Potential Use of Quantum Dots in Flow Cytometry

QDs may offer significant advantages in environmental and bead-based applications where the target cells need to be discriminated above background fluorescence. We have examined the possible applications of QDs for flow cytometric measurements (FCM) by studying their excitation - emission spectra and their binding to paramagnetic beads. We labelled beads with either QDs or a commonly-used fluorochrome (FITC) and studied their fluorescence intensity by FCM. Flow cytometric comparisons indicated that the minimum fluorophore concentration required for detection of QDs above autofluorescent background was 100-fold less than for FITC.     

Synthesis and properties of water-soluble 1,9-dialkyl-substituted BF2 azadipyrromethene fluorophores

Bis-alkylsulfonic acid and polyethylene glycol (PEG)-substituted BF₂ azadipyrromethenes have been synthesized by an adaptable and versatile route. Only four synthetic stages were required to produce the penultimate fluorophore compounds, containing either two alcohol or two terminal alkyne substituents. The final synthetic step introduced either sulfonic acid or polyethylene glycol groups to impart aqueous solubility. Sulfonic acid groups were introduced by reaction of the bis-alcohol-substituted fluorophore with sulfur trioxide, and a double Cu(I)-catalyzed cycloaddition reaction between the bis-alkyne fluorophore and methoxypolyethylene glycol azide yielded a neutral bis-pegylated derivative. Both fluorophores exhibited excellent near-infrared (NIR) photophysical properties in methanol and aqueous solutions. Live cell microscopy imaging revealed efficient uptake and intracellular labelling of cells for both fluorophores. Their simple synthesis, with potential for last-step structural modifications, makes the present NIR-active azadipyrromethene derivatives potentially useful as NIR fluorescence imaging probes for live cells.     

1,8‐Naphthalimide Derivative Dyes with Large Stokes Shifts for Targeting Live‐Cell Mitochondria

An ideal fluorescent dye for staining cell organelles should have multiple properties including specificity, stability, biocompatibility, and a large Stokes shift. Tunable photophysical properties enable 1,8‐naphthalimide to serve as an excellent fluorophore in biomedical applications. Many naphthalimide derivatives have been developed into drugs, sensors, and other dyes. In this study, a series of 1,8‐naphthalimide derivatives targeting live cell mitochondria were synthesized. Among these probes, Mt‐4 was characterized as the best one, with highly specific mitochondrial localization, low cytotoxicity, and a large Stokes shift. More importantly, Mt‐4 stood out as a potential mitochondrial dye for living‐cell experiments involving induced mitochondrial stress arising from the treatments because Mt‐4 shows enhanced fluorescence in mitochondrial stress situations.     

Fluorescent N2,N3-epsilon-adenine nucleoside and nucleotide probes: synthesis, spectroscopic properties, and biochemical evaluation

N1,N(6)-ethenoadenine, epsilon-A, nucleos(t)ides have been previously applied as fluorescent probes in numerous biochemical systems. However, these epsilon-A analogues lack the H-bonding capability of adenine. To improve the fluorescence characteristics while preserving the H-bonding pattern required for molecular recognition, we designed a novel probe: N(2),N3-etheno-adenosine, (N(2),N3-epsilon-A). Here, we describe four novel syntheses of the target epsilon-nucleoside and related analogues. These methods are short, facile, and provide the product regiospecifically. In addition, we report the absorption and emission spectra of N(2),N3-epsilon-A and the dependence of the spectral features on the pH and polarity of the medium. Specifically, maximum emission of N(2),N3-epsilon-A in water is observed at 420 nm (phi=0.03, excitation at 290 nm). The biochemical relevance of the new probe was evaluated with respect to the P2Y(1) receptor and NTPDases 1 and 2. N(2),N3-epsilon-ATP was found to be almost equipotent with ATP at the P2Y(1) receptor and was hydrolyzed by NTPDases 1 and 2 at about 80 % of the rate of ATP. Furthermore, protein binding does not seem to shift the fluorescence of N(2),N3-epsilon-ATP. Based on the fluorescence and full recognition by ATP-binding proteins, we propose N(2),N3-epsilon-ATP and related nucleo(s)tides as unique probes for the investigation of adenine nucleo(s)tide-binding proteins as well as for other biochemical applications.     

含吡嗪、喹啉荧光探针的合成及其对金属离子的识别研究

设计合成了3个含有吡嗪基团的荧光探针分子和喹啉基团的荧光探针分子,其结构通过红外(FT-IR)、质谱(MS)、核磁共振氢谱(1HNMR)进行表征,通过吸收光谱和荧光光谱研究了不同金属离子对目标探针分子的影响。结果表明:Cd2+使401 nm处荧光蓝移了20 nm;Co2+、Hg2+、Pb2+、Zn2+使357 nm处荧光分别红移了20、50、1401、40 nm;Cd2+、Hg2+使349 nm处荧光分别红移了707、5 nm。     

Following in situ photoinitiated polymerization of multifunctional acrylic monomers by fluorescence and photocalorimetry simultaneously

The photoinitiated polymerization of multifunctional (meth)acrylic monomers was simultaneously monitored in real time by fluorescence and differential scanning calorimetry. As the curing proceeds, the fluorescence emission changes due to an increase in the viscosity of the microenvironment. A good correlation between fluorescence intensity and degree of conversion was established by using two different fluorescent probes, each having the same fluorophore, dialkylamino derivatives of 7-nitro-2-oxa-1,3-diazol. One of the probes contains an acrylic moiety which can react with the monomers labelling the formed network. The use of the first moment of fluorescence is presented as a suitable methodology to avoid experimental fluctuations. Furthermore, the influence of the length of the spacer between the acrylic groups in the polymerization reaction kinetics and fluorescence changes has been also discussed in terms of reaction diffusion controlled termination kinetics and free volume fraction. It was concluded that the fluorescence method is a powerful tool to study the kinetics of photopolymerization of multifunctional monomers.     

Single Labeled DNA FIT Probes for Avoiding False‐Positive Signaling in the Detection of DNA/RNA in qPCR or Cell Media

Oligonucleotide hybridization probes that fluoresce upon binding to complementary nucleic acid targets allow the real‐time detection of DNA or RNA in homogeneous solution. The most commonly used probes rely on the distance‐dependent interaction between a fluorophore and another label. Such duallabeled oligonucleotides signal the change of the global conformation that accompanies duplex formation. However, undesired nonspecific binding events and/or probe degradation also lead to changes in the label–label distance and, thus, to ambiguities in fluorescence signaling. Herein, we introduce singly labeled DNA probes, “DNA FIT probes”, that are designed to avoid false‐positive signals. A thiazole orange (TO) intercalator dye serves as an artificial base in the DNA probe. The probes show little background because the attachment mode hinders 1) interactions of the “TO base” in cis with the disordered nucleobases of the single strand, and 2) intercalation of the “TO nucleotide” with double strands in trans. However, formation of the probe–target duplex enforces stacking and increases the fluorescence of the TO base. We explored open‐chain and carbocyclic nucleotides. We show that the incorporation of the TO nucleotides has no effect on the thermal stability of the probe–target complexes. DNA and RNA targets provided up to 12‐fold enhancements of the TO emission upon hybridization of DNA FIT probes. Experiments in cell media demonstrated that false‐positive signaling was prevented when DNA FIT probes were used. Of note, DNA FIT probes tolerate a wide range of hybridization temperature; this enabled their application in quantitative polymerase chain reactions.     

A sensitive and selective near-infrared fluorescent probe for mercuric ions and its biological imaging applications

A new mercury(II) near-infrared region fluorescent probe 3,9-dithia-6-monoazaundecane-tricarbocyanine has been designed and synthesized. It consists of two functional moieties: the tricarbocyanine performs as the near-infrared region fluorophore, and the 3,9-dithia-6-monoazaundecane acts as the selected binding site for metal ions. The near-IR excitation and emission profiles of the probe can minimize cell and tissue damage and avoid native fluorescence from natural cellular species. It exhibits fluorescence increase upon the binding of the Hg(2+) based on the inhibition of the photoinduced electron transfer quenching mechanism. Excellent sensitivity and selectivity for mercuric ions are observed with this probe. The value of the system is demonstrated by its use in monitoring the real-time uptake of Hg(2+) within HepG2 cells and five day old zebrafish. The synthesis and remarkable properties of it help to extend the development of metal ions fluorescent probes for biological applications.     

Design and synthesis of caged fluorescent nucleotides and application to live-cell RNA imaging

A binary photocontrolled nucleic acid probe that contains a nucleotide modified with one photolabile nitrobenzyl unit and two hybridization-sensitive thiazole orange units has been designed for area-specific fluorescence imaging of RNA in a cell. The synthesized probe emitted very weak fluorescence regardless of the presence of the complementary RNA, whereas it showed hybridization-sensitive fluorescence emission at 532 nm after photoirradiation at 360 or 405 nm for uncaging. Fluorescence suppression of the caged probe was attributed to a decrease in the duplex-formation ability. Caged fluorescent nucleotides with other emission wavelengths (622 and 724 nm) were also synthesized in this study; they were uncaged by 360 nm irradiation, and emitted fluorescence in the presence of the complementary RNA. Such probes were applied to area-specific RNA imaging in a cell. Only probes in the defined irradiation area were activated by uncaging irradiation, and subnuclear mRNA diffusion in a living cell was monitored.     

In situ monitoring of sorption and drying of polymer films and coatings: self-referencing, nearly temperature-independent fluorescence sensors

Sorption and drying processes were monitored in situ in polymer films by a fluorescence rotor probe, 4-tricyanovinyl-[N-(2-hydroxyethyl)-N-ethyl]aniline (TC1), a solvatochromatic fluorescence probe, 4-(N,N-dimethylamino)-4′-nitrostilbene (DANS), and pyrene. Taking advantage of an intensity ratio to monitor sorption, these fluorescence probes were found to serve as self-referencing sensors of water sorption in polyvinylacetate, with DANS being the most sensitive followed by TC1 and pyrene. Additionally, the shapes of the emission spectra (and thus intensity ratios) for TC1 and DANS were independent of temperature over a range of reasonable expected use temperatures. Covalent attachment of these fluorescence dyes was shown to enable the determination of sorbate levels within particular layers of multilayer films or coatings. Finally, these probes were also shown to provide sensitivity to desorption or drying of both water and organic sorbates.     

Two-photon fluorescent probes for biomembrane imaging: effect of chain length

Two-photon fluorescent probes for the cellular membrane, derived from 6-acyl-2-aminonaphthalene as the fluorophore and hexanoyl (CH), lauryl (CL), and stearyl (CS) groups as the receptor, have been synthesized. Their photophysical properties and utility as membrane probes were also studied. Whereas CH cannot be used as a membrane probe due to its high water solubility, CL and CS are useful two-photon probes for membrane lateral heterogeneity, as they can easily stain cells, emit fluorescence with high sensitivity to the environment polarity, and are capable of imaging the membrane lateral heterogeneity in live cells. Moreover, CS is more likely to be located in the plasma membrane due to its negligible water solubility. Our results show that the liquid ordered-like domain covers 31-35% of the cellular surface.     

稀土/支化聚芳酰胺复合高分子的制备与表征

采用合成的Eu-NTA和对二氨基联苯(ODA)为单体,亚磷酸三苯酯(TPP)和吡啶(Py)为浓缩剂,用溶液聚合的方法制备出具有荧光性能的聚合物Eu-NTA-co-ODA。对所合成聚合物利用红外光谱、溶解性能测试、XRD、紫外光谱与荧光光谱等手段进行了表征。通过红外光谱分析表明,聚合物Eu-NTA-co-ODA成功合成,Eu3+与聚合物体系中羰基发生了作用。通过荧光光谱分析表明Eu-NTA-co-ODA与Eu-NTA的荧光谱带的位置和形状基本相同,都反映了Eu3+的特征发射,位于592nm处的荧光发射为Eu3+的5D0→7F1磁偶极跃迁峰,出现在618nm处的跃迁为Eu3+的5D0→7F2电偶极跃迁峰,强度高于磁偶极跃迁峰,表现出红色荧光。     

Experimental methods in chemical engineering: Fluorescence emission spectroscopy

Fluorescence is a luminescence phenomenon in which a compound emits light after absorption of electromagnetic irradiation. Specialized terms such as photoluminescence, cathodoluminescence, anodoluminescence, radioluminescence, and x‐ray fluorescence sometimes are used to indicate the type of exciting radiation. Fluorescence spectroscopy provides reliable quantitative and qualitative data. It precisely tracks chemical reactions from fluorescent materials compounds with aromatic groups, or conjugated planar, or cyclic molecules. It is up to 1000 times more sensitive than UV‐vis or infrared spectroscopy. Fluorescence intensity depends on the fluorophore (compound that fluoresces), its concentration, excitation and emission wavelengths, temperature and contamination. We adjust the slit dimensions, photomultiplier tube voltage and bandpass filter cutoff to maximize the signal while avoiding saturating the detector. Together with x‐ray diffraction, it is the most common spectroscopic technique with applications in geology, chemistry, medicine, and astronomy. A bibliometric analysis of the top 10 000 cited papers identified 5 clusters based on keywords centered around: (1) cancer, cells, and proteins; (2) aggregation induced emission, LED, and complexes; (3) live cells, sensors, and probes; (4) quantum dots, DNA, and biosensors; and (5) nanoparticles, in vivo, and drug delivery. Chemical engineers have yet to fully embrace fluorescence spectroscopy as the category is ranked 16th among all scientific categories that exploit it.     

咪唑并吩嗪基苯硼酸衍生物的合成与荧光特性研究

合成了2种含有1 H-咪唑[4,5-b]并吩嗪荧光团的苯硼酸探针分子。它们均有大的斯托克斯位移(分别为136nm和141nm)和荧光发射波长(>540nm)。其中化合物3b:(2-(1H-咪唑[4,5-b]并吩嗪-2-基)苯硼酸)的荧光强度小于3a:(4-(1H-咪唑[4,5-b]并吩嗪-2-基)苯硼酸),这是由于分子内的B-N间的相互作用影响了分子内电荷发生转移。研究表明设计合成的咪唑并吩嗪基苯硼酸类荧光糖探针可选择性识别不同结构的糖。     

Fluorogenic polydiacetylene supramolecules: immobilization, micropatterning, and application to label-free chemosensors

This Account describes a new strategy for the preparation of label-free sensor systems based on the fluorogenic properties of the conjugated polymer, polydiacetylene (PDA). PDA has been extensively investigated as a sensor matrix, owing to a brilliant blue-to-red color transition that takes place in response to environmental perturbations. It has been known for some time that "blue-phase" PDAs are nonfluorescent while their "red-phase" counterparts fluoresce. For the most part, however, the significance of the different fluorogenic properties of PDAs has been ignored in the context of sensor applications. In the course of developing PDA-based sensors, we discovered that PDA vesicles can be readily immobilized on solid substrates. This is an attractive property of PDAs since it leads to the combined advantages of the vesicle sensors (which have three-dimensional interactions between sensor and target molecules) and film sensors (which are applicable to a two-dimensional array or chip format). Stable blue-phase immobilized PDAs can be prepared by employing one of three strategies involving the formation of covalent adducts, biotin-avidin complexes, or complexes formed through nonspecific physical adsorption. A procedure for generating well-patterned fluorescence images is necessary for the immobilized PDAs to function in chip-based sensor systems. Patterned fluorescence images are readily constructed by employing (1) the photolithographic technique, (2) the micromolding in capillaries (MIMIC) method, or (3) an array spotting system. Heat treatment of the patterned "blue-phase" PDA vesicles transforms the nonfluorescent images into their fluorescent red forms. The observation that finely resolved fluorescence patterns can be generated by heat treatment of microarrayed PDAs is highly significant in that it indicates that fluorescence signals might be produced by specific molecular recognition events. Indeed, red fluorescence emission is observed when immobilized PDAs are subjected to specific molecular recognition events, such as ligand--cyclodextrin or protein-protein interactions. The facile immobilization of PDA vesicles on solid substrates and the affinity-induced fluorescence emission combine to make this system applicable to the fabrication of label-free PDA sensors. Since in theory any molecular recognition event that promotes the blue-to-red color transition of PDAs should result in the generation of fluorescence, it should be possible to reformat a variety of previously described colorimetric PDA sensors into fluorescence-based sensor systems. The fluorescence properties of PDAs, when combined with modern methods for the fabrication of microarrays, should stimulate the development of a number of new label-free chemosensor systems.     

Synthesis of random copolymers of styrene and arylamino substituted styrenes and their sensitivities towards nitroaromatics

Random copolymers of styrene and substituted styrenes bearing arylamino substituents as fluorophore units have been obtained. Their photophysical properties have been investigated by measuring absorption and emission spectra as in solutions as solid-state. All copolymers proved to possess absolute quantum yields up to 0.39 in solution and up to 0.05 in solid-state, depending on their fluorophore substituents. Fluorescence studies have shown that these copolymers show a highly sensitive response towards a diversity of nitroaromatic compounds, both in solutions and in a vapor phase. The detection limits for these compounds towards model nitroaromatic explosives in dichloromethane solution proved to be in the range from 10⁻⁶ to 10⁻⁷ mol/L. The fluorescent materials prepared by electrospinning of synthesized copolymers have been evaluated as sensor materials for detecting nitrobenzene vapor for our hand-made sniffer with detection limits of 0.5 ppm during 100-s exposure to the vapor.     

苯丙烯酸邻菲罗啉铕钇配合物的合成及荧光特性

在乙醇介质中，合成了标题化合物，测定了其组成，红外光谱及荧光光谱，结果表明，羧酸的羟基氧及邻菲罗啉中的氮原子均与稀土离子配位，铕钇配合物的荧光强度值高于铕配合物。     

Fluorescent probes for rapid screening of potential drug-drug interactions at the CYP3A4 level

Steroid derivatives bearing fluorescent groups such as anthracene, dansyl, deazaflavin, and pyrene attached to C6 were synthesized. These compounds are unique inhibitors of cytochrome P450 3A4 (CYP3A4) and display similar IC(50) values in the microM range for the CYP3A4 substrates midazolam, testosterone, and nifedipine. On binding to CYP3A4, the fluorescence of the dansyl, deazaflavin, and pyrene probes is quenched by photophysical interaction of the fluorophore with the heme. The addition of drug candidates with binding constants in the nM-microM range causes displacement of the probes from the active site, and hence leads to restoration of fluorescence. Accordingly, relative affinities of drug candidates to CYP3A4 can be easily and accurately determined by fluorescence measurements.     

Syntheses and electrochromic and fluorescence properties of three double dithienylpyrroles derivatives

Three double dithienylpyrroles derivatives have been successfully prepared by performing a Knorr–Paal condensation between 1,4-di(thiophen-2-yl) butane-1,4-dione and various aromatic diamines. Additionally, their corresponding polymer films were synthesized via electropolymerization. Their electrochemical, spectroelectrochemical and electrochromic behaviors were further investigated by thermogravimetric analysis, scanning electron microscopy, cyclic voltammetry, UV–vis absorption and fluorescence emission spectra. Scanning electron microscopy and thermogravimetric analysis demonstrated that the polymer films possessed homogeneous, compact and smooth layer structures and thermal stabilities (up to nearly 180 °C). Cyclic voltammograms and UV–vis absorption spectra studies showed that the polymer films have stable, well-defined, reversible redox processes, low optical band gaps (E_g < 2.2 eV) and multicolor electrochromic behaviors. Additionally, the fluorescence spectra study showed that all of the monomers and polymers exhibited different intensity emission bands at different wavelengths.     

Plasmon-induced modulation of the emission spectra of the fluorescent molecules near gold nanorods

Both the excitation and emission processes of a fluorescent molecule positioned near a noble metal nanocrystal can interact strongly with the localized surface plasmon resonance of the metal nanocrystal. While the effects of this plasmon-fluorophore interaction on the intensity, polarization, and direction of the fluorescence emission have been intensively investigated, the plasmonic effect on the emission spectrum has barely been explored. We show, on the single-particle level, that the localized surface plasmon resonance of Au nanorods can strongly alter the spectral profile of the emission from adjacent fluorescent molecules. The fluorescent molecules are embedded in a mesostructured silica shell that is uniformly coated on each Au nanorod. The longitudinal plasmon resonance wavelengths of the nanorods are deliberately shifted away from the intrinsic fluorescence emission peak wavelength by synthetically tuning the nanorod aspect ratio. The resultant emission spectra of the fluorescent molecules are found to be remarkably modulated. Besides the intrinsic fluorescence peak, a plasmon-induced new peak emerges at the plasmon resonance wavelength. The intensity of this plasmon-induced fluorescence peak increases as the size of the Au nanorod is increased. This spectral modulation can be understood by depicting the decay process of the fluorophore with multiple vibrational energy levels. The plasmon with a specific resonance energy will enhance the transition rate between the energy levels that have the transition energy approximately equal to the plasmon energy. This plasmon-enhanced transition rate results in a modulated spectral profile of the fluorescence emission.