甲川链修饰菁染料的合成研究进展

在小分子荧光探针中,菁染料由于其良好的光学特性,在成像及生物传感等领域具有广泛应用.甲川链的功能化可以显著提高染料的稳定性并改变其光物理性能和生物选择性,已成为近年来菁染料结构修饰的热点.总结了甲川链修饰菁染料的合成方法,根据甲川链取代基的不同来源,按照甲川链不同长度对甲川链修饰菁染料的研究进展分别进行综述.     

选择性识别平行性G-四联体的方酸类近红外荧光探针的研究

G-四联体是抗肿瘤药物筛选的一个重要靶点.开发针对某些拓扑结构的G-四联体荧光探针对于研究其结构和生物学功能具有十分重要的意义.设计、合成了四个方酸类花菁荧光染料即CSTS,CSBE,CSEM和CSBM,并检测了其对不同类型DNA的选择性识别作用.结果表明,所合成的四个化合物在缓冲溶液中几乎没有荧光发射,加入正平行G-四联体之后荧光增强大约1 000倍;而加入反平行G-四链体或单双链DNA荧光仅仅增强几十倍,说明其可以特异性识别平行G-四联体.但流式实验结果显示,CSTS不能透过细胞膜,同时存在高荧光背景的缺点,因此无法应用于活体分析.而另外三个不带阴离子侧链的衍生物则容易进入细胞,进入细胞的难易顺序为CSBECSEMCSBMCSTS.高选择性、低背景荧光和易进入细胞等优点使CSBE具有作为近红外荧光探针检测生物样品中正平行G-四联体的潜力.     

冠醚菁染料的研究——Ⅰ.冠醚菁染料的合成

Dix等^[1]曾讨论冠醚偶氮染料、冠醚二苯甲烷染料和冠醚三苯甲烷染料的研究结果,关于冠醚菁染料迄今还未见报道.鉴于菁染料发色团之甲川链的可调性[2a]和冠醚空腔大小的可调性^[3],作者设想将菁染料结构因子和冠醚结构因子结合于同一分子之中,可望延伸出一系列富于颜色变化的离子选择性络合的冠醚菁染料;也可能用于分析化学和生物膜的研究等.     

比率检测高/半胱氨酸荧光染料的合成及活细胞成像研究

经一步简单反应获得了一例长波长尼罗蓝衍生物荧光染料RB-S. 该染料分子RB-S对高/半胱氨酸具有明显的荧光响应,且随着高/半胱氨酸浓度（0.03～0.33 μmol/L）逐渐增加,染料荧光强度在685 nm处减弱,相应地在755 nm处增强,在685与755 nm处的荧光变化具有良好线性关系的比率荧光变化. 通过高效液相色谱-质谱联用技术以及核磁滴定氢谱研究了染料分子检测高/半胱氨酸的机理. 试验结果表明：染料分子中的醛基与高/半胱氨酸发生成环反应生成噻唑烷. 在体外溶液测试中（pH=7.4）,染料分子RB-S对高/半胱氨酸的检出限为0.025 μmol/L,且不存在其他物质干扰,成功用于血清样品中内高/半胱氨酸荧光比率检测. 染料分子RB-S具有良好的活细胞膜通透性,使其能够对活细胞内高/半胱氨酸荧光比率成像.     

吲哚七甲川类荧光探针在生物体应用的研究现状

菁染料是一种商品化的近红外荧光染料, 其光谱范围位于近红外区域, 此光谱区域内生物基体光吸收或荧光强度较小, 因此利用菁染料对生物体进行成像, 可以降低背景干扰. 吲哚七甲川菁染料是一类具有代表性的菁染料, 其由吲哚杂环、七甲川链和N-取代基侧链组成, 由于具有溶解性好, 最大吸收波长可调, 摩尔消光系数大等优良的光学性质, 被广泛用于肿瘤靶向治疗、蛋白标记、痕量金属离子检测等生物方面. 以吲哚七甲川为母体, 通过向母体中引入活性基团或改变它们的结构, 使探针具有不同的功能, 这已成为生物领域荧光成像的研究热点. 分别从检测金属阳离子、pH变化、小分子和靶向标记肿瘤细胞、蛋白质等方面综述了近年来用于生物体荧光成像的吲哚七甲川类荧光探针的研究进展, 其中也对荧光探针在生物体内(模型)分布、光学成像及代谢方面进行了介绍, 最后讨论了这类荧光探针存在的问题及发展趋势.     

近红外吸收菁染料分子链结构对其光氧化稳定性能的影响

本文对具有不同分子链结构及不同链长,而母核结构分别为吲哚类及喹啉类的六种菁染料在溶液中的光氧化稳定性能进行了研究。结果表明菁染料的光褪色主要是由光氧化反应所致,当在分子链上引入不饱和环体结构时,可以使菁染料分子的光稳定性能增加;而随着分子链长的增加菁染料的光氧化稳定性能则明显下降。通过顺磁共振谱测定结果表明,在菁染料的自敏光氧化反应原初过程中,既存在单重态氧过程又存在超氧负离子过程。     

基于菁染料高选择性检测半胱氨酸的近红外荧光探针

本文设计合成了以菁染料为荧光团,以4-(三氟甲基)苯硫基为半胱氨酸响应识别基团的近红外荧光探针(Cy-CF_3)。利用探针分子Cy-CF_3与半胱氨酸和谷胱甘肽反应发生的机理不同,实现了对半胱氨酸特异性识别。探针分子Cy-CF_3与半胱氨酸发生芳香亲核取代反应生成巯基取代产物,进一步通过分子内重排反应生成氨基取代产物Cy-Cys。光谱研究结果表明,探针分子Cy-CF_3与半胱氨酸作用后发生明显的吸收波长蓝移(160nm),并且可观察到明显的颜色变化;荧光光谱中,随着半胱氨酸的加入,探针分子Cy-CF_3在780nm处的近红外荧光显著增强。Cy-CF_3能高选择性检测半胱氨酸,并且不受其它氨基酸尤其是结构类似的谷胱甘肽干扰。探针分子Cy-CF_3被成功地应用于活体细胞中检测半胱氨酸。     

感半导体激光菁染料稳定性的研究

菁染料因其具有容易合成、价格便宜、光学特性好等优点,在感光胶片中已大量应用,在有机光盘中也越来越受到人们的重视。但是,由于长链的菁染料在光照下极易发生光氧化还原反应,稳定性较差,因此研究和解决菁染料的光稳定性对其在激光照排材料和有机光盘中的应用都具有非常重要的意义。     

近红外双发射荧光探针比率检测高/半胱氨酸

合成的吲哚菁染料Cy5-A对高/半胱氨酸展现显著的荧光响应.高/半胱氨酸浓度的增加引发其荧光强度在660 nm处减弱,相应地在700 nm处增强,且700 nm与660 nm处的比率荧光变化与高胱氨酸(40.0~200.0 μM)及半胱氨酸(10.0~100.0 μM)浓度呈现良好的线性关系,最低检测限分别为13.2 μM与2.67 μM.通过核磁共振谱和质谱研究了Cy5-A与高/半胱氨酸的作用机制.研究表明,Cy5-A中的醛基与高/半胱氨酸发生环合反应,从而诱导产生近红外双发射光谱,探针响应不受其他干扰物的影响.因此,Cy5-A是一种简单高效的反应型荧光探针,可成功应用于血清样品中高/半胱氨酸的准确检测.     

方酸菁荧光探针的应用研究进展

功能性方酸菁染料具有独特的D-A-D共轭结构,其在可见光和近红外区域有强烈的吸收和荧光发射。方酸菁染料作为近红外荧光探针母体被应用于蛋白质、氨基酸、一些生物小分子、环境污染物及金属离子等的检测。本文结合课题组近几年工作综述了方酸菁染料在蛋白质、氨基酸、生物小分子、阳离子和其它物质检测方面的应用。     

Interaction of cyanine dyes with nucleic acids. XXV. Influence of affinity-modifying groups in the structure of benzothiazol-4-[2,6-dimethylpyridinium] dyes on the spectral properties of the dyes in the presence of nucleic acids.

Novel monomethine pyridinium cyanine dyes of similar structure and containing 'affinity-modifying' groups of different chemical nature were studied by spectral-luminescent methods as possible fluorescent probes for the nucleic acids detection. It was shown that the nature of the functional groups in the dye linker influences the fluorescent properties of the dye-nucleic acids complexes. Incorporation of a hydroxyl group into the linker structure leads to a significant increase in the fluorescence intensity of the dye--double-stranded DNA complexes relative to the parent dye Cyan 40.     

菁染料薄膜的光谱性能及稳定性的研究

本文研究了6种长链菁染料薄膜的光谱特性与光稳定性,以及两种抗氧剂对其薄膜光稳定性的影响。研究表明,结构相近的菁染料利用旋涂法成膜后,光稳定性与其母核的结构有关,依吲哚>喹啉> 唑>噻唑>硒唑顺序而变,与溶液状态下相类似,两种抗氧剂均为有效的单重态氧猝灭剂,都能提高菁染料的光稳定性。且抗氧剂的用量对染料的稳定性也会产生一定的影响     

利用花青染料转化DNA杂交信号的研究

基于花青染料3-乙基-2-[7-(3-乙基-2-苯并噻唑啉)-1,3,5-庚三烯]碘化苯并噻唑与DNA杂交双链的特异性结合作用,将DNA的杂交信号转化为染料在可见光区的光吸收信号。实验结果表明,与DNA杂交双链结合后,该染料在760nm处产生强吸收峰,吸光度数值及其稳定性受杂交链长度和体系温度的影响。在25℃条件下,采用24-mer探针,可以检测mg·mL-1数量级的互补DNA链,比DNA自身杂交信号灵敏度提高两个数量级。在优化实验条件下,利用该染料还可识别短链DNA中存在的单核苷酸多态性。与其它DNA片段的检测以及单核苷酸多态性的识别方法相比,该方法更为简便迅速,在临床诊疗中具有潜在的应用价值。     

i-Motif-modulated fluorescence detection of silver(I) with an ultrahigh specificity

A novel Ag⁺ sensor has been designed based on the mechanism that i-motif formation induced by Ag⁺ was sensitively recognized by a cyanine dye. The sensor exhibited an over 130–16,000 fold selectivity toward Ag⁺ than that toward other metal ions. This research not only provides a step forward toward the development of Ag⁺ detection but also represents a new application for i-motif DNA.     

Handling and detection of 0.8 amol of a near-infrared cyanine dye by capillary electrophoresis with laser-induced fluorescence detection

We are interested in the detection of DNA adducts and other trace analytes by labeling them with a fluorescent tag followed by use of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for high resolution and sensitivity. Towards this goal, here we report the following: (1) synthesis and handling properties of a near-IR, carboxyl-substituted heptamethine cyanine dye; (2) modification of an existing ball lens LIF detector to provide near-LIF detection with excitation at 785 nm for CE; and (3) corresponding handling and detection of as little as 0.8 amol of the dye by enrich-injection of 4.7 microl of 1 x 10(-13) mol/l dye in methanol from an 8-microl volume into a corresponding CE-LIF system. The electrolyte for the separation was methanol-40 mmol/l aqueous sodium borate (98:2, v/v). This finding encourages further exploration of the dye by functionalization of its carboxyl group for chemical labeling purposes.     

合成的近红外花菁染料测定痕量血清蛋白质

染料结合法测定蛋白质在生化及临床分析中已有广泛应用,常用的染料有溴甲酚绿、溴酚蓝、考马斯亮蓝及铬天青S等,这些染料的最大吸收波长均处于可见光区.近年来,近红外染料越来越引起人们的注意.     

Multiplex detection of endonucleases by using a multicolor gold nanobeacon

A highly sensitive and selective assay based on a novel enzyme‐responsive multicolor gold nanobeacon has been developed for the multiplex detection of endonucleases, a group of very important nucleases. The nanobeacon takes advantage of the high specificity of DNA cleavage reactions combined with the unique fluorescence‐quenching property of gold nanoparticles (AuNPs). To prepare the nanobeacon, three hairpin DNA reporters, each labeled at the 5′ terminus with a fluorescent dye (i.e., fluorescein amidite (FAM), carboxy‐X‐rhodamine (ROX), cyanine dye (Cy5)), that respond to one of three different endonucleases are co‐assembled at the surface of AuNPs (15 nm). This assembly brings the dyes into very close proximity with the AuNP, which leads to significant quenching of the fluorescence due to the nanosurface energy‐transfer (NSET) effect. When the nanobeacon is exposed to the targeted endonucleases, specific DNA cleavage occurs and pieces of DNA fragments are released from the AuNP surface along with the fluorescent dye, which results in the fluorescence recovery that provides the basis for a quantitative measurement of endonuclease activity. Three endonucleases, namely HaeIII, EcoRI, and EcoRV, were studied as the proof‐of‐concept analytes. These endonucleases in homogeneous mixture solutions were simultaneously quantified by the proposed assay with high sensitivity and specificity. The limits of detection obtained were in the range of 5.0×10^?4 U mL^?1 to 1.0×10^?3 U mL^?1 of endonuclease; these limits are at least 100 times more sensitive than the previously reported endonuclease assays. Endonuclease inhibitors impair the DNA cleavage, so it is anticipated that the present method has great potential for screening inhibitors of endonucleases. To demonstrate this application, the inhibitory effects of certain anticancer drugs on HaeIII, EcoRI, and EcoRV activities were studied. The present protocol proved to be sensitive, reliable, and easy to carry out.     

Fluorescent signal amplification of carbocyanine dyes using engineered viral nanoparticles

We report enhancement in the fluorescent signal of the carbocyanine dye Cy5 by using an engineered virus as a scaffold to attach >40 Cy5 reporter molecules at fixed locations on the viral capsid. Although cyanine dye loading is often accompanied by fluorescence quenching, our results demonstrate that organized spatial distribution of Cy5 reporter molecules on the capsid obviates this commonly encountered problem. In addition, we observe energy transfer from the virus to adducted dye molecules, resulting in a highly fluorescent viral nanoparticle. We have used this enhanced fluorescence for the detection of DNA-DNA hybridization. When compared with the most often used detection methods in a microarray-based genotyping assay for Vibrio cholerae O139, these viral nanoparticles markedly increased assay sensitivity, thus demonstrating their applicability for existing DNA microarray protocols.     

Cyanine‐Like Dyes with Large Bond‐Length Alternation

Herein, the synthesis and properties of alkyne‐bridged carbocations, which are analogous in structure to cyanine dyes, are reported. An alkene‐bridged dye, linked at the third position of the indole, was also synthesized as a reference compound. These new carbocations are stable under ambient conditions, allowing characterization by UV/Vis and NMR (¹H and ¹³C) spectroscopies. These techniques revealed a large degree of delocalization of the positive charge, similar to a previously reported porphyrin carbocation. The linear and nonlinear optical properties are compared with cyanine dyes and triarylmethyl cations, to investigate the effects of the bond‐length alternation and the overall molecular geometry. The value of Re(γ), the real part of the third‐order microscopic polarizability, of ?1.3×10^?33 esu for the alkyne‐linked cation is comparable to that of a cyanine dye of similar length. Nondegenerate two‐photon absorption spectra showed that the alkene‐bridged dye exhibited characteristics of cyanines, whereas the alkyne‐bridged dye is reminiscent of octupolar chromophores, such as the triarylmethyl carbocation brilliant green. Such attributes were confirmed and rationalized by quantum chemical calculations.     

Closing the Ring to Bring Up the Light: Synthesis of a Hexacyclic Acridinium Cyanine Dye

The synthesis of a geometrically constrained and near‐planar hexacyclic acridinium cyanine dye 9 is reported. When compared to its unlocked and non‐fluorescent monomethine cyanine dye analogue 3 , this photostable dye emits in the green area of the spectrum with a remarkable quantum yield close to unity in organic solvents and above 0.5 in water. A detailed steady‐state and time‐resolved spectroscopic study revealed that dye 9 forms emissive aggregates in water, which are responsible for a red‐shifted and broadened emission band and longer emission lifetime, τ≈33 compared to 6.5–7.0 ns for the monomeric dye. Dye 9 also binds strongly to DNA (both duplex and quadruplex) in its monomeric form and is very efficiently taken up by cells, in which it accumulates primarily into the nucleus.