A coumarin-containing Schiff base fluorescent probe with AIE effect for the copper(ii) ion

A novel coumarin-derived Cu²⁺-selective Schiff base fluorescent “turn-off” chemosensor CTPE was successfully obtained, which showed an AIE effect. It could identify Cu²⁺ by quenching its fluorescence. The lower limit of detection was 0.36 μM. CTPE can act as a highly selective and sensitive fluorescence probe for detecting Cu²⁺.

A novel coumarin-derived Schiff base fluorescent “turn-off” chemosensor with AIE effect showed selectivity towards Cu²⁺. The recognition mechanism is presented.

The specific detection of transition metal ions has received considerable attention because of their significant roles in the fields of biological, chemical, medical and environmental processes.¹ Among these metal ions, copper(ii), acting as one of the most important micronutrients, is of particular interest due to its essential role in a variety of fundamental physiological processes. In spite of the fact that copper(ii) is essential in living systems, accumulation of copper(ii) can lead to serious environmental and health problems.² For instance, the metabolic balance of Cu²⁺ in the body being destroyed can lead to neurodegenerative diseases, such as Alzheimer''s, Parkinson''s, Menkes, and Wilson''s disease.^3–8 Besides, free Cu²⁺ is regarded as a significant environmental pollutant.^9,10 Therefore, an increasingly convenient and fast method to detect Cu²⁺ ion existing in environmental and biological resources is currently of considerably important. By far there existing many methods make it possible to detect and quantify Cu²⁺ ion, such as atomic absorption spectrometry,^11,12 inductively coupled plasma mass spectrometry,¹³ inductively coupled plasma-atomic emission spectrometry¹⁴ and voltammetry,^15,16etc. However, these methods require tedious sample preparation procedures and complex instrumentation, which limit their prosperous applications. Alternatively, more and more attention has been attracted to the analytical methods based on fluorescent chemosensors for the detection of the metal ions owing to their high sensitivity, selectivity, and simplicity.¹⁷ As a result, various types of fluorescent probes such as organic dyes, magnetic nanoparticles (MNPs), semiconductor quantum dots (QDs), carbon dots (CDs), fluorescent metal nanoclusters (NCs), and fluorescent metal organic frameworks (MOFs) have been designed and prepared to determine Cu²⁺ ion.^18–27 Since first reported in 2000, MNPs are extensively used in the field of nanotechnology for its nonhazardous feature, strong magnetization values, superparamagnetic property, active surface that can easily assembled of biological soluble structure and targeting, imaging, and therapeutic molecules.²² Unlike organic fluorescent dyes, QDs are semiconductor particles which exhibit high photochemical stability, excellent resistance to chemical degradation, outstanding photodegradation and extremely large Stokes shift.²³ CDs is a comprehensive term for various nanosized carbon materials including graphene quantum dots (GQDs), carbon nanodots (CNDs), and polymer dots (PDs)²⁴ which shows excellent properties including high stability, bio-compatibility, low photo bleaching and toxicity and so on.²⁵ Despite successfully applying in detecting Cu²⁺, traditional fluorescent probes are still puzzled by the toxicity, poor solubility, “aggregation-caused quenching” (ACQ) effect, etc. Thus, preparing fluorescent probes that can compensate for these deficiencies is still imminently desired in the Cu²⁺ ion detection.Coumarin derivatives are well known excellent fluorophores in the fluorescent probe design and synthesis with the advantages of high fluorescence intensity, excellent solubility, efficient cell permeation, high quantum yield and ease of preparation.^28,29 As a result, an increasing number of papers concerning coumarin derivatives fluorescent chemosensors for copper(ii) ion have emerged.^30–33 In 2009, Lee Jin Yong and co-workers developed a novel coumarin-based fluorogenic probe, which can act as a fluorescent chemosensor with high selectivity and suitable affinity in biological systems toward Cu²⁺ (ref. 34). Besides, in 2019, Yin Jiqiu and co-workers obtained a fluorescent chemosensor concerning coumarin derivatives for copper(ii) which exhibited good sensitivity, fast response time and high selectivity for Cu²⁺ ion in the presence of other important relevant metal ions.³⁵In 2001, Tang Benzhong and co-workers proposed the concept of “aggregation-induced emission” (AIE).³⁶ Due to their effective in circumventing the ACQ effect, AIE-active materials provide a new path for the design and synthesis of fluorescent probes. Tetraphenylethene (TPE), one of the most commonly used aggregation-induced emission luminogens (AIEgens), has been widely favored by researchers.^37,38 Owing to its simple structure, simple synthesis, easy modification, and obvious AIE effect, TPE is usually used as an ideal model for construction of various fluorescent sensors.³⁹ In 2019, Zhao Feng and co-workers designed and synthesized a new tetraphenylethene-based Schiff base ligand with AIE effect, and it could be utilized as optical recording materials.⁴⁰ The same year, Ni Zhonghai and co-workers obtained two different polymorphs of a new tetraphenylethene-based Schiff base, which exhibited totally different photochromic and fluorescence properties.⁴¹Many Schiff base ligands have been synthesized for the detection of Cu²⁺, which have general fluorescence characteristics. To solve the ACQ problem, we are going to design and synthesize a Schiff base fluorescent probe with AIE effect. For this purpose, a Schiff base fluorescent probe (CTPE) incorporating the TPE group into coumarin framework has been designed and synthesized, which exhibits fast response time, simple synthetic step, lower cost and AIE effect. And it can rapidly recognize Cu²⁺ in a mixed THF/H₂O system. Scheme 1 was the synthetic route for CTPE. Other compounds and intermediates were labelled and displayed in Scheme S1 in the ESI.^† As shown in Scheme 1, CTPE was prepared in high yield via Maillard reaction of 8-formyl-7-hydroxy-4-methylcoumarin (M1) and 1-(4′-aminophenyl)-1,2,2-triphenylethene (TPE-NH₂) in absolute ethanol. M1, TPE-NH₂ and CTPE were prepared according to the literatures.^42,43 Their chemical structures were confirmed by nuclear magnetic resonance (NMR), high resolution mass spectrometry (HRMS) and Fourier transform infrared (FTIR) (Fig. S1–S10, ESI^†). In Fig. S10,^† compared with TPE-NH₂ and M1, the spectrum of CTPE appeared an in-plane bending vibration absorption peak of C N bond at 1620 cm⁻¹, and a C O bond stretching vibration absorption peak on the aldehyde group at 1740 cm⁻¹, indicating that CTPE has been successfully synthesized.Open in a separate windowScheme 1Synthesis and structure of CTPE.As an AIE-based fluorescent probe, the AIE effect of CTPE was confirmed by its fluorescence spectra in THF/H₂O mixtures with various water contents. CTPE was in a good dispersion state in THF solvent, but CTPE gradually aggregated as the volume fraction of water (f_w) increased. As shown in Fig. 1A and B, CTPE had good dispensability and exhibited weak luminescence in dilute THF solution (f_w = 0%). The weak emissive nature of the luminogens in THF and aqueous mixtures with low f_w values should be ascribed to active intramolecular rotations of the genuinely dissolved compounds, which effectively consumed the energy of the excitons non-radiatively.⁴⁴ When f_w increased from 10% to 70%, the fluorescence intensity of CTPE gradually decreased, which was attributed to the intramolecular charge transfer (ICT) effect with the increasing polarity of the solution for CTPE.^40,45 When f_w was above 70%, CTPE displayed a significantly sudden increase in the fluorescence intensity owing to molecular aggregation. The fluorescence intensity at 565 nm was about 60 in THF/H₂O (30/70, v/v) mixtures, while it increased to about 1288 in THF/H₂O (1/99, v/v) mixtures with about 21-fold enhancement. Simultaneously, and the maximum emission wavelength showed a slight red shift from 565 nm to 575 nm with the increase of fluorescence intensity. The fluorescence enhancement phenomenon could be ascribed to the restriction of the intramolecular rotations. In the aggregated state, intramolecular motion (mainly including C N isomerization and rotation of the benzene ring) was limited, and the attenuation of non-radiative energy was correspondingly blocked, so that a clear enhancement and a slight red shift of the fluorescence of CTPE were observed.⁴⁰ The above results clearly implied that CTPE exhibited a significant AIE effect.Open in a separate windowFig. 1(A) PL spectra of CTPE in THF/H₂O mixtures with different f_w. (B) Plots of PL intensity of CTPEversus f_w in THF/H₂O mixtures at 565 nm. Inset: photograph of CTPE in THF/H₂O mixtures under a hand-held UV lamp illumination (λ_ex = 365 nm). (C) PL spectra of CTPE after adding Cu²⁺ in THF/H₂O (1/99, v/v) at different reaction time. Inset: relationship between fluorescence intensities of CTPE in THF/H₂O (1/99, v/v) at 575 nm and time of addition of Cu²⁺. (D) UV-vis absorption and (E) fluorescence spectra of CTPE with different metal ions in THF/H₂O (10/90, v/v). (F) Fluorescence responses at 565 nm of CTPE to various metal cations in THF/H₂O (10/90, v/v) solution.The Cu²⁺-specific binding of CTPE with various competitive metal ions was then investigated under the same experimental condition. Different from these AIE sensors that worked in the aggregated state, there was not complete fluorescence quenching even after about 1 h of adding 20 equivalent Cu²⁺ in THF/H₂O (1/99, v/v) solution (shown in Fig. 1C). However, at the moment of adding Cu²⁺, the fluorescence was immediately quenched in THF/H₂O (10/90, v/v) solution. Therefore the THF/H₂O (10/90, v/v) system was adopted to study the selectivity of CTPE to all metal ions.Metal ion selectivity studies were performed on absorption and fluorescence spectra. Absorption spectra of CTPE recorded after the addition of each metal ion (20 eq.) was shown in Fig. 1D. In Fig. 1D, upon addition of a constant amount (20 eq.) of Cu²⁺ ion to CTPE, a significant hypsochromic shift from 395 nm to 371 nm in absorption spectrum was observed. No other metal ion induced any change under the identical conditions except Fe³⁺ and Fe²⁺ ions. These results demonstrated the specificity of the CTPE for selective binding interaction with Cu²⁺ ion.To further investigate the selectivity of CTPE to Cu²⁺, we also studied the fluorescence response of CTPE in THF/H₂O (10/90, v/v) solution. The behaviour of fluorescence “turn off” merely shown by Cu²⁺ was observed in the Fig. 1E. Surprisingly, the fluorescence was immediately quenched in THF/H₂O (10/90, v/v) solution at the moment of adding Cu²⁺. And the fluorescence intensities decreased after adding Fe³⁺ and Fe²⁺, respectively. No obvious changes were observed upon addition of 20 eq. other competitive metal ions. The interferences from the other metal ion with CTPE in its response to Cu²⁺ were performed. As shown in Fig. 1F, the red bars represent the emission changes of CTPE in the presence of metal ions of interest (all are 20 eq.). The black bars represent the changes of the emission that occurs upon the subsequent addition of Cu²⁺ to the above solution. The fluorescent intensity of CTPE in the presence of any of the other metal ions tested after adding Cu²⁺ was decreased significantly, demonstrating little interference from the other metal ions.Fluorescent titration experiments of CTPE in THF/H₂O (10/90, v/v) solution with Cu²⁺ ion were carried out and the changes in the fluorescence intensity at 565 nm of CTPE solution with the concentration of Cu²⁺ ion were measured and showed in Fig. 2A. It was found that the fluorescence intensity at 565 nm of CTPE solution decreased with the increase in the concentration of Cu²⁺ ion. As showed in Fig. 2B, F₀ and F were the fluorescence intensities at 565 nm without adding Cu²⁺ and adding different concentrations of Cu²⁺, respectively. In addition, when the Cu²⁺ concentration in the solution to be tested was less than 1.2 μM, the F₀/F of CTPE at 565 nm had a good linear relationship with the concentration of Cu²⁺.Open in a separate windowFig. 2(A) Fluorescence titration spectra of CTPE (10 μM) in THF/H₂O (10/90, v/v) solution. Inset: fluorescence intensities of CTPE (10 μM) at 565 nm as a function of Cu²⁺ concentration (0–3.2 μM). (B) Linear relationship between F₀/F and Cu²⁺ concentration.By taking that change in fluorescence intensity in micro molar range we have calculated the lower limit of detection (LOD) from standard deviation and the slope of calibration plot (Fig. 2B) using the equation.⁴⁶ It was deduced from the fluorescence titration profile that the LOD of CTPE toward Cu²⁺ ion reached 0.36 μM.To gain a better understanding the sensing mechanism of CTPE towards Cu²⁺, ¹H-NMR titration experiments were performed in DMSO-d₆ at room temperature (Fig. 3A). The ¹H-NMR spectra of CTPE showed considerable variation with the increasing of Cu²⁺ in DMSO-d₆ solvent. Cu²⁺ is a paramagnetic ion that affects the NMR resonance frequency of protons that are close to the Cu²⁺ binding site.⁴⁷ The downfield value of H_a (of –OH̲) at δ = 14.86 ppm in CTPE was due to the intramolecular hydrogen bond between the imine-N atom of CTPE with H_a forming a six-membered transition state.⁴⁸ On addition of Cu²⁺ ion, the intramolecular hydrogen bonding was disturbed.⁴⁹ As Cu²⁺ gradually increased, the proton signal of H_a almost disappeared. The proton signal of H_b (of –CH̲ N–) at δ = 9.16 ppm significantly decreased indicating the participation of the nitrogen atom in the binding with Cu²⁺ ion. The proton signals of the aromatic ring became broader and weaker with the increasing amount of Cu²⁺. These results confirmed that Cu²⁺ binded to the CTPE chemosensor through the N atom of the imine and the O atom of phenolic hydroxyl, which were directly connected to the aromatic rings.⁵⁰ Thus, combined with the FTIR spectra of CTPE before and after the addition of Cu²⁺ (Fig. S11^†), it was clearly illustrated that the fluorescence “turn off” of CTPE was attributed to the chelation between imine-N atom, phenolic hydroxyl-O atom and Cu²⁺ ion. The possible coordination modes of Cu²⁺ and CTPE were shown in Fig. 3B.Open in a separate windowFig. 3(A) ¹H-NMR data of CTPE in DMSO-d₆ solution in the absence and presence of Cu²⁺. (B) Proposed mechanism for CTPE upon addition of Cu²⁺.In summary, a novel Schiff base fluorescent probe CTPE based on coumarin and TPE showing an AIE effect has been successfully synthesized and characterized. The fluorescence of CTPE was rapidly quenched by Cu²⁺, but no significant influence was observed for other metal ions tested. CTPE can distinguish Cu²⁺ from other metal ions via the fluorescence “turn off”. The LOD of CTPE for Cu²⁺ can reach 0.36 μM. Recognition mechanism between CTPE and Cu²⁺ was given. Therefore, CTPE can act as a potential fluorescence probe to selectively and rapidly identify Cu²⁺.     

食管癌颈部吻合口瘘与感染关系的研究

目的 探讨导致食管癌颈部吻合口瘘发生的主要原因.方法 为预防颈部切口感染对围手术期的处理进行改进.设定改进前为污染组,改进后为清洁组.两组吻合口瘘发生率的比较采用χ2检验.结果 污染组发生率为12.5%,清洁组发生率为2.4%,χ2＝9.258,P＝0.002,差异有显著性.结论 颈部切口感染是食管癌颈部吻合口瘘发生的主要原因.     

我院药学服务现状调查

目的调查我院临床药师工作的现状,为改进药学服务提出合理化建议。方法对我院5名临床药师和部分临床医生进行访谈,了解医院临床药学工作的现状。结果药学服务工作已经开展,但仍需要改进。结论临床药学工作尚需相关部门重视以及药师的自身努力。     

对由于系统单相接地引发的两起电缆事故的分析

本文介绍了两起典型的由于系统单相接地引发的电缆事故,在深入剖析故障原因的基础上,总结分析了在不同接地方式下发生事故的类型,并对电缆附件安装过程的关键部位提出了一些建议,为电缆同行业人员提供了一定的借鉴作用。     

斯奇康足三里穴位注射治疗慢性荨麻疹的效果观察与护理

[目的]探讨穴位注射治疗慢性荨麻疹的临床疗效及护理。[方法]将98例慢性荨麻疹病人随机分为两组。对照组40例,每日睡前口服氯雷他定10 mg;治疗组58例,除每日睡前口服氯雷他定10 mg外,并加用斯奇康双侧足三里穴位注射,隔日1次。连续治疗36 d并随访3个月,观察近远期疗效。[结果]治疗组总有效率为91.4%,复发率为25.0%;对照组总有效率为75.0%,复发率为64.3%;两组比较均有统计学意义。[结论]穴位注射能提高药物治疗慢性荨麻疹的疗效,给予有效的护理指导,促进病人康复,降低复发率。     

颈椎前路融合术后邻近节段退变性疾病的原因分析及治疗策略

 目的 探讨颈椎前路融合术后邻近节段退变性疾病的发生原因及治疗策略。方法 对2005年12月至2012年8月颈椎前路减压融合术后并发邻近节段退变性疾病的14例进行回顾性分析。男10例,女4例,初次手术年龄36~68岁,平均（52.0±11.0）岁。测量初次手术后颈椎整体曲度、手术节段局部曲度、钢板与上、下间隙的距离。二次手术时对节段数量≤2个且不伴严重后纵韧带或黄韧带骨化的9例采用颈椎前路减压融合内固定术,对节段数量≥3个且不伴明显后凸畸形、排列不稳及严重黄韧带骨化的3例采用颈椎后路减压椎管扩大成形术,对伴有明显后凸畸形、排列不稳且因技术原因无法行前路减压及合并严重黄韧带骨化的2例采用颈椎后路全椎板切除减压融合术。结果 初次术后邻近节段退变性疾病的发生时间为（9.3±4.4）年,二次手术时患者年龄（61.3±12.4）岁。初次术后颈椎整体曲度与手术节段局部曲度分别为12.4°±10.8°、1.5°±6.8°;钢板与上、下间隙的距离分别为（0.9±1.8）mm、（3.8±3.2）mm。二次术后平均随访（30.4±17.8）个月,末次随访时日本骨科学会颈椎病疗效评分改善率为73.9%±9.7%。结论钢 板与邻近间隙距离过小、手术节段局部曲度恢复不良可能是前路融合术后邻近节段退变性疾病的发生原因;二次手术方案的选择应根据病变累及节段、致压因素、颈椎曲度等因素进行综合分析。     

除湿1号洗剂治疗婴儿渗出型湿疹的临床观察

目的:观察除湿1号洗剂外用治疗婴儿渗出型湿疹的临床疗效。方法:以3%硼酸溶液、尤卓尔软膏西药外用治疗为对照组,除湿1号洗剂外用治疗为治疗组。治疗观察92例婴儿渗出型湿疹患者。结果:治疗组的痊愈显效率为89.13%,与对照组的65.22%比较有显著差异性(P<0.01);治疗后随访2周,治疗组的复发率均低于对照组,比较有显著差异性(P<0.01)。结论:除湿1号洗剂外用治疗婴儿渗出型湿疹效果良好,且复发率低,具有很好的临床应用价值。     

成人寰枢正中关节间隙正常值的CT多平面重组测量

目的 测量MSCT影像成人寰枢正中关节间隙(MADI)的正常值范围,探讨MADI值与年龄和性别的关系。方法 选取2009年8月—2014年7月山东省医学影像研究所18岁以上行寰齿关节MSCT扫描的700例患者影像资料行回顾性分析,排除外伤、累及上颈椎各类疾病者,按年龄分为18～24岁、25～29岁、30～39 岁、40～49岁、50～59 岁、60～69 岁 和≥70 岁7组,每组入选100例。对寰齿关节正中矢状位CT影像进行多平面重组,并测量寰椎前后结节中点连线经过寰齿关节间隙的长度,即MADI。计算不同年龄组患者MADI值,并采用线性回归方法进一步分析MADI值与年龄、性别的关系。 结果 7组由低至高不同年龄患者的MADI值依次为(1.77±0.39)mm、(1.61±0.37)mm、(1.58±0.36)mm、(1.41±0.29) mm、(1.34±0.28)mm 、(1.31±0.29)mm、和(1.06±0.47)mm,MADI 随年龄增加而递减 (r=-0.511, P＜0.01)。男性MADI(1.50±0.36)mm,女性MADI(1.38±0.34)mm,MADI 在不同性别之间无相关性 (r=0.000, P＞0.05)。结论 在MSCT正中矢状位上MADI值与年龄呈负相关,与性别无关。     

口腔黏膜下纤维性变癌变中Survivin的表达及意义

目的:检测口腔黏膜下纤维性变(OSF)及其癌变组织中凋亡抑制蛋白Survivin的表达,探讨Survivin在OSF癌变过程中的临床意义.方法:应用免疫组织化学SP法检测10例正常口腔黏膜组织、40例OSF组织和42例OSF癌变组织中Survivin的表达情况.结果:正常口腔黏膜组织上皮中无Survivin阳性表达,OSF癌变组织中Survivin阳性表达率(95.2%)显著高于OSF组(47.5%)(P＜0.01),而早、中、晚期OSF 3组间Survivin阳性表达率无显著性差异(P＞0.05).结论:Survivin蛋白的过度表达在OSF癌变过程中具有重要作用;Survivin有望成为OSF癌变的早期诊断标志物.