双光子荧光探针

双光子吸收是指在强光激发下,介质分子同时吸收两个光子,从基态跃迁到两倍光子能量的激发态的过程。荧光显微成像是研究活体生物的重要工具,而最通常的细胞成像方法则是使用单光子激发荧光团的单光子显微成像。近红外光源激发的双光子荧光探针克服了单光子荧光探针的光漂白与光致毒而更适于生物检测与成像,为生命科学研究提供了更为锐利的工具。双光子荧光探针的作用机理包括分子内电荷迁移(ICT)、荧光共振能量迁移(FRET)、光诱导电子迁移(PET)与基团转换(GC) 4种方式。该文综述了双光子阳离子探针(Mg²⁺, Ca²⁺, Pb²⁺, Hg²⁺, Ag⁺, Fe³⁺, Zn²⁺, Na⁺, Cr³⁺)、双光子阴离子探针(F^-)、pH探针、双光子葡萄糖示踪器、双光子脂筏探针、双光子巯基探针、双光子半胱氨酸探针和双光子生物标记探针,以及双光子荧光探针在生物成像方面的应用,展望了双光子荧光探针的发展趋势与应用前景。     

高灵敏线粒体靶向近红外二氧化硫荧光探针的开发及细胞、小鼠成像研究

二氧化硫(SO₂)及其衍生物(亚硫酸盐、亚硫酸氢盐等)作为活性硫物种的重要组成部分,与生命体的生理过程有着密切的关系.此外,摄入过量的SO₂会对呼吸系统造成不可修复的损伤,例如呼吸系统损伤、心脑血管和神经疾病等.近红外发射荧光探针具有较强的组织穿透能力,可以有效地检测活体内的生物分子.因此,设计了一种基于迈克尔加成(Michael Addition)机理的线粒体靶向近红外SO₂荧光探针XA-SO₂.溶液光谱实验表明,该探针XA-SO₂对SO₂有着显著的选择性和灵敏度,并且具有快速检测SO₂的能力.值得注意的是,该荧光探针XA-SO₂具有较好的细胞膜渗透能力,成功检测到了细胞外/内源性SO₂.更重要的是,该探针XA-SO₂能够有效地在细胞的线粒体上富集,有望实现细胞线粒体内SO₂的动态检测.     

基于萘酰亚胺-苯并咪唑鎓的荧光传感器对H2PO-4的高选择性识别

设计合成了基于萘酰亚胺-咪唑鎓的受体分子1和2,通过荧光发射光谱研究了受体分子1和2对阴离子F^-、Cl^-、Br^-、I^-、AcO^-、HSO^-₄、H₂PO^-₄、NO^-₃、ClO^-₄的识别性能。 研究发现,在受体分子1和2的乙腈溶液(5.0×10^-6 mol/L)中加入10倍化学计量的H₂PO^-₄时,受体分子1的荧光猝灭率高达98%,受体分子2的荧光猝灭率仅为24%。 表明具有多重识别位点的受体分子1在构型上与H₂PO^-₄更匹配,可作为H₂PO^-₄的荧光关闭型(turn-off)探针。 受体分子1与H₂PO^-₄的结合比为1∶1,结合常数为(1.25±0.11)×10⁵ L/mol,检出限为6.90×10^-6 mol/L。     

近红外二区发光稀土纳米材料的设计及生物成像应用

近年来,近红外二区(NIR-II,1000~1700 nm)荧光成像因其较高的空间分辨率、较深的组织穿透能力,在分子影像领域引起了广泛的关注。常见的NIR-II发光材料(如有机小分子、共轭聚合物、量子点等)通常具有光稳定性差、荧光量子产率低、斯托克斯位移小、荧光峰宽等问题,限制了这一新型成像技术的进一步发展与应用。稀土纳米材料由于其独特的发光特性,能够较好地克服这些不足,近年来不同结构的稀土纳米材料也逐渐被设计开发并应用于近红外二区荧光成像与检测,展示出了巨大的应用潜力。本综述首先介绍了稀土纳米材料的光学特性,然后按敏化离子的不同(Yb³⁺、Nd³⁺、Er³⁺、Tm³⁺)详细介绍了近红外二区稀土纳米材料的设计方法及相关应用,最后对稀土纳米材料在近红外二区成像领域的进一步发展进行了展望。     

检测汞离子的比色-荧光双通道探针的设计合成及应用

以7-羟基吩恶嗪酮(试卤灵)为荧光团、硫代甲酸苯酯为汞离子(Hg²⁺)识别基团,经一步简单有机合成反应,制备了比色-荧光双通道探针NMP。采用核磁共振氢谱(¹H NMR)、核磁共振碳谱(¹³C NMR)和高分辨率质谱(HRMS)表征了探针结构,利用吸收光谱和荧光发射光谱测试了探针对Hg²⁺的选择性和灵敏度。光谱测试结果表明,探针对Hg²⁺具有较好的选择性和检测灵敏度,检出限(3σ)为17 nmol/L。反应溶液由淡黄色变为紫红色,可对溶液中Hg²⁺进行比色检测,并可用于实际水样中Hg²⁺的检测。采用CCK-8法测试了探针的细胞毒性,结果表明,在探针浓度低于10μmol/L时,细胞存活率高于90%。共聚焦荧光显微成像结果表明,探针具有较好的细胞通透性,可对细胞内Hg²⁺进行荧光成像。     

检测硫化氢分子的荧光探针

硫化氢（H₂S）是继一氧化碳和一氧化氮之后,第三种可在生命体内发挥生理作用的内源性气体信号分子。该气体分子在心血管和神经系统中担负着重要的生理病理调节作用。因此,选择性识别和高灵敏检测生物体内的H₂S具有十分重要的生物医学意义。在生物检测技术手段中,荧光探针法具有选择性好、灵敏度高、对生物样品损伤小以及可实现实时原位检测等独特的优势,故应用荧光探针法检测细胞内H₂S浓度的变化是近年来研究热点之一。本文依据荧光探针与H₂S之间的化学反应类型,将近三年来所研发的H₂S荧光探针按照其母体荧光团进行分类和总结,综述了H₂S荧光探针的研究进展,概述了相关荧光探针的设计理念、检测机理及生物应用,探讨了探针的结构和性能之间的关系,最后展望了H₂S荧光探针的发展趋势和应用前景。     

2-[3-氰基呋喃-2(5H)-亚基]丙二腈衍生物的合成及其对Pd2+的识别

通过两步反应合成了一种2-(3-氰基呋喃-2(5H)-亚甲基)丙二腈衍生物TCF-Pyn,该化合物在MeCN/磷酸缓冲容溶液(PBS)(V∶V=2∶8,PBS 2×10^-2 mol/L,pH=7.4)混合溶液中能高选择性荧光"OFF-ON"识别Pd²⁺,发射波长达到近红外区(653 nm).该探针具有较好的抗干扰能力、较宽的pH适用范围(3~8)和较低的检测限(6.92μmol/L).应用研究表明,TCF-Pyn能检测真实水样中的Pd²⁺,并可对活细胞中的Pd²⁺进行荧光成像.     

一种基于二氰基异佛尔酮的近红外汞离子荧光探针的合成与应用

以二氰基异佛尔酮和4-二乙氨基水杨醛为原料,通过两步反应合成了一种具有近红外发射(675 nm)的荧光探针SAM-S,并对其结构进行了表征。此探针在甲醇/4-羟乙基哌嗪乙磺酸(HEPES)(1∶1,V/V,pH=7.4)溶液中表现出弱荧光,加入Hg²⁺后荧光显著增强,基于此建立了检测Hg²⁺的新方法。本方法具有选择性好、抗干扰能力强、检出限低(0.49μmol/L)等优点。采用此探针检测湖水和自来水中Hg²⁺的含量,加标回收率在99.9%～104.8%之间。探针SAM-S可制作成荧光墨水,有望用于商标包装;由于SAM-S的毒性较低,因此可用于活细胞中Hg²⁺的荧光成像。     

高特异性的La3+靶向多肽探针及其在细胞和斑马鱼成像中的应用

研究报道了一种新型多肽探针(TPE-Glu-Glu-Pro-Gly-Glu-Glu-NH₂),该多肽探针单独存在时没有荧光，稀土元素镧离子(La³⁺)加入后，与多肽探针特异性结合，限制了四苯乙烯(TPE)分子内的旋转，导致溶液产生强的荧光信号。该探针具有特异性，16种常见金属的存在也几乎不影响其对La³⁺的靶向检测。我们研究发现，谷氨酸在多肽探针结合La³⁺的过程中发挥重要作用，减少谷氨酸个数会影响其特异性识别。滴定和Job工作曲线结果表明，该多肽探针与La³⁺结合比为1∶2,结合常数为1.29×10¹⁰(L/mol)²。计算机模拟结果表明，La³⁺与多肽探针酰胺键的氧和谷氨酸羧基中羟基的氧结合。该探针可以高灵敏检测水溶液和唾液中的La³⁺,检测限分别为0.12μmol/L和0.32μmol/L。该高灵敏、高特异性多肽探针具有良好的生物相容性，可以用于细胞和斑马鱼中La³⁺     

N-乙基咔唑-3-甲醛席夫碱荧光探针的合成及其检测Cu2+性质

以咔唑为原料,经过两步反应制备得到N-乙基咔唑-3-甲醛,其结构经X射线单晶衍射测定属于单斜晶系,空间群为P2₁/n。再以N-乙基咔唑-3-甲醛与1,3-二氨-2-丙醇为原料,设计、合成了一种新型双席夫碱荧光探针分子CMP。借助荧光光谱在体积比为6∶4的DMSO/H₂O缓冲溶液(Tris-HCl,p H=7.0)中研究了探针CMP对Cu²⁺的选择性识别。研究结果表明,探针CMP与Cu²⁺以1∶2的比例配位,结合常数为1.52×10⁵L·mol^-1,检出限为0.205μmol·L^-1。回收实验表明,探针分子CMP可应用于环境水样中Cu²⁺的检测。     

A pinacol boronate caged NIAD-4 derivative as a near-infrared fluorescent probe for fast and selective detection of hypochlorous acid

A pinacol boronate caged NIAD-4 derivative was demonstrated to be a near-infrared fluorescent probe for fast and selective detection of hypochlorite over other ROS species.     

Organelle targetable fluorescent probes for hydrogen peroxide

The review summarized organelle target strategies, structures, fluorescence behavior and biological applications of H₂O₂ fluorescent probes with mitochondria, nuclei and lysosomes-targetable ability.     

Synthesis of Highly Sensitive Fluorescent Probe Based on Tetrasubstituted Imidazole and Its Application for Selective Detection of Ag+ Ion in Aqueous Media

A small molecule fluorescent probe, 4-[2-(4-chlorophenyl)-4,5-diphenyl-1H-imidazol-1-yl]aniline(L) for detecting Ag⁺ ion was gently synthesized via one-pot multi-component reaction catalyzed by H₃[PW₁₂O₄₀] under solvent-free microwave irradiation. When the concentration of Ag⁺ ion changed from 0 to 8.0×10⁻⁶ mol/L in the solution of H₂O/CH₃OH(19:1, volume ratio), the fluorescence emission spectrum was blue-shifted and accompanied by a gradual increase in fluorescence intensity with a low detection limit of 3.0×10⁻¹¹ mol/L. Moreover, UV-Vis absorption titration experiment demonstrated a 1:1 stoichiometric ratio and an association constant of (9.95±0.44)×10⁵ L/mol between probe L and Ag⁺ ion, and thus their complexation mechanism was also proposed and verified. More importantly, this fluorescent probe was remarkably specific for Ag⁺ ion under the interference of other metal ions and exhibited a wide pH application range of 4.0-8.0. Additionally, preliminary application of this probe was also carried out and satisfactory results were shown.     

A near-infrared fluorescent probe for monitoring fluvastatin-stimulated endogenous H2S production

Most reported fluorescent probes have limitations in practical applications in living systems due to the strong autofluorescence background,construction of probes with near-infrared(NIR) fluorescence emission is an accessible approach for addressing this challenge.We here designed a NIR fluorescent probe for monitoring the endogenous production of H_2S in living cells.The designed probe showed significant NIR fluorescence turn-on response to H_2S with high selectivity,enabling the sensitive detection H_2S.Importantly,the probe could be applied in monitoring the endogenous production of H_2S in raw 264.7 macrophages.This study showed that fluvastatin can promote the activity of cystathionineγ-lyase(CSE) for generation H_2S.     

气体分子在二维石墨烯纳米孔中的选择性渗透特性

二维石墨烯纳米孔中气体分子的选择性渗透对多孔石墨烯分离膜非常重要。本文采用分子动力学方法研究了气体分子在氮氢修饰石墨烯纳米孔中的渗透特性,从分子的大小和结构、纳米孔的构型以及分子与石墨烯之间的作用强度等角度阐明了分子出现选择性渗透的原因。结果表明,不同分子的渗透率不同,即H₂O＞H₂S＞CO₂＞N₂＞CH₄。渗透率跟分子的质量和直径以及分子在石墨烯表面上的吸附密度有关;根据气体分子动理学理论,渗透率跟分子质量成反比关系;而分子在石墨烯表面上的高吸附密度对渗透起促进作用。对于H₂O和CH₄分子,分子直径起主导作用;H₂O分子直径最小,其渗透率最大;同理,CH₄分子的渗透率最小。对于H₂S和CO₂分子,H₂S分子的直径较大,但其与石墨烯之间的作用强度较大(吸附密度较高),导致渗透率较高;对于CO₂和N₂分子,CO₂分子的直径较小,并且与石墨烯之间的作用强度较大,渗透率较高。同时发现,分子在纳米孔中的渗透使得其在石墨烯表面的密度分布极不均匀。纳米孔左右两侧的功能化氮原子使CH₄分子容易从孔两侧区域穿过,而其它分子由于直径较小在纳米孔中心区域穿过的概率最大。分子与石墨烯之间的作用越强,导致分子在石墨烯表面区域内停留的时间越长,最终使其在渗透纳米孔的过程中所经历的时间越长。本文所采用的氮氢修饰石墨烯纳米孔中,分子渗透速率达到~10^-3 mol·s^-1·m^-2·Pa^-1,并且其它分子相对于CH₄分子的选择性也很高,说明基于该类型纳米孔的多孔石墨烯分离膜在天然气处理等工业气体分离领域具有很好的应用前景。     

基于香豆素343的荧光探针快速检测亚硫酸根

以香豆素343和2-苯并噻唑乙腈为原料,合成了香豆素343-亚硫酸根离子探针(Coumarin 343-SO₂),并用¹H NMR、¹³C NMR、MS和HRMS等技术手段对合成的化合物进行了表征。 该探针与亚硫酸根离子(SO₃^2-)发生亲核加成反应后,阻断了苯并噻唑与香豆素的共轭结构,从而引起荧光强度的变化,达到检测亚硫酸根离子的目的。 此探针对SO₃^2-具有响应快、高灵敏度、高选择性及检测限低至0.08 μmol/L的特点,其它常见的阴离子及还原性物质对SO₃^2-的检测均无干扰。 此外,该探针具有良好的细胞膜通透性,可用于活细胞中对SO₃^2-进行荧光成像。     

Design of a novel mitochondria targetable turn-on fluorescence probe for hydrogen peroxide and its two-photon bioimaging applications

Considering that hydrogen peroxide (H₂O₂) plays significant roles in oxidative stress, the cellular signal transduction and essential biological process regulation, the detection and imaging of H₂O₂ in living systems undertakes critical responsibility. Herein, we have developed a novel two-photon fluorescence turn on probe, named as Pyp-B for mitochondria H₂O₂ detection in living systems. Selectivity studies show that probe Pyp-B exhibit highly sensitive response toward H₂O₂ than other reactive oxygen species (ROS) and reactive nitrogen species (RNS) as well as biologically relevant species. The fluorescence colocalization studies demonstrate that the probe can localize in the mitochondria solely. Furthermore, as a bio-compatibility molecule, the highly selective and sensitive of fluorescence probe Pyp-B have been confirmed by its cell imaging application of H₂O₂ in living A549 cells and zebrafishes under the physiological conditions.     

Rational Design of Ratiometric Fe3+ Fluorescent Probes Based on FRET Mechanism

As the most abundant transition metal element in mammals, iron(Fe) plays a vital role in life activities. It is of great significance to study the variation of Fe³⁺ level in living organisms. In virtue of the advantages of high sensitivity, good selectivity and low damage to living systems, the fluorescence detection of Fe³⁺ has attracted much attention. Compared with the intensity-based fluorescent probe, the ratiometric fluorescent probe has less interference of environmental and can realize quantitative detection. In this study, four ratiometric Fe³⁺ fluorescent probes, R1, R2, R3 and R4_, were designed and synthesized using fluorescence resonance energy transfer(FRET) mechanism to achieve quantitative detection of Fe³⁺. In the FRET systems, 1,8-naphthalimide fluorophore derivatives were adopted as donors while rhodamine B derivatives were selected as receptors. The connection sites of the donor and acceptor in R3 and R4 are different from those in R1 and R2. All the four probes showed good response and selectivity to Fe³⁺. The energy transfer efficiencies of R3 and R4 were obviously higher than those of R1 and R2. This work provided a promising strategy for the development of fluorescent ratiometic Fe³⁺sensors.     

一种具有荧光性质的阳离子Ga⁃MOF用于Fe3+和硝基化合物识别

通过溶剂热法合成了一种新型阳离子镓金属-有机框架Ga-MOF, 其化学式为[Ga₃O(H₂O)₃(TCA)₂]·NO₃·6DMF·2H₂O(JOU-27, H₃TCA=4,4′,4″-三苯胺三羧酸). 结构分析表明, JOU-27是基于氧心三核镓簇的三维微孔结构. 由于荧光配体H₃TCA的引入, JOU-27具有强的荧光发射强度, 因此可用于检测Fe³⁺离子和硝基芳香族化合物. 结果表明, 其对Fe³⁺的检出限低至2.22×10^-6 mol/L(Stern-Volmer常数K_SV=52823 L/mol); 当硝基苯(NB)浓度仅为3.27×10^-3 mol/L时, 荧光猝灭效率可达91%. 进一步的研究表明, JOU-27的猝灭性能可能与荧光共振能量转移(FRET)效应、 激发光吸收竞争以及骨架激发态与硝基芳烃化合物之间的电子转移有关.     

Design,Synthesis, and Evaluation of Near-Infrared Fluorescent Molecules Based on 4H-1-Benzopyran Core

A series of novel fluorescent 4H-1-benzopyrans was designed and developed as near-infrared fluorescent molecules with a compact donor–acceptor-donor architecture. Spectral intensity of the fluorescent molecules M-1, M-2, M-3 varied significantly with the increasing polarities of solvents, where M-3 showed high viscosity sensitivity in glycerol-ethanol system with a 3-fold increase in emission intensity. Increasing concentrations of compound M-3 to 5% BSA in PBS elicited a 4-fold increase in fluorescence intensity, exhibiting a superior environmental sensitivity. Furthermore, the in vitro cellular uptake behavior and CLSM assay of cancer cell lines demonstrated that M-3 could easily enter the cell nucleus and bind to proteins with low toxicity. Therefore, the synthesized near-infrared fluorescent molecules could provide a new direction for the development of optical imaging probes and potential further drugs.