亲水性二芳基乙烯荧光分子开关的研究进展

二芳基乙烯荧光分子开关因具有优良的抗疲劳性和双稳态特征而被广泛地研究与应用,亲水化成为其作为荧光开关探针走向应用的关键点之一。本文综述了亲水性二芳基乙烯荧光分子开关当前的研究进展,归纳了实现亲水性的几种重要途径和结构,分析了各种亲水化方法的优缺点,并着重介绍了亲水性二芳基乙烯荧光分子开关作为荧光开关探针在化学传感、生物传感、生物成像以及超分辨成像等领域的应用现状,并指出当前应用研究中存在的一些问题,同时也对其未来的应用前景进行了展望。     

Live Cell Imaging Using Photoswitchable Diarylethene‐Doped Fluorescent Polymer Dots

Fluorescence photoswitching using nanomaterials has recently emerged as a promising approach for the imaging of biological targets. However, despite intensive research efforts during the last decade, practical microscopy of biological targets using photoswitchable nanoparticles in real time remains challenging. To address this problem, we have developed live macrophage cell imaging and single particle imaging methods, using photoswitchable fluorescent diarylethene‐doped polymer nanoparticles (P‐dots) under Xe lamp irradiation. We established a 34‐times prolonged “off‐state”, using P‐dots doped with a diarylethene‐containing methoxy substituent, upon visible‐light irradiation using a Xe lamp and a green fluorescent protein filter cube. To demonstrate the practicality of doped P‐dots imaging, we imaged lysosomes in macrophage cells, and observed 11‐times slower recovery of the fluorescence from the “off‐state” to the “on‐state”, indicating their potential for cellular imaging.     

Super-resolution fluorescence nanoscopy applied to imaging core-shell photoswitching nanoparticles and their self-assemblies

Using photo-actuated unimolecular logical switching attained reconstruction (PULSAR) nanoscopy, the structures of photoswitchable polymeric nanoparticles self-assembled on the surfaces of CaCl(2) crystals at the nanoscale were revealed; the photoswitching events and the locations of the photoswitchable fluorescent dyes inside the hydrophobic cores of the core-shell type polymeric nanoparticles were determined.     

Photoswitchable nanoparticles enable high-resolution cell imaging: PULSAR microscopy

Beyond-diffraction-limit optical imaging of cells will reveal biological mechanisms, cellular structures, and physiological processes in nanometer scale. Harnessing the photoswitching properties of spiropyran fluorophores, we achieved nanoresolution fluorescence imaging using photoactuated unimolecular logical switching attained reconstruction (PULSAR) microscopy. The PULSAR microscope successfully resolved nanostructures and subcellular organelles when the photoswitchable nanoparticles containing spiropyran dyes were used as fluorescent probes.     

A visible light responsive azobenzene‐functionalized polymer: Synthesis,self‐assembly,and photoresponsive properties

A novel visible light responsive random copolymer consisting of hydrophobic azobenzene‐containing acrylate units and hydrophilic acrylic acid units has been prepared. The azobenzene molecule bearing methoxy groups at all four ortho positions is readily synthesized by one‐step conversion of diazotization. The as‐prepared polymer can self‐assemble into nanoparticles in water due to its amphiphilic nature. The tetra‐o‐methoxy‐substituted azobenzene‐functionalized polymer can exhibit the trans‐to‐cis photoswitching under the irradiation with green light of 520 nm and the cis‐to‐trans photoswitching under the irradiation with blue light of 420 nm in both solution and aggregate state. The morphologies of the self‐assembled nanoparticles are revealed by TEM and DLS. The controlled release of loaded molecules from the nanoparticles can be realized by adjusting pH value since the copolymer possesses pH responsive acrylic acid groups. The fluorescence of loaded Nile Red in the nanoparticles can be tuned upon the visible light irradiation. The reversible photoswitching of the azobenzene‐functionalized polymer under visible light may endow the polymer with wide applications without using ultraviolet light at all. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 2768–2775     

Giant Amplification of Photoswitching by a Few Photons in Fluorescent Photochromic Organic Nanoparticles

Controlling or switching the optical signal from a large collection of molecules with the minimum of photons represents an extremely attractive concept. Promising fundamental and practical applications may be derived from such a photon‐saving principle. With this aim in mind, we have prepared fluorescent photochromic organic nanoparticles (NPs), showing bright red emission, complete ON–OFF contrast with full reversibility, and excellent fatigue resistance. Most interestingly, upon successive UV and visible light irradiation, the NPs exhibit a complete fluorescence quenching and recovery at very low photochromic conversion levels (<5 %), leading to the fluorescence photoswitching of 420±20 molecules for only one converted photochromic molecule. This “giant amplification of fluorescence photoswitching” originates from efficient intermolecular energy‐transfer processes within the NPs.     

A FRET-Facilitated Photoswitching Using an Orange Fluorescent Protein with the Fast Photoconversion Kinetics

Fluorescent proteins photoswitchable with noncytotoxic light irradiation and spectrally distinct from multiple available photoconvertible green-to-red probes are in high demand. We have developed a monomeric fluorescent protein, called PSmOrange2, which is photoswitchable with blue light from an orange (ex./em. at 546 nm/561 nm) to a far-red (ex./em. at 619 nm/651 nm) form. Compared to another orange-to-far-red photoconvertable variant, PSmOrange2 has blue-shifted photoswitching action spectrum, 9-fold higher photoconversion contrast, and up to 10-fold faster photoswitching kinetics. This results in the 4-fold more PSmOrange2 molecules being photoconverted in mammalian cells. Compared to common orange fluorescent proteins, such as mOrange, the orange form of PSmOrange has substantially higher photostability allowing its use in multicolor imaging applications to track dynamics of multiple populations of intracellular objects. The PSmOrange2 photochemical properties allow its efficient photoswitching with common two-photon lasers and, moreover, via F?rster resonance energy transfer (FRET) from green fluorescent donors. We have termed the latter effect a FRET-facilitated photoswitching and demonstrated it using several sets of interacting proteins. The enhanced photoswitching properties of PSmOrange2 make it a superior photoconvertable protein tag for flow cytometry, conventional microscopy, and two-photon imaging of live cells.     

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.     

Carboxylated Photoswitchable Diarylethenes for Biolabeling and Super‐Resolution RESOLFT Microscopy

Reversibly photoswitchable 1,2‐bis(2‐ethyl‐6‐phenyl‐1‐benzothiophene‐1,1‐dioxide‐3‐yl)perfluorocyclopentenes (EBT) having fluorescent “closed” forms were decorated with four or eight carboxylic groups and attached to antibodies. Low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and good photophysical properties were demonstrated. Alternating light pulses of UV and blue light induce numerous reversible photochemical transformations between two stables states with distinct structures. Using relatively low light intensities, EBTs were applied in biology‐related super‐resolution microscopy based on the reversible saturable (switchable) optical linear fluorescence transitions (RESOLFT) and demonstrated optical resolution of 75 nm.     

Ultrafast Photoswitching in a Copper‐Nitroxide‐Based Molecular Magnet

Molecular compounds with photoswitchable magnetic properties have been intensively investigated over the last decades due to their prospective applications in nanoelectronics, sensing and magnetic data storage. The family of copper‐nitroxide‐based molecular magnets represents a new promising type of photoswitchable compounds. We report the first study of these appealing systems using femtosecond optical spectroscopy. We unveil the mechanism of ultrafast (<50 fs) spin state photoswitching and establish its principal differences compared to other photoswitchable magnets. On this basis, we propose potential advantages of copper‐nitroxide‐based molecular magnets for the future design of ultrafast magnetic materials.     

Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: numerical analysis and application to the fluorescent protein Dronpa

The fast and reversible on/off switching of the fluorescence emission of the GFP-like fluorescent protein Dronpa has attracted considerable interest for applications in subdiffraction imaging. In this paper we study the use of a donut-mode beam in combination with two more overlapping laser beams to increase the imaging resolution through selective switching to the nonfluorescent photoswitched state. We devise and run a series of numerical simulations to determine suitable photophysical parameters of prospective, thermally stable photoswitchable molecules, in terms of photoswitching quantum yields, fatigue resistance, and possible presence of transient nonfluorescent states. Many of our findings are applicable to other measurements that make use of donut beams, and these guidelines can be used in the synthesis and screening of novel photoswitchable compounds. We experimentally demonstrate the possibility of obtaining increased resolution by making use of the efficient and thermally stable Dronpa photoswitching, using equipment that is commonly available.     

Preparation and Characterization of Uniform Near IR Polystyrene Nanoparticles

Biomaterials for in vivo fluorescence imaging are required to be biocompatible, nontoxic, photostable and highly fluorescent. Fluorescence must be in the near infrared (NIR) region of the electromagnetic spectrum to avoid absorption and autofluorescence of endogenous tissues. NIR fluorescent polystyrene nanoparticles may be considered ideal biomaterials for in vivo imaging applications. These NIR nanoparticles were prepared by a swelling process of polystyrene template nanoparticles with a hydrophobic NIR dye dissolved in a water‐miscible swelling solvent, a method developed for preparation of nonbiodegradable nanoparticles, for NIR fluorescent bioimaging applications. This method overcomes common problems that occur with dye entrapment during nanoparticle formation such as loss of fluorescence and size polydispersity. Fluorescence intensity of the nanoparticles was found to be size dependent, and was optimized for differently sized nanoparticles. The resulting NIR nanoparticles were also found to be more fluorescent and highly photostable compared to the free dye in solution, showing their potential as biomaterials for in vivo fluorescence imaging.     

Photoswitchable Adsorption in Metal–Organic Frameworks Based on Polar Guest–Host Interactions

Reversible remote‐controlled switching of the properties of nanoporous metal–organic frameworks (MOFs) is enabled by incorporating photoswitchable azobenzene. The interaction of the host material with different guest molecules, which is crucial for all applications, is precisely studied using thin MOF films of the type Cu₂(BDC)₂(AzoBipyB). A molecule‐specific effect of the photoswitching, based on dipole–dipole interactions, is found.     

Photoswitchable Fluorescent Self-Assembled Metallacycles with High Photostability

In this study, photoswitchable fluorescent supramolecular metallacycles with high fatigue-resistance have been constructed by coordination-driven self-assembly by using bithienylethene with dipyridyl units ( BTE ) as a coordination donor and a fluorescent di-platinum(II) ( Pt-F ) as a coordination acceptor. The photo-triggered reversible transformation between the ring-open and ring-closed form of the metallacycles was confirmed by ¹H NMR, ³¹P NMR, and UV/Vis spectroscopy. This unique property enabled a reversible noninvasive “off–on” switching of fluorescence through efficient Förster resonance energy transfer (FRET). Importantly, the metallacycles remained structurally intact after up to 10 photoswitching cycles. The photoresponsive property and exceptional photostability of the metallacycles posit their potential promising application in optical switching, image storage, and super-resolution microscopy.     

Photoswitchable and Water‐Soluble Fluorescent Nano‐Aggregates Based on a Diarylethene–Dansyl Dyad and Liposome

In this work, a unique approach is developed to generate photoswitchable and water‐soluble fluorescent nano‐aggregates. Initially, a new light‐controlled diarylethene–dansyl dyad DAE 1 is formed by linking two dansyl fluorophores covalently to a symmetrical dithienylethene backbone, whose photophysical properties can be reversibly switched by optical stimuli. Subsequently, the water insolubility of the molecular switch 1 is overcome by incorporating it into the bilayer of liposome DPPC (1,2‐dihexadecanoyl‐sn ‐glycero‐3‐phosphocholine) in water. This strategy creates stable fluorescent nano‐aggregates OF‐1@DPPC (≈25 nm diameter) that are soluble in an aqueous medium. The nano‐aggregates OF‐1@DPPC retain and even improve the photoswitchable fluorescence properties of DAE 1 . More importantly, OF‐1@DPPC exhibits a remarkable photostability and fatigue resistance after 5 cycles of irradiation with UV and visible light, which is crucial for its practical application.     

Synthesis and characterization of photoswitchable fluorescent SiO2 nanoparticles

Switchable fluorescent silica nanoparticles have been prepared by covalently incorporating a fluorophore and a photochromic compound inside the particle core. The fluorescence can be switched reversibly between an on‐ and off‐state via energy transfer. The particles were synthesized using different amounts of the photoswitchable compound (spiropyran) and the fluorophore (rhodamine B) in a size distribution between 98 and 140 nm and were characterized in terms of size, switching properties, and fluorescence efficiency by TEM, and UV\Vis and fluorescence spectroscopy.     

A Photoswitchable Agonist for the Histamine H3 Receptor,a Prototypic Family A G‐Protein‐Coupled Receptor

Spatiotemporal control over biochemical signaling processes involving G protein‐coupled receptors (GPCRs) is highly desired for dissecting their complex intracellular signaling. We developed sixteen photoswitchable ligands for the human histamine H₃ receptor (hH₃R). Upon illumination, key compound 65 decreases its affinity for the hH₃R by 8.5‐fold and its potency in hH₃R‐mediated G_i protein activation by over 20‐fold, with the trans and cis isomer both acting as full agonist. In real‐time two‐electrode voltage clamp experiments in Xenopus oocytes, 65 shows rapid light‐induced modulation of hH₃R activity. Ligand 65 shows good binding selectivity amongst the histamine receptor subfamily and has good photolytic stability. In all, 65 (VUF15000) is the first photoswitchable GPCR agonist confirmed to be modulated through its affinity and potency upon photoswitching while maintaining its intrinsic activity, rendering it a new chemical biology tool for spatiotemporal control of GPCR activation.     

A Photoswitchable Agonist for the Histamine H3 Receptor,a Prototypic Family A G‐Protein‐Coupled Receptor

Spatiotemporal control over biochemical signaling processes involving G protein‐coupled receptors (GPCRs) is highly desired for dissecting their complex intracellular signaling. We developed sixteen photoswitchable ligands for the human histamine H₃ receptor (hH₃R). Upon illumination, key compound 65 decreases its affinity for the hH₃R by 8.5‐fold and its potency in hH₃R‐mediated G_i protein activation by over 20‐fold, with the trans and cis isomer both acting as full agonist. In real‐time two‐electrode voltage clamp experiments in Xenopus oocytes, 65 shows rapid light‐induced modulation of hH₃R activity. Ligand 65 shows good binding selectivity amongst the histamine receptor subfamily and has good photolytic stability. In all, 65 (VUF15000) is the first photoswitchable GPCR agonist confirmed to be modulated through its affinity and potency upon photoswitching while maintaining its intrinsic activity, rendering it a new chemical biology tool for spatiotemporal control of GPCR activation.     

Synthesis of Redshifted Azobenzene Photoswitches by Late‐Stage Functionalization

Azobenzenes are versatile photoswitches that can be cycled between their trans‐ and cis‐configuration with light. The wavelengths required for this isomerization are substantially shifted from the UV to the visible range through tetra‐ortho‐chlorination. These halogenated azobenzenes display unique photoswitching characteristics, but their syntheses remain limited and inefficient. A new general method for the synthesis of tetra‐ortho‐chloro azobenzenes has been developed, which relies on direct palladium(II)‐catalyzed C?H activation of pre‐existing standard azobenzenes. This late‐stage functionalization has a broad substrate scope and can be used to create a variety of useful building blocks for the construction of more elaborate redshifted photopharmaceuticals. This method is used to prepare red‐ AzCA‐4 , a photoswitchable vanilloid that enables optical control of the cation channel TRPV1 with visible light.     

Unexpected Acid-Triggered Formation of Reversibly Photoswitchable Stenhouse Salts from Donor-Acceptor Stenhouse Adducts

Donor-acceptor Stenhouse adducts (DASAs) are reversibly photoswitchable dyes, which are able to interconvert between a red/NIR absorbing triene-like state and a colorless cyclic state. Although optically attractive for multiple applications, their low solubility and lack of photoswitching in water impede their use in aqueous environments. We developed water-soluble DASAs based on indoline as donor and methyl, or trifluoromethyl, pyrazolone-based acceptors. In acetonitrile, photophysical analysis and photochemical studies, accounted with a three-state kinetic model, confirmed the reversible photoswitching mechanism previously proposed. In water, the colorless cyclic state is a thermodynamic sink at neutral pH values. In contrast, in acidic conditions, we observed a fast scrambling of DASAs’ end-group resulting in the in situ formation of Stenhouse salts (StS), which are in turn capable of reversible photoswitching. We believe that this unexpected result is of interest not only for the future design of DASAs with improved stability, but also for further development and applications of StS as photoswitchable probes.