underlying diseases such as diabetes and vascular insufficiencies being on the rise. Because chronic wounds’ etiology and specific traits differ, there is widespread polypragmasia in targeting non-healing conditions. Reactive oxygen and nitrogen species (ROS/RNS) are an overarching theme accompanying wound healing and its biological stages. ROS are signaling agents generated by phagocytes to inactivate pathogens. Although ROS/RNS’s central role in the biology of wound healing has long been appreciated, it was only until the recent decade that these agents were explicitly used to target defective wound healing using gas plasma technology. Gas plasma is a physical state of matter and is a partially ionized gas operated at body temperature which generates a plethora of ROS/RNS simultaneously in a spatiotemporally controlled manner. Animal models of wound healing have been vital in driving the development of these wound healing-promoting technologies, and this review summarizes the current knowledge and identifies open ends derived from in vivo wound models under gas plasma therapy. While gas plasma-assisted wound healing in humans has become well established in Europe, veterinary medicine is an emerging field with great potential to improve the lives of suffering animals.     

A 3,7‐Dihydroxyphenoxazine‐based Fluorescent Probe for Selective Detection of Intracellular Hydrogen Peroxide

A novel N‐borylbenzyloxycarbonyl‐3,7‐dihydroxyphenoxazine fluorescent probe (NBCD) for detecting H₂O₂ in living cells is described. The probe could achieve high selectivity for detecting H₂O₂ over other biological reactive oxygen species (ROS). In addition, upon addition of H₂O₂, NBCD exhibited color change from colorless to pink, which makes it a “naked‐eye” probe for H₂O₂ detection. NBCD could not only be used to detect enzymatically generated H₂O₂ but also to detect H₂O₂ in living systems by using fluorescence spectroscopy, with a detection limit of 2 μm . Importantly, NBCD enabled the visualization of epidermal growth factor (EGF)‐stimulated H₂O₂ generation inside the cells.