N-乙基Cy5菁染料的合成及光谱性能测试

通过中间体1-乙基-2,3,3-三甲基-3H-吲哚啉-5-磺酸钾和中间体1-乙基-2,3,3-三甲基-3H-吲哚啉分别与缩合剂3-丙烯醛缩苯胺合成两种N-乙基Cy5菁染料。采用柱层析法分离提纯,并用液质色谱仪分析结构,紫外可见及荧光光谱检测其荧光性能;在乙醇、甲醇、丙酮、乙腈和乙酸乙酯等14种溶剂中,通过紫外可见分光光度计和荧光光谱仪,检测不同离子对其光谱性能的影响。结果表明:在1×10~(-5)mol/L的N-乙基Cy5菁染料乙腈溶液中,Cu~(2+)离子对菁染料紫外-可见吸收峰有着显著的影响,加入Cu~(2+)离子后,溶液由蓝色变成了透明无色,而其他离子并无此现象。     

含羟基氧杂蒽荧光染料的合成及其荧光成像应用

近年来,氧杂蒽类荧光染料具有很多优点而被广泛应用。由于近红外荧光染料用于荧光成像,具有光损伤小、组织穿透性大和背景荧光干扰小的优点,设计合成了具有羟基的1-(2-羟基-4-二乙氨-苯基)-氧杂蒽类荧光染料(NIR1)和1-(6-羟基-萘基-2)-氧杂蒽类荧光染料(NIR2)。通过核磁共振谱和质谱确认了荧光染料的结构。利用紫外-可见吸收光谱和荧光光谱对其光谱性能进行研究,结果表明,NIR-1和NIR-2最大吸收波长分别为642 nm和607 nm,最大发射波长分别为748 nm和752 nm。最后,对含羟基氧杂蒽荧光染料用于细胞和活体的荧光标记成像进行研究,结果表明NIR-1具有良好的细胞和活体荧光标记成像效果。     

新型近红外氧杂蒽荧光染料用于线粒体荧光成像

荧光成像具有操作简单、灵敏度高和实时等优点,而近红外荧光成像能够有效避免生物组织自发荧光干扰.通过设计和合成3个新型近红外氧杂蒽荧光染料NXD-1～NXD-3用于细胞荧光成像.首先,3个氧杂蒽荧光染料分子结构得到核磁共振谱和高分辨质谱的确认.其次,详细研究了这些荧光染料的吸收光谱和荧光光谱,实验数据表明3个荧光染料的发...     

生物基可降解荧光聚酯的制备及性能研究

以天然生物基材料蓖麻油(CO)、柠檬酸(CA)和L-半胱氨酸(L-Cys)或L-丝氨酸(L-Ser)为原料,合成一系列生物基可降解荧光聚酯(BBPP-Cys、BBPP-Ser)。采用FTIR、¹HNMR、紫外-可见光谱和荧光光谱对其结构和荧光特性进行表征。同时,测量了聚酯的水接触角、粒径大小、力学性能、降解性能以及细胞毒性。研究结果表明,悬挂在聚酯主链上独特六元环状结构是其产生荧光特性的原因。BBPP-Cys在265 nm处有最强紫外吸收,在445 nm处有最强荧光发射;BBPP-Ser在266 nm处有最强紫外吸收,在460 nm处有最强荧光发射。所合成聚酯具有良好的亲水性和较小粒径,拉伸强度范围为1.21～1.95 MPa,在0.1 mol/L NaOH溶液中7 d降解率达到60%以上。采用MTT法对荧光聚酯进行细胞毒性实验的结果表明,细胞增殖率均大于90%,证明其具有良好的生物相容性。     

PEG化羟基磷灰石纳米体系的制备及双通道荧光成像

PEG化的药物递送系统(DDSs)可以通过增强药物的渗透性和滞留性(EPR)效应克服传统化疗的副作用。利用共沉淀法和水热法制备纳米粒子DOX@HAP,进一步通过偶联反应修饰菁染料(Cy),通过铜(I)催化的炔-叠氮化物环加成反应修饰PEG链,构建了纳米制剂DOX@HAP-Cy-PEG。通过透射电子显微镜(TEM)、扫描电子显微镜(SEM)、粒度分析仪、傅里叶红外光谱仪(FTIR)、X射线光电子能谱仪(XPS)和X射线衍射仪(XRD)对该纳米载药体系的形貌、粒径、物相组成进行表征分析。利用紫外-可见(UV-Vis)分光光度法测定了该纳米材料的药物负载量以及体外药物释放曲线。进一步,利用DOX和Cy双通道荧光成像,监测DDSs在Hela和HepG2细胞中的摄取行为。表明DOX@HAP-Cy-PEG纳米载药体系有望作为一种新型的治疗与示踪一体化的抗癌纳米制剂。     

基于萘酰亚胺母体的棉用荧光活性染料的合成及其应用性能研究

本文以萘酰亚胺为荧光母体,设计并合成出3支水溶性的荧光活性染料,并通过红外和核磁共振谱对其结构进行表征。研究了3支染料在棉织物上的上染率、固色率、提升力以及荧光反射率曲线,并讨论了染料结构与性能之间的关系。结果表明:3支染料的上染率、固色率以及提升力都有较好,并且染色织物具有良好的各项牢度性能。     

1,1′-联萘类荧光染料的合成及性能研究

本文介绍了两支1,1′-联萘类荧光染料的合成方法,通过核磁和质谱等表征了染料化合物的结构,并测试了其在固体和溶液中的紫外吸收、荧光发射、荧光寿命以及荧光量子产率等性能。     

一种新型AIE/AIEE特征荧光染料的合成与光谱性质研究

聚集诱导发光增强(AIEE)现象,因其特殊的荧光性质在已成为国内外研究的热点。本论文设计并合成了一种新型AIEE的荧光染料,首先以2-羟基-6-萘甲醛为骨架,在1号位引入醛基,其次,醛基在与1-氨基苯硫醇反应,合成新型聚集诱导发光增强AIEE荧光染料,经分离纯化后获得产物Np-COH。进一步对产物通过质谱确定其分子量为410.05,并对此荧光染料的紫外光谱和不同条件下的荧光光谱进行了测定。测定结果显示该新型荧光染料在水比二甲基亚砜为30%条件下,表现了较好的荧光性能,在水比二甲基亚砜大于50%条件下,荧光较弱。Np-COH荧光染料在515 nm处的荧光受p H值影响有微弱的变化,说明染料能在较宽的pH值范围内使用。     

三苯胺类荧光探针的合成与细胞成像

以三苯胺为原料,通过Vilsmeier-Haack和McMurry反应合成了一个碳碳双键连接两个三苯胺的荧光分子1,2-二(三苯胺基)乙烯(TPAS)。采用紫外-可见光谱和荧光光谱对TPAS的光学性质进行了考察。结果表明:TPAS有很强的荧光发射性能,荧光发射为蓝光,荧光强度与浓度的线性关系较好,且溶剂化效应不强,光稳定性良好。采用扫描电镜测试了TPAS的聚集态行为。结果显示:当水的体积分数增大后,TPAS分子聚集,形貌改变。采用MTT法对TPAS进行了细胞毒性测试。结果表明,不同TPAS浓度下,细胞的存活度都在80%以上,说明TPAS对细胞的毒性很低,生物相容性好。并实现了TPAS对A549的活细胞成像。     

基于萘酰肼的铜离子选择性荧光探针在溶液和薄膜中的应用

对萘酰肼衍生物(HHMN)的铜离子识别特性进行了研究。紫外-可见光谱和荧光光谱结果表明,该衍生物能够选择性识别铜离子,检出限达到8.9×10~(-9)mol/L。在溶液中加入铜离子后,溶液颜色由无色变为黄色,在紫外灯照射下溶液荧光发生了明显淬灭。因此,借助该化合物可以实现对铜离子的肉眼辨识,并且将该化合物制备得到荧光薄膜材料后,该荧光薄膜材料也能实现对铜离子的选择性识别,这为进一步制备铜离子识别器件奠定了基础。     

Bioorthogonally Applicable Fluorescence Deactivation Strategy for Receptor Kinetics Study and Theranostic Pretargeting Approaches

The availability of a receptor for theranostic pretargeting approaches was assessed by use of a new click‐chemistry‐based deactivatable fluorescence‐quenching concept. The efficacy was evaluated in a cell‐based model system featuring both membranous (available) and internalized (unavailable) receptor fractions of the clinically relevant receptor chemokine receptor 4 (CXCR4). Proof of concept was achieved with a deactivatable tracer consisting of a CXCR4‐specific peptide functionalized with a Cy5 dye bearing a chemoselective azide handle (N₃‐Cy5‐AcTZ14011). Treatment with a Cy7 quencher dye (Cy7‐DBCO) resulted in optically silent Cy7‐[click]‐Cy5‐AcTZ14011. In situ, a >90 % FRET‐based reduction of the signal intensity of N₃‐Cy5‐AcTZ14011 [K_D=(222.4±25.2) nm ] was seen within minutes after quencher addition. In cells, discrimination between the membranous and the internalized receptor fraction could be achieved through quantitative assessment of quenching/internalization kinetics. Similar evaluation of an activatable tracer variant based on the same targeting moiety (Cy5‐S‐S‐Cy3‐AcTZ14011) was unsuccessful in vitro. As such, using the described deactivatable approach to screen membrane receptors and their applicability in receptor‐(pre‐)targeted theranostics can become straightforward.     

生物标示用3H-吲哚菁染料

随着生物技术和荧光标示技术的飞速发展,3H-吲哚菁染料已成为在DNA、蛋白质及核酸等分析检测中使用的主要荧光探针。其中代表性化合物Cy3.、Cy5.作为新一代商品化荧光标示剂在生物芯片、分子信标等方面得到了重要应用。本文综述了该类碳菁染料在生物领域中取得的重大应用进展、并探讨了其结构与性能之间的关系。     

Protein A Detection Based on Quantum Dots-Antibody Bioprobe Using Fluorescence Coupled Capillary Electrophoresis

In this report, fluorescence detection coupled capillary electrophoresis (CE-FL) was used to detect Protein A. Antibody was first labeled with Cy5 and then mixed with quantum dots (QDs) to form QDs-antibody bioprobe. Further, we observed fluorescence resonance energy transfer (FRET) from QDs donor to Cy5 acceptor. The bioprobe was formed and brought QDs and Cy5 close enough to allow FRET to occur. After adding protein A, the FRET system was broken and caused the FRET signal to decrease. Thus, a new method for the determination of protein A was proposed based on the FRET signal changes. This study provides a new trail of thought for the detection of protein.     

A spectroscopic study of the self-association and inter-molecular aggregation behaviour of pH-responsive poly(l-lysine iso-phthalamide)

The pH mediated intra-molecular association and inter-molecular aggregation of a range of amphiphilic poly(l-lysine iso-phthalamide) polymers have been investigated in aqueous solution over a range of pH values and concentrations. The desired functionality of these novel bioresponsive amphiphilic polymers was achieved by incorporating pendant hydrophilic carboxyl groups along the polymer backbone, via the l-lysine moiety, balanced by a degree of hydrophobicity introduced via the iso-phthaloyl moiety. Incorporation of low levels of bis-functional Cy3 (poly-Cy3) and/or Cy5 dye (poly-Cy3/5 or poly-Cy5) co-monomers in the responsive polymer backbone allowed detailed probing of the pH mediated hydrophobic association using a combination of optical spectroscopic techniques. Both steady-state fluorescence spectroscopy and fluorescence lifetime measurements of poly-Cy3 revealed a conformational transition at pH 4.5. Thus, below a critical pH the polymer collapsed into a compact globular structure (hypercoil) bringing the fluorophore molecules into close proximity with one another. This resulted in a dramatic reduction in fluorescence intensity and fluorescent lifetime in the single fluorophore systems (poly-Cy3) accompanied by a red shift in the maximum emission wavelength. Observed redshifts in the emission maxima and enhancements of fluorescent lifetimes with increasing polymer concentration suggested the formation of polymer aggregates. Fluorescence resonance energy transfer (FRET) was measured in mixtures of single fluorophore containing poly-Cy3 (donor) and poly-Cy5 (acceptor) and dual fluorophore containing poly-Cy3 (donor)/Cy5 (acceptor) in an effort to distinguish between intra-molecular versus inter-molecular association. The relevance of the results with respect to potential in vivo applications (drug delivery and biodiagnostics) is discussed.     

Theranostic Polyaminocarboxylate–Cyanine–Transferrin Conjugate for Anticancer Therapy and Near‐Infrared Optical Imaging

Iron chelation therapy has been recognized as a promising antitumor therapeutic strategy. Herein we report a novel theranostic agent for targeted iron chelation therapy and near‐infrared (NIR) optical imaging of cancers. The theranostic agent was prepared by incorporation of a polyaminocarboxylate‐based cytotoxic chelating agent (N‐NE3TA; 7‐[2‐[(carboxymethyl)amino]ethyl]‐1,4,7‐triazacyclononane‐1,4‐diacetic acid) and a NIR fluorescent cyanine dye (Cy5.5) onto a tumor‐targeting transferrin (Tf). The N‐NE3TA–Tf conjugate (without Cy5.5) was characterized and evaluated for antiproliferative activity in HeLa, HT29, and PC3 cancer cells, which have elevated expression levels of the transferrin receptor (TfR). The N‐NE3TA–Tf conjugate displayed significant inhibitory activity against all three cancer cell lines. The NIR dye Cy5.5 was then incorporated into N‐NE3TA–Tf, and the resulting cytotoxic and fluorescent transferrin conjugate N‐NE3TA–Tf–Cy5.5 was shown by microscopy to enter TfR‐overexpressing cancer cells. This theranostic conjugate has potential application for dual use in targeted iron chelation cancer therapy and NIR fluorescence imaging.     

Fluorescence from fluorescent dye based polyurethane ionomer(III)

A series of reactive fluorescent dyes were successfully synthesized and their structure was proven by IR spectra, NMR spectra, elemental analysis, and mass spectra. The fluorescence performance 6‐amino‐2(‐3‐phenyl‐propyl)‐benzo[de]isoquinoline‐1,3‐dione and 2‐benzyl‐6‐hydroxy‐benzo[de]isoquinoline‐1,3‐dione appears at around 276 and 437.4 nm, respectively, and their quantum yields are 0.662 and 0.562, respectively. It is important to indicate that the fluorescence performance is better for 6‐amino‐2(‐3‐phenyl‐propyl)‐benzo[de]isoquinoline‐1,3‐dione than for as a result of more electron donating groups linked to the 6‐amino‐2(‐3‐phenyl‐propyl)‐benzo[de]isoquinoline‐1,3‐dione molecule. These fluorescent dyes further react with toluene diisocyanate and other additives to form fluorescent dye based polyurethane (PU) ionomer molecules, and their structures are demonstracted by IR spectra. In aqueous solution, the fluorescence performance appears to be better for 6‐amino‐2(‐3‐phenyl‐propyl)‐benzo[de]isoquinoline‐1,3‐dione based PU ionomer than for 6‐amino‐2‐phenyl‐ethyl‐benzo[de]isoquinoline‐1,3‐dione based PU ionomer. For the fluorescent dye based PU ionomer molecule system, the number‐average particle sizes of the fluorescent dye based PU ionomer molecules in water increase with increasing concentration of the fluorescent dye, as a result of the increased free volume of the ionomer molecule. This may be the result of increased intermolecular interactions between ionomer– molecules themselves. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 455–465, 2005     

Fast detection of single nucleotide polymorphisms (SNPs) by primer elongation with monitoring of supercritical-angle fluorescence

We describe the rapid detection of single nucleotide polymorphisms (SNPs) by real-time observation of primer elongation. The enzymatic elongation of surface-bound primers is monitored by detecting the increase of surface-bound fluorescence caused by the incorporation of Cy5-labelled deoxycytidine 5'-triphosphate residues (Cy5-dCTPs) into the corresponding strand. In order to discriminate against the fluorescence from unbound Cy5-dCTPs, the detection volume was restricted to the surface by collecting supercritical-angle fluorescence. The efficiency of enzymatic double-stranded DNA synthesis is governed by the complementarity of the primer and template. An SNP in the sequence of the primer obstructs its elongation increasingly with decreased distance of the mismatch to the 3' end of the primer. By real-time fluorescence detection during primer elongation, SNPs can be detected within a few minutes, which is significantly faster than in experiments where the fluorescence is measured after completion of the reaction. We demonstrate the efficiency of the method by detecting an SNP in the ErbB2 gene that is involved in causing a higher risk of breast cancer.     

Live-Cell Copper-Induced Fluorescence Quenching of the Flavin-Binding Fluorescent Protein CreiLOV

CreiLOV is a flavin-binding fluorescent protein derived from the blue-light photoreceptor protein family that contains light-oxygen-voltage (LOV) sensing domains. Flavin-binding fluorescent proteins represent a promising foundation for new fluorescent reporters and biosensors that can address limitations of the well-established green fluorescent protein (GFP) family. Flavin-binding fluorescent proteins are smaller than GFPs, are stable over a wider pH range, offer rapid chromophore incorporation, and are oxygen-independent so can be applied to live anaerobic organisms. Among the flavin-binding fluorescent proteins, CreiLOV has a high quantum yield and excellent photophysical properties, making it promising for cellular applications. Here, we investigated the suitability of CreiLOV as an intensity- and fluorescence-lifetime-based metal sensor. CreiLOV selectively binds copper(II) over other biologically relevant metals with low-micromolar affinity, resulting in fluorescence quenching and a decrease in the fluorescence lifetime that can be observed in cuvettes and live bacterial cells.     

Fluorescence Activation with Photochromic Auxochromes

The inherent reversibility of photochromic transformations can be exploited to switch on and off the fluorescence of appropriate organic chromophores under optical control. In turn, the photoactivation of fluorescence permits the monitoring of dynamic processes in real time as well as the reconstruction of images with spatial resolution at the nanometer level. Thus, the identification of viable structural designs to construct and operate photoactivatable fluorophores on the basis of photochromic processes can translate into the realization of valuable analytical tools for biomedical research. In this context, a strategy was designed to connect a simple photochromic oxazine to essentially any fluorescent chromophore with a pendant formyl group. In the resulting fluorophore photochrome dyads, the photoinduced interconversion of the photochromic component between its two states controls that ability of the fluorescent component to absorb exciting radiation and emit as a result. Under these conditions, the interplay of two illuminating beams, designed to operate the photochromic component and excite the fluorescent one, respectively, offers the opportunity to switch fluorescence reversibly for multiple cycles. Furthermore, the fluorophore photochrome dyads described herein can be entrapped within the hydrophobic core of polymer micelles and operated under these conditions in aqueous solutions and within the intracellular environment.     

Fluorescence technique for the determination of low critical micelle concentrations

A technique for determining low critical micelle concentrations (CMC) by means of a hydrophobic fluorescence probe has been developed. The amount of the fluorescent probe at the CMC is so small that the effect of the probe on micelle formation is negligible. The fluorescence intensity was measured at fixed dye/surfactant ratios, and it decreased with concentration. A quantity proportional to fluorescent quantum yield was calculated and found to be high for concentrations of surfactant above the CMC and almost zero below the CMC, giving a distinct break in the quantum yield vs. the concentration curve.