近红外荧光探针检测金属离子、小分子和生物大分子

荧光探针技术是近年来发展迅速的一种荧光分析方法,具有灵敏度高、选择性好、操作简便和响应迅速等特点,受到环境及生命科学领域的青睐.随着荧光探针技术的发展,近红外一区荧光探针由于具有发射波长长(600～ 900 nm)、对细胞损伤小、组织穿透性强和自发荧光背景低等优点,被广泛应用于细胞、组织等复杂生物体系中生物分子的检测、...     

A Selective and Sensitive "Turn-on" Fluorescent Chemosensor for Recognition of Hg(2+) Ions in Water

Based upon highly selective and irreversible Hg(2+) -promoted deprotection of the dithioacetal reaction, a new water-soluble "turn-on" fluorescent chemosensor (1) was prepared and exhibited high selectivity and sensitivity towards the Hg(2+) ion over other heavy and transition-metal ions in pure water by transforming a weakly fluorescent precursor (colorless) to a highly fluorescent aldehyde (yellow-green; see figure) with a 155-fold increase in fluorescent intensity.     

Fluorescent β-cyclodextrins modified by isomeric aminobenzamides: synthesis, conformational analysis, and fluorescent behaviors

Three isomeric fluorescent β-cyclodextrins bearing 2-, 3-, and 4-(2-aminoethyl)amino-N-butylbenzamide, respectively (1-3) have been synthesized. The conformations of these fluorescent CDs have been investigated by 2D NMR and induced circular dichromism. It is confirmed that the ortho isomer 1 takes a butyl-included conformation, while the other two isomers 2 and 3 display a phenyl-included conformation, respectively. The three fluorescent CDs 1-3 exhibited totally different self- and guest-inclusion fluorescence behavior. In the presence of adamantane carboxylate sodium (ADA) or deoxycholate sodium (DCA), the fluorescence intensity of 1 showed an enhancement over 1-fold, while 2 exhibited dramatic fluorescence quenching. Interestingly, the fluorescent responses of 3 toward two guests respectively were highly distinguishable. The fluorescence intensity of 3 only showed a slight increase upon the addition of ADA, but the addition of DCA led to a large decrease in fluorescence intensity. The investigations have been further carried out by 2D NMR, induced circular dichromism, fluorescence spectroscopy and molecular modeling to explore the relationships between the conformations and the fluorescence characteristics of CDs 1-3 in the absence and presence of guest molecules. On the basis of the above investigations, the origins for the different fluorescence behaviors have been proposed.     

Photoacoustic Effect of Near-Infrared Absorbing Organic Molecules via Click Chemistry

Near-infrared dyes were developed to be contrast agents due to their ability to improve the productivity of photoacoustic (PA) imaging and photothermal therapy (PTT) treatments. During the article, we described in detail the PA and PT effects of a category of organic molecules. F₄-TCNQ could potentially cause a red-shift in the peak PA intensity. The results show that the PTT intensity of the near-infrared dyes with phenyl groups were higher than near-infrared dyes with thiophene groups. We also investigated the photodynamic treatment effect of C1b to demonstrate that these dyes are highly desirable in biochemistry. The high photoacoustic intensity of the organic molecules and the good yield of reactive oxygen species could indicate that these dyes have good potential for a wide range of imaging applications. Finally, we embedded the dye (C1b) in a liposomal hydrophobic phospholipid bilayer (C1b⊂L) to facilitate the application of hydrophobic dyes in biomedical applications, which can be absorbed by cells with good compatible and high stability for the imaging of cellular PA.     

一种新型Zn2＋近红外荧光探针的研究

Zn^2＋是多种酶和转录因子的必要成分, 在生物体内起着重要的作用, 检测Zn²⁺具有十分重要的生物学意义. 我们合成了一种新型近红外荧光探针TAEA-IR-780, 该探针通过配位作用与Zn^2＋结合后, 荧光显著增强. 通过质谱和氢谱对该探针进行了表征, 并探讨了pH值、其它金属离子对Zn^2＋检测的影响. 该探针的激发波长为683 nm, 发射波长为750 nm, 可避免对生物活体的损伤, 同时生物体内常见的金属离子对其干扰较小. 该探针对Zn^2＋的检测下限达1.0× 10^－9 mol•L^－1, 具有较高的检测灵敏度.     

First artificial receptors and chemosensors toward phosphorylated peptide in aqueous solution

The first fluorescent chemosensors toward a native phosphorylated peptide are successfully synthesized. Dinuclear zinc(II)-dipicolylamine-based anthracene (1, 2) can selectively recognize and sense phosphorylated species with an increase in the fluorescence intensity. We also demonstrated that these artificial receptors fluorometrically detect a phosphorylated peptide with high affinity (>107 M-1) in aqueous solution.     

新型水溶性不对称五甲川菁染料的合成、光稳定性及蛋白质的荧光标记

合成并表征了系列水溶性五甲川菁染料, 研究了其在不同溶剂中的光谱性能. 结果表明, 染料在水中的最大吸收和荧光光谱在647~665 nm波长范围内, 荧光量子产率达到0.1左右. 考察了N位取代基对染料水溶液光稳定性的影响, 结果表明, 在N原子上引入带有苯环结构和大体积的磺酸基, 可以提高染料的光稳定性. 高效液相色谱(HPLC)分析结果表明, 染料4a的N-羟基琥珀酰亚胺(NHS)活性酯标记牛血清白蛋白(BSA)的检测限为1.2×10^-8 mol/L, 与紫外检测相比, 检测灵敏度提高了近2个数量级.     

Spectral properties of pro-multimodal imaging agents derived from a NIR dye and a metal chelator

Monomolecular multimodal imaging agents (MOMIAs) are able to provide complementary diagnostic information of a target diseased tissue. We developed a convenient solid-phase approach to construct two pro-MOMIAs (before incorporating radiometal) derived from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and cypate, a near-infrared (NIR) fluorescent dye analogous to indocyanine green (ICG). The possible interaction between d orbitals of transition metal DOTA complexes or free metals and the p orbitals of cypate chromophore could quench the fluorescence of pro-MOMIAs. However, we did not observe significant changes in the spectral properties of cypate upon conjugation with DOTA and subsequent chelation with metals. The fluorescence intensity of the chelated and nonmetal-chelated PRO-MOMIAs remained fairly the same in dilute 20% aqueous dimethylsulfoxide (DMSO) solution (1 x 10(-6) M). Significant reduction in the fluorescence intensity of pro-MOMIAs occurred in the presence of a large excess of metal ions (>1 molar ratio for indium and 20-fold for a copper relative to pro-MOMIA). This study suggests the feasibility of using MOMIAs for combined optical and radioisotope imaging.     

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17β-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17β-estradiol is 1.9 pg mL⁻¹, which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.     

A highly enantioselective chiral Schiff-base fluorescent sensor for mandelic acid

A chiral Schiff-base compound, 4-methyl-2,6-bis-[(2-hydroxy-1-phenylethylimino)methyl]phenol, is found to act as highly enantioselective fluorescent agent for α-hydroxycarboxylic acid, e.g., mandelic acid. It is observed that, within a certain concentration range, one enantiomer of the chiral acid can increase the fluorescence intensity of the Schiff-base compound 122-fold while the other enantiomer enhances the intensity only 42-fold. Such highly enantioselective responses towards the chiral acid make the unusual Schiff-base compound attractive as a fluorescent sensor for determining the enantiomeric composition of α-hydroxycarboxylic acids.     

Zn2+-triggered excited-state intramolecular proton transfer: a sensitive probe with near-infrared emission from bis(benzoxazole) derivative

Near-infrared (NIR) emission can offer distinct advantages for biological applications. A fluorescent sensor, Zinhbo-1, based on bis(benzoxazole) ligand with 2,2'-dipicolylamine (DPA) as receptor, was synthesized. In aqueous solution, Zinhbo-1 demonstrates high sensitivity and selectivity for sensing Zn(2+) with about 10-fold enhancement and nanomolar sensitivity (K(d) = 0.29 nM). Moreover, sensor Zinhbo-1 can detect Zn(2+) in near-infrared region (over 700 nm) with large Stokes shift (ca. 230 nm) attributing to the Zn(2+)-induced excited state intramolecular proton transfer (ESIPT).     

A highly selective turn-on fluorescent probe for hypochlorous acid based on hypochlorous acid-induced oxidative intramolecular cyclization of boron dipyrromethene-hydrazone

A BODIPY-based fluorescent probe, HBP, was developed for the detection of hypochlorous acid based on the specific hypochlorous acid-promoted oxidative intramolecular cyclization of heterocyclic hydrazone in response to the amount of HOCl. The reaction is accompanied by a 41-fold increase in the fluorescent quantum yield (from 0.004 to 0.164). The fluorescence intensity of the reaction between HOCl and HBP is linear in the HOCl concentration range of 1–8 μM with a detection limit of 2.4 nM (S/N = 3). Confocal fluorescence microscopy imaging using RAW264.7 cells showed that the new probe HBP could be used as an effective fluorescent probe for detecting HOCl in living cells.     

HSA-Lys-161 covalent bound fluorescent dye for in vivo blood drug dynamic imaging and tumor mapping

The specific combination of human serum albumin and fluorescent dye will endow superior performance to a coupled fluorescent platform for in vivo fluorescence labeling. In this study, we found that lysine-161 in human serum albumin is a covalent binding site and could spontaneously bind a ketone skeleton quinoxaline–coumarin fluorescent dye with a specific turn-on fluorescence signal for the first time. Supported by the abundant drug binding domains in human serum albumin, drugs such as ibuprofen, warfarin and clopidogrel could interact with the fluorescent dye labeled human serum albumin to feature a substantial enhancement in fluorescence intensity (6.6-fold for ibuprofen, 4.5-fold for warfarin and 5-fold for clopidogrel). The drug concentration dependent fluorescence intensity amplification realized real-time, in situ blood drug concentration monitoring in mice, utilizing ibuprofen as a model drug. The non-invasive method avoided continuous blood sample collection, which fundamentally causes suffering and consumption of experimental animals in the study of pharmacokinetics. At the same time, the coupled fluorescent probe can be efficiently enriched in tumors in mice which could map a tumor with a high-contrast red fluorescence signal and could hold great potential in clinical tumor marking and surgical resection.

HSA lysine-161 covalent bound quinoxaline–coumarin based fluorescent dye realized in situ blood drug concentration monitoring and tumor visualization.     

Preparation of luminescent Cy5 doped core-shell SFNPs and its application as a near-infrared fluorescent marker

Cy5 dye is widely used as a biomarker in the research fields of life science because of its excitation at wavelengths above 600 nm where autofluorescence of bio-matter is much reduced. However, Cy5 dye could not be encapsulate into silica directly to form stable nanoparticles by using of the traditional methods. In this paper, an improved method had been developed to prepare Cy5 dye doped core-shell silica fluorescent nanoparticles (SFNPs), employing biomolecules conjugated Cy5 as the core material and silica coating produced from the hydrolysis TEOS (tetraethyl orthosilicate) in the water-in-oil microemulsion. To obtain stable Cy5 dye doped SFNPs with core-shell structure, five kinds of biomolecules with different iso-electric point (pI) have been selected to conjugate Cy5 for preparation of core-shell SFNPs. Results demonstrated that very bright and photostable Cy5 doped core-shell SFNPs could be both prepared by use of positive polysine conjugated Cy5 or IgG conjugated Cy5 as the core material, respectively. IgG conjugated Cy5 doped core-shell SFNPs was selected as a demonstration to be characterized and applied as a near-infrared fluorescent marker in cell recognition. The results showed that Cy5 doped core-shell SFNPs prepared by conjugating with a positive biomolecules IgG as the core material were luminescent and stable. About 110 Cy5 dye molecules could be doped in one nanoparticle with size of 42 ± 5 nm. The breast cancer cells had been selectively recognized by use of the near-infrared fluorescent marker based on the Cy5-IgG doped core-shell SFNPs. And the results demonstrated that this Cy5 doped core-shell SFNPs fluorescence marker was superior to the pure Cy5 dye marker for cell recognition in photostability and detection sensitivity.     

Conjugated polymer-based fluorescence turn-on sensor for nitric oxide

A turn-on fluorescent sensor for NO (g) in solution was synthesized using a bipyridyl-substituted poly(p-phenylene vinylene) derivative (CP1) as the sensory scaffold. The action of NO (g) upon the CP1-Cu(II) complex reduces it to the CP1-Cu(I) complex with a concomitant 2.8-fold increase in emission intensity. The reagent is selective for NO (g) versus other biological reactive nitrogen species, except for nitroxyl, and has a detection sensitivity limit of 6.3 nM. [structure: see text]     

Highly enantioselective fluorescent recognition of α-amino acid derivatives

Bisbinaphthyl-based macrocycles are found to carry out enantioselective fluorescent recognition of α-amino acid derivatives. It is observed that one enantiomer of a N-protected phenyl glycine can increase the fluorescence intensity of the binaphthyl fluorophores by over 4-fold but the other enantiomer does not cause much fluorescence enhancement. This highly enantioselective fluorescent response makes the binaphthyl macrocycles practically useful for the enantioselective fluorescent recognition of the amino acid substrate.     

一种具有双重功能的可视化pH荧光探针的合成及光谱研究

以二烯单酮结构为荧光团,酚羟基为脱质子基团,合成了一种具有双重功能的可视化pH荧光分子探针.pH滴定实验表明,探针的紫外吸收和荧光光谱均对溶液的pH值有很强的依赖性,当体系溶液由酸性变为碱性时,探针的紫外吸收光谱发生明显的红移,并伴有溶液颜色的显著变化;荧光光谱强度在酸性条件下随pH值的变化不大,而在碱性条件下随pH值...     

一种香豆素类荧光探针的合成及其性能研究

本文设计合成了一种基于香豆素的荧光探针L,通过氢谱、质谱对其结构进行表征。该探针在DMSO/H_2O(体积比9∶1)体系中对Co~(2+)和Ni~(2+)具有较好的选择性和灵敏度。Co~(2+)和Ni~(2+)的加入使得探针L的荧光发射发生猝灭,其他金属离子未对探针的荧光产生明显的影响。探针L与Co~(2+)和Ni~(2+)的配位比均为1∶2,其对Co~(2+)和Ni~(2+)的检出限分别为1.002×10~(-7)和9.78×10~(-6) mol/L,结合常数分别是1.06×10~6和9.84×10~5 L·mol~(-1)。     

Sensitivity enhancement of CE and CE-MS for the analysis of peptides by a dynamic pH junction

An analytical method of CE-MS and CE with an online preconcentration technique induced by a dynamic pH junction, addition of organic solvent and large volume injection was developed for sensitive determination of peptides in biological samples. Leucine enkephalin, methionine enkephalin, dynorphin A, β-endorphin and angiotensin II were used as model peptides. The optimal online preconcentration conditions were obtained at a sample matrix consisting of 100?mM borate buffer (pH 10.0) with 50% v/v acetonitrile and a BGE containing 1?M formic acid at pH 2.0, along with a 25-cm injection length. Under the optimized conditions, a 4.0×10(3)-1.1×10(4)-fold increase in peak intensity was achieved without degrading the peak shape. This online preconcentration method was applied to analyze the intracellular angiotensin II within the peptides extracted from HL1 cells and approximately increase of 1×10(4)-fold sensitivity was achieved compared to normal condition. Thus, the developed method could be applied to the analysis of various peptides for peptidomics study in biological samples.     

Novel Fluorescent Methods for Biotechnological and Biomedical Sensoring: Assessing Antioxidants,Reactive Radicals,NO Dynamics,Immunoassay, and Biomembranes Fluidity

We proposed and developed a series of fluorescent methods for analysis and investigation of biological systems with a view of future biotechnological and biomedical applications. The methods we describe have been built upon several photochemical and photophysical phenomena including fluorescent quenching, photochrome photoisomerization, and energy transfer. Three new types of molecular probes have been developed and employed for such studies: (1) dual fluorophore–nitroxide compounds, (2) fluorescence–photochrome molecules, and (3) super molecules containing both fluorescence and fluorescent quenching segments. The fluorescent properties of the new probes were intensively exploited for several practical applications including a real-time analysis of antioxidants, nitric oxide, superoxide, reactive radicals, trinitrotoluene, and metal ions, investigation of molecular dynamics of biomembranes in a wide range characteristic times, detection of protein conformational transition, and characterization of surface system. Owning high sensitivity, simplicity, and availability of fluorescent techniques, these methods can be widely employed and are adaptable to fibrooptic sensoring. A general survey of the physical principles and application of the new fluorescent methods has been provided.