Fabrication of Novel Polymer Nanoparticle-Based Fluorescence Resonance Energy Transfer Systems and their Tunable Fluorescence Properties

In this study, two hydrophobic fluorescent dyes, nitrobenzoxadiazolyl (NBD) and 9-(diethylamino)benzo[a]phenoxazin-5-one (NR) with different doping ratios were incorporated into polymer nanoparticles to constitute novel polymer nanoparticle-based fluorescence resonance energy transfer (FRET) systems via a facile one-step mini-emulsion polymerization. Spectroscopic characteristics demonstrate that the two fluorophores have been successfully embedded into the nanoparticles, and the fluorescence emission intensity of the two hydrophobic dyes can be greatly enhanced in aqueous media. The as-prepared fluorescent nanoparticles also display a uniform small size (ca. 55 nm), high dye load, intense fluorescence, as well as controllable amount and ratio of the two dyes. The observed FRET efficiencies (16.0–75.2%), as well as the distance (r) between NBD (donor) and NR (acceptor), is closely correlated to the doping ratio of two dyes. Moreover, by varying the doping ratio of two dyes, the fluorescent nanoparticles would exhibit multicolor through FRET upon a single wavelength excitation, and the fluorescence emission signals of the dye-doped nanoparticles could be accurately tuned. These results indicate that the as-prepared uniform FRET-mediated nanoparticles are of high interest in multiplexed bioanalysis.     

A core-shell nanoparticle approach to photoreversible fluorescence modulation of a hydrophobic dye in aqueous media

Amphiphilic core-shell nanoparticles containing spiropyran moieties have been prepared in aqueous media. The nanoparticles consist of hydrophilic and biocompatible poly(ethyleneimine) (PEI) chain segments, which serve as the shell, and a hydrophobic copolymer of methyl methacrylate (MMA), a spiropyran-linked methacrylate, and a cross-linker, which forms the core of the nanoparticles. A hydrophobic fluorescent dye based on the nitrobenzoxadiazolyl (NBD) group was introduced into the nanoparticles to form NBD-nanoparticle complexes in water. The nanoparticles not only greatly enhance the fluorescence emission of the hydrophobic dye NBD in aqueous media, probably by accommodating the dye molecules in the interface between the hydrophilic shells and the hydrophobic cores, but also modulate the fluorescence of the dye through intraparticle energy transfer. This biocompatible and photoresponsive nanoparticle complex may find applications in biological areas such as biological diagnosis, imaging, and detection. In addition, this nanoparticle approach will open up possibilities for the fluorescence modulation of other hydrophobic fluorophores in aqueous media.     

Fabrication of Novel Reversible Photoswitchable Fluorescent Nanoparticles

Novel photoswitchable fluorescent nanoparticles were fabricated by a facile one-step mini-emulsion polymerization, in which fluorescence resonance energy transfer (FRET) donor, 9,10-diphenylanthracene (DPA), and photoswitchable acceptor, spiropyran derivate, were simultaneously embedded in polymer matrix during the polymerization process. The prepared fluorescent nanoparticles exhibit the typical absorption properties of both DPA dye and spiropyran moiety, indicating that the two chromophores have been incorporated into the polymer nanoparticles. The obtained fluorescent nanoparticles exhibited superior photoswitchable fluorescent performance due to the effective photoinduced interparticle FRET. Moreover, the novel photoswitchable fluorescent nanoparticles also revealed small size (ca. 60 nm), high intensity, relatively fast photoresponsive property and good photoreversibility in aqueous media.     

Fighting Aggregation‐Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion

Dye‐loaded polymer nanoparticles (NPs) emerge as a powerful tool for bioimaging applications, owing to their exceptional brightness and controlled small size. However, aggregation‐caused quenching (ACQ) and leakage of dyes at high loading remain important challenges of these nanomaterials. The use of bulky hydrophobic counterions has been recently proposed as an effective approach to minimize ACQ and dye leakage, but the role of counterion structure is still poorly understood. Here, a systematic study based on ten counterions, ranging from small hydrophilic perchlorate up to large hydrophobic tetraphenylborate derivatives, reveals how counterion nature can control encapsulation and emission of a cationic dye (rhodamine B octadecyl ester) in NPs prepared by nanoprecipitation of a biodegradable polymer, poly‐lactide‐co‐glycolide (PLGA). We found that increase in counterion hydrophobicity enhances dye encapsulation efficiency and prevents dye adsorption at the particle surface. Cellular imaging studies revealed that ≥95 % encapsulation efficiency, achieved with most hydrophobic counterions (fluorinated tetraphenylborates), is absolutely required because non‐encapsulated dye species at the surface of NPs are the origin of dye leakage and strong fluorescence background in cells. The size of counterions is found to be essential to prevent ACQ, where the largest species, serving as effective spacer between dyes, provide the highest fluorescence quantum yield. Moreover, we found that the most hydrophobic counterions favor dye–dye coupling inside NPs, leading to ON/OFF fluorescence switching of single particles. By contrast, less hydrophobic counterions tend to disperse dyes in the polymer matrix favoring stable emission of NPs. The obtained structure‐property relationships validate the counterion‐based approach as a mature concept to fight ACQ and dye leakage in the development of advanced polymeric nanomaterials with controlled optical properties.     

Core‐shell type dually fluorescent polymer nanoparticles for ratiometric pH‐sensing

The synthesis and pH‐sensing properties of fluorescent polymer nanoparticles (NPs) in the 20 nm diameter range with a sensitive dye covalently attached to the particle surface and a reference dye entrapped within the particle core are presented. Fluorescein‐functionalized NPs were readily obtained by conjugation of fluorescein isothiocyanate (FITC) to amine‐coated crosslinked polystyrene‐based nanoparticles prepared by microemulsion polymerization followed by postfunctionalization. This all water‐based method gave access to stable aqueous suspensions of pH‐sensing fluorescent NPs. The encapsulation of the insensitive reference fluorescent dye (1,9‐diphenylanthracene, DPA) was then conveniently achieved by soaking leading to dual fluorescent NPs containing about 20 DPA and 55 fluorescein, as deduced from spectroscopic analyses. This core‐shell type architecture maximizes the interactions of the sensing dye with the medium while protecting the reference dye. The variations of the ratio of the fluorescence emission intensities of the sensitive dye (fluorescein) to the reference dye (DPA) with pH show that the dual fluorescent NPs act as a ratiometric pH sensor with a measuring range between pH 4 and pH 8. This pH nanosensor was found to be fast, fully reversible, and robust without any leaching of dye over a long period of time. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 6206–6213, 2008     

Synthesis and Characterization of Diketopyrrolopyrrole-Based Aggregation-Induced Emission Nanoparticles for Bioimaging

Conventional fluorescent dyes have the property of decreasing fluorescence due to aggregation-caused quenching effects at high concentrations, whereas aggregation-induced emission dyes have the property of increasing fluorescence as they aggregate with each other. In this study, diketopyrrolopyrrole-based long-wavelength aggregation-induced emission dyes were used to prepare biocompatible nanoparticles suitable for bioimaging. Aggregation-induced emission nanoparticles with the best morphology and photoluminescence intensity were obtained through a fast, simple preparation method using an ultrasonicator. The optimally prepared nanoparticles from 3,6-bis(4-((E)-4-(bis(40-(1,2,2-triphenylvinyl)-[1,10-biphenyl]-4-yl)amino)styryl)phenyl)-2,5-dihexyl-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (DP-R2) with two functional groups having aggregation-induced emission properties and additional donating groups at the end of the triphenylamine groups were considered to have the greatest potential as a fluorescent probe for bioimaging. Furthermore, it was found that the tendency for aggregation-induced emission, which was apparent for the dye itself, became much more marked after the dyes were incorporated within nanoparticles. While the photoluminescence intensities of the dyes were observed to decrease rapidly over time, the prepared nanoparticles encapsulated within the biocompatible polymers maintained their initial optical properties very well. Lastly, when the cell viability test was conducted, excellent biocompatibility was demonstrated for each of the prepared nanoparticles.     

Spiropyran-based photochromic polymer nanoparticles with optically switchable luminescence

Polymer nanoparticles of 40-400 nm diameter with spiropyran-merocyanine dyes incorporated into their hydrophobic cavities have been prepared; in contrast to their virtually nonfluorescent character in most environments, the merocyanine forms of the encapsulated dyes are highly fluorescent. Spiro-mero photoisomerization is reversible, allowing the fluorescence to be switched "on" and "off" by alternating UV and visible light. Immobilizing the dye inside hydrophobic pockets of nanoparticles also improves its photostability, rendering it more resistant than the same dyes in solution to fatigue effects arising from photochemical switching. The photophysical characteristics of the encapsulated fluorophores differ dramatically from those of the same species in solution, making nanoparticle-protected hydrophobic fluorophores attractive materials for potential applications such as optical data storage and switching and biological fluorescent labeling. To evaluate the potential for biological tagging, these optically addressable nanoparticles have been delivered into living cells and imaged with a liquid nitrogen-cooled CCD.     

Synthesis and characterization of dually labeled Pickering-type stabilized polymer nanoparticles in a downscaled miniemulsion system

Dual fluorescently labeled polymer particles were prepared in a downscaled Pickering-type miniemulsion system. Stable dispersions were obtained and the size of the hybrid particles could be varied between ca. 180 and 430 nm. Silica nanoparticles were employed as sole emulsifier, which were labeled by a fluorescein dye (FITC) or (encapsulated) quantum dots, and the polymer core was labeled by a perylene derivative. Downscaling of the Pickering-type miniemulsion system is intriguing by itself as it allows the use of precious nanoparticles as emulsifiers. Here, silica particles with a fluorescent core and an overall diameter between 20 and 40 nm were prepared and employed as stabilizer. The dual excitation and emission of both dyes was tested by fluorescence measurements and confocal laser scanning microscopy (cLSM).     

Photo‐ and pH‐Tunable Multicolor Fluorescent Nanoparticle‐Based Spiropyran‐ and BODIPY‐Conjugated Polymer with Graphene Oxide

We report a stimuli‐responsive fluorescent nanomaterial, based on graphene oxide coupled with a polymer conjugated with photochromic spiropyran (SP) dye and hydrophobic boron dipyrromethane (BODIPY) dye, for application in triggered target multicolor bioimaging. Graphene oxide (GO) was reduced by catechol‐conjugated polymers under mildly alkaline conditions, which enabled to formation of functionalized multicolor graphene nanoparticles that can be induced by irradiation with UV light and by changing the pH from acidic to neutral. Investigation of these nanoparticles by using AFM, fluorescence emission, and in vitro cell and in vivo imaging revealed that they show different tunable colors in bioimaging applications and, more specifically, in cancer‐cell detection. The stability, biocompatibility, and quenching efficacy of this nanocomposite open a different perspective for cell imaging in different independent colors, sequentially and simultaneously.     

Supramolecular Translation of Enzymatically Triggered Disassembly of Micelles into Tunable Fluorescent Responses

The need for advanced fluorescent imaging and delivery platforms has motivated the development of smart probes that change their fluorescence in response to external stimuli. Here a new molecular design of fluorescently labeled PEG–dendron hybrids that self‐assemble into enzyme‐responsive micelles with tunable fluorescent responses is reported. In the assembled state, the fluorescence of the dyes is quenched or shifted due to intermolecular interactions. Upon enzymatic cleavage of the hydrophobic end‐groups, the labeled polymeric hybrids become hydrophilic, and the micelles disassemble. This supramolecular change is translated into a spectral response as the dye–dye interactions are eliminated and the intrinsic fluorescence is regained. We demonstrate the utilization of this molecular design to generate both Turn‐On and spectral shift responses by adjusting the type of the labeling dye. This approach enables transformation of non‐responsive labeling dyes into smart fluorescent probes.     

Oligonucleotide-stabilized Ag nanocluster fluorophores

Single-stranded oligonucleotides stabilize highly fluorescent Ag nanoclusters, with emission colors tunable via DNA sequence. We utilized DNA microarrays to optimize these scaffold sequences for creating nearly spectrally pure Ag nanocluster fluorophores that are highly photostable and exhibit great buffer stability. Five different nanocluster emitters have been created with tunable emission from the blue to the near-IR and excellent photophysical properties. Ensemble and single molecule fluorescence studies show that oligonucleotide encapsulated Ag nanoclusters exhibit significantly greater photostability and higher emission rates than commonly used cyanine dyes.     

A fluorescence ratiometric nano-pH sensor based on dual-fluorophore-doped silica nanoparticles

We have synthesized dual-fluorophore-doped core-shell silica nanoparticles used as ratiometric pH sensor. The nanoparticles were prepared with a reverse microemulsion technique by simultaneously encapsulating two different fluorophores, the pH-sensitive dye fluorescein as a pH indicator and the pH-insensitive dye phenosafranine as an internal reference for fluorescence ratiometric measurement, into silica shell. The nanoparticles prevent the fluorescence dyes leaching from the silica matrix when immersed inside water. The hydrophilic silica shells were made by hydrolysing and polymerizing tetraethoxysilane (TEOS) in water-in-oil microemulsion. The fluorescence intensity ratio of the two dyes varied linearly as a function of pH in the range from 4.0 to 8.0. The sensor was also applied to measure pH of real water samples. The results are in good agreements with that using the conventional glass electrode method. The as-prepared fluorescent nanoparticles showed rapid response, excellent stability and high reproducibility as pH sensors.     

Artificial light-harvesting arrays: electronic energy migration and trapping on a sphere and between spheres

A sophisticated model of the natural light-harvesting antenna has been devised by decorating a C(60) hexa-adduct with ten yellow and two blue boron dipyrromethene (Bodipy) dyes in such a way that the dyes retain their individuality and assist solubility of the fullerene. Unusually, the fullerene core is a poor electron acceptor and does not enter into light-induced electron-transfer reactions with the appended dyes, but ineffective electronic energy transfer from the excited-state dye to the C(60) residue competes with fluorescence from the yellow dye. Intraparticle electronic energy transfer from yellow to blue dyes can be followed by steady-state and time-resolved fluorescence spectroscopy and by excitation spectra for isolated C(60) nanoparticles dissolved in dioxane at 293 K and at 77 K. The decorated particles can be loaded into polymer films by spin coating from solution. In the dried film, efficient energy transfer occurs such that photons absorbed by the yellow dye are emitted by the blue dye. Films can also be prepared to contain C(60) nanoparticles loaded with the yellow Bodipy dye but lacking the blue dye and, under these circumstances, electronic energy migration occurs between yellow dyes appended to the same nanoparticle and, at higher loading, to dye molecules on nearby particles. Doping these latter polymer films with the mixed-dye nanoparticle coalesces these multifarious processes in a single system. Thus, long-range energy migration occurs among yellow dyes attached to different particles before trapping at a blue dye. In this respect, the film resembles the natural photosynthetic light-harvesting complexes, albeit at much reduced efficacy. The decorated nanoparticles sensitize amorphous silicon photocells.     

Anti-Her-2 monoclonal antibody conjugated polymer fluorescent nanoparticles probe for ovarian cancer imaging

Fluorescent nanoparticles (FNPs) with unique optical properties may be useful as biosensors in living cancer cell imaging and cancer targeting. A novel kind of polymer fluorescent nanoparticles (PFNPs) was synthesized and its application for ovarian cancer imaging with fluorescence microscopy imaging technology was presented in this study. The PFNPs were synthesized with precipitation polymerization by using methacrylic acid (MAA) as monomer, trimethylolpropane trimethacrylate (Trim) as cross-linker, azobisisobutyronitrile (AIBN) as radical initiator and butyl rhodamine B (BTRB) as fluorescent dye. And the fluorescent dye was embedded into the three-dimensional network of the polymer when the polymer was produced. With this method the PFNPs can be prepared easily. And then the PFNPs were successfully modified with anti-Her-2 monoclonal antibody. The fluorescence probe based on anti-Her-2 monoclonal antibody conjugated PFNPs has been used to detect ovarian cancer cells with fluorescence microscopy imaging technology. The experimental results demonstrate that the anti-Her-2 monoclonal antibody conjugated PFNPs can effectively recognize ovarian cancer cells and exhibit good sensitivity and exceptional photostability, which would provide a novel way for the diagnosis and curative effect observation of ovarian cancer cells.     

Amphiphilic ESIPT benzoxazole derivatives as prospective fluorescent membrane probes

Fluorescent amphiphilic benzoxazole derivatives were synthesized and used to produce photoactive phosphatidylcholine (PC) liposomes by reserve-phase evaporation. The dyes absorbed in the UV region and were fluorescent in the blue-green region (determined by solvent polarity). The alkyl chain length seemed to play a fundamental role in the photophysics of the benzoxazole fluorophore in reverse liposomes, and despite the same ESIPT core and phospholipid building block, each amphiphilic dye had a particular emission profile related to the dye location in the liposome. The fluorescence emission spectra from dye 5 showed that its fluorophore experienced a polar environment, due to the single normal emission, while dyes 6–7 had (in part) a normal emission, and the main fluorescent band ascribed to the ESIPT emission indicated a more hydrophobic environment. Despite the complex fluorescent profiles, the benzoxazole derivatives could be successfully introduced into the reverse liposome structure due to the interaction between the alkyl chain and PC bilayer.     

基于聚合物核壳纳米粒子的Hg~(2+)比率型荧光检测传感器

采用一步聚合的方法,制备了以疏水的聚甲基丙烯酸甲酯(PMMA)为核、亲水性的聚电解质支化聚乙烯亚胺(PEI)为壳的纳米粒子分散液.将供体荧光团4-胺基-7-硝基-N-辛基苯并[1,2,5]噁二唑(NBD)以包埋的方式在聚合过程中直接引入PMMA核内部,而受体荧光团罗丹明衍生物SRHB通过吸附作用进入PEI-PMMA核壳界面,构成了含有两种不同荧光分子且可对Hg2+进行荧光比率检测的传感器.考察了含荧光分子的聚合物粒子光谱学性质,证明两种荧光分子均被引入了聚合物粒子体系.在汞离子的荧光检测试验中,加入Hg2+后,体系中的NBD荧光强度下降,而罗丹明的特征发射峰在579 nm处出现,并随着Hg2+浓度的增加,受体/供体的荧光强度比值呈现增长趋势.研究还发现,聚合物粒子基荧光探针对于Hg2+具有较好的选择性,且最佳使用范围是体系pH值在5～8之间,其检测Hg2+的最低浓度可达到1μmol/L.     

One‐pot synthesis of carbon dots@ZrO2 nanoparticles with tunable solid‐state fluorescence

In recent years, fluorescent carbon dots (CDs) have been developed and showed potential applications in biomedical imaging and light‐emitting diodes (LEDs) for their excellent fluorescent properties. However, it still remains a challenge to incorporate fluorescent CDs into the host matrix in situ to overcome their serious self‐quenching. Herein, a one‐pot hydrothermal method is used to prepare nano‐zirconia with CDs (CDs@ZrO₂) nanoparticles. During the reaction, CDs and nano‐zirconia are generated simultaneously and connected with silane coupling agent. The CDs@ZrO₂ nanoparticles exhibit tunable emission wavelength from 450 to 535 nm emission by regulating the content of citric acid in the feed. The quantum yield of the CDs@ZrO₂ is up to 23.8%. Furthermore, the CDs@ZrO₂ nanoparticles with regulable fluorescence emission can be used for the fluorescent material to prepare white LEDs. The prepared LED has significant white light emission with color coordinates of (0.30, 0.37) and its color rendering index (CRI) is 67.1. In summary, we have developed the solid‐state CDs@ZrO₂ nanoparticles with tunable emission by a valuable strategy, that is, one‐pot method, for white LEDs.     

嵌合异硫氰酸罗丹明B核壳荧光纳米颗粒制备的新方法研究

随着纳米技术的发展,结合了纳米技术与材料制备技术而发展起来的荧光染料嵌合的核壳荧光纳米颗粒的制备为生物医学领域的研究提供了新的材料、技术和方法。何晓晓等以联钉吡啶配合物为核材料,制备了嵌合无机金属配合物的核壳荧光纳米颗粒,段菁华等用异硫氰酸荧光素FITC与蛋白质IgG相结合,     

Efficient Excitation‐Energy Transfer in Ion‐Based Organic Nanoparticles with Versatile Tunability of the Fluorescence Colours

Organic nanoparticles consisting of 3,3′‐diethylthiacyanine (TC) and ethidium (ETD) dyes are synthesized by ion‐association between the cationic dye mixture (10 % ETD doping) and the tetrakis(4‐fluorophenyl)borate (TFPB) anion, in the presence of a neutral stabilizing polymer, in aqueous solution. Doping with ETD makes the particle size smaller than without doping. Size tuning can also be conducted by varying the molar ratio (ρ) of the loaded anion to the cationic dyes. The fluorescence spectrum of TC shows good overlap with the absorption of ETD in the 450–600 nm wavelength region, so efficient excitation‐energy transfer from TC (donor) to ETD (acceptor) is observed, yielding organic nanoparticles whose fluorescence colours are tunable. Upon ETD doping, the emission colour changes significantly from greenish‐blue to reddish or whitish. This change is mainly dependent on ρ. For the doped nanoparticle sample with ρ=1, the intensity of fluorescence ascribed to ETD is ～150‐fold higher than that from pure ETD nanoparticles (efficient antenna effect). Non‐radiative Förster resonance‐energy transfer (FRET) is the dominant mechanism for the ETD fluorescence enhancement. The organic nanoparticles of a binary dye system fabricated by the ion‐association method act as efficient light‐harvesting antennae, which are capable of transferring light energy to the dopant acceptors in very close proximity to the donors, and can have multi‐wavelength emission colours with high fluorescence quantum yields.     

Bulky Barbiturates as Non-Toxic Ionic Dye Insulators for Enhanced Emission in Polymeric Nanoparticles

Bulky hydrophobic counterions (weakly coordinating anions) can insulate ionic dyes against aggregation-caused quenching (ACQ) and enable preparation of highly fluorescent dye-loaded nanoparticles (NPs) for bioimaging, biosensing and light harvesting. Here, we introduce a family of hydrophobic anions based on fluorinated C-acyl barbiturates with delocalized negative charge and bulky non-polar groups. Similarly to fluorinated tetraphenylborates, these barbiturates prevent ACQ of cationic dye alkyl rhodamine B inside polymer NPs made of biodegradable poly(lactic-co-glycolic acid) (PLGA). Their efficiency to prevent ACQ increases for analogues with higher acidity and bulkiness. Their structure controls dye-dye communication, yielding bright NPs with on/off switching or stable emission. They enhance dye encapsulation inside NPs, allowing intracellular imaging without dye leakage. Compared to fluorinated tetraphenylborates known as cytotoxic transmembrane ion transporters, the barbiturates display a significantly lower cytotoxicity. These chemically available and versatile barbiturate derivatives are promising counterion scaffolds for preparation of bright non-toxic fluorescent nanomaterials.