Molecular engineering of fluorescent dyes for long-term specific visualization of the plasma membrane based on alkyl-chain-regulated cell permeability.

Long-term visualization of changes in plasma membrane dynamics during important physiological processes can provide intuitive and reliable information in a 4D mode. However, molecular tools that can visualize plasma membranes over extended periods are lacking due to the absence of effective design rules that can specifically track plasma membrane fluorescent dye molecules over time. Using plant plasma membranes as a model, we systematically investigated the effects of different alkyl chain lengths of FMR dye molecules on their performance in imaging plasma membranes. Our findings indicate that alkyl chain length can effectively regulate the permeability of dye molecules across plasma membranes. The study confirms that introducing medium-length alkyl chains improves the ability of dye molecules to target and anchor to plasma membranes, allowing for long-term imaging of plasma membranes. This provides useful design rules for creating dye molecules that enable long-term visualization of plasma membranes. Using the amphiphilic amino-styryl-pyridine fluorescent skeleton, we discovered that the inclusion of short alkyl chains facilitated rapid crossing of the plasma membrane by the dye molecules, resulting in staining of the cell nucleus and indicating improved cell permeability. Conversely, the inclusion of long alkyl chains hindered the crossing of the cell wall by the dye molecules, preventing staining of the cell membrane and demonstrating membrane impermeability to plant cells. The FMR dyes with medium-length alkyl chains rapidly crossed the cell wall, uniformly stained the cell membrane, and anchored to it for a long period without being transmembrane. This allowed for visualization and tracking of the morphological dynamics of the cell plasma membrane during water loss in a 4D mode. This suggests that the introduction of medium-length alkyl chains into amphiphilic fluorescent dyes can transform them from membrane-permeable fluorescent dyes to membrane-staining fluorescent dyes suitable for long-term imaging of the plasma membrane. In addition, we have successfully converted a membrane-impermeable fluorescent dye molecule into a membrane-staining fluorescent dye by introducing medium-length alkyl chains into the molecule. This molecular engineering of dye molecules with alkyl chains to regulate cell permeability provides a simple and effective design rule for long-term visualization of the plasma membrane, and a convenient and feasible means of chemical modification for efficient transmembrane transport of small molecule drugs.

Target-activated multicolor fluorescent dyes for 3D imaging of plasma membranes and tracking of apoptosis.

Real-time tracking of dynamic changes in the three-dimensional morphology of the cell plasma membrane is of great importance for a deeper understanding of physiological processes related to the cell plasma membrane. However, there is a lack of imaging dyes that can specifically be used for a long term labelling of plasma membranes, especially for plant cells. Here, we have used molecular engineering strategies to develop a series of target-activated multicolour fluorescent dyes that can be used for long-term and three-dimensional imaging of plant cell plasma membranes. By combining different electron acceptors and donors, four molecular backbones with different emission colours from green to NIR have been obtained. In the designed styrene-based dyes, referred to as the SD dyes, several functional groups were introduced into the backbones to achieve the properties of target-activated fluorescence, rapid and wash-free staining, high plasma membrane targeting ability and long-term imaging function. Using onion epidermal cells as a platform, these dye molecules can provide high-quality imaging of the plasma membrane for up to 6 hours, providing a powerful tool for long-term monitoring of plasma membrane-related biological events. Calcium-mediated apoptosis of plant cells has been tracked for the first time by monitoring the morphological changes of the plasma membrane in real time using SD dyes. These dyes also exhibit excellent 3D imaging performance of the plasma membrane and were further used to track in real time the 3D morphological changes of the plasma membrane during plasmolysis of plant cells, providing a powerful imaging tool for three-dimensional (3D) biology. This work provides a set of multi-colour dye tools for long-term and three-dimensional imaging of plant cell plasma membranes, and also provides molecular design principles for guiding the transmembrane transport of small molecules.

Photoinduced fluorescence modulation through controllable intramolecular [2+2] photocycloaddition in single molecules and molecular aggregates.

We report a general molecular design strategy of spatial proximity, which allows intramolecular [2+2] photocycloaddition reaction to take place in both single molecules and molecular aggregates. Sharply contrasting photoinduced fluorescence changes in solution and in the solid state were found and attributed to the aggregation-induced quenching property of the monomers and the aggregation-induced emission nature of the photodimers.

Photoactivation of Solid-State Fluorescence through Controllable Intermolecular [2+2] Photodimerization.

Intermolecular [2+2] photodimerization provides a distinctive approach to construct photoresponsive fluorescent materials in a manner of switching on solid-state fluorescence. Herein, we report efficient photoactivation of bright solid-state fluorescence based on controllable intermolecular [2+2] photodimerization reaction of benzo[b]thiophene 1,1-dioxide (BTO) derivatives, which provides a simple and effective way to construct smart photoresponsive solid-state fluorescent materials. Rational choice of substituents in BTO molecular skeleton enables them to efficiently undergo photodimerization through regulating molecular stacking in crystal, and also leads to photoactivation of solid-state fluorescence due to the generation of brightly fluorescent photodimers. This intermolecular photodimerization reaction also offers an effective method to synthesize photostable AIEgens with purely through-space conjugation.

Facial Preparation of Cyclometalated Iridium (III) Nanowires as Highly Efficient Electrochemiluminescence Luminophores for Biosensing.

In this study, highly efficient ECL luminophores composed of iridium complex-based nanowires (Ir-NCDs) were synthesized via covalently linking bis(2-phenylpyridine)-(4-carboxypropyl-2,2'-bipyridyl) iridium(III) hexafluorophosphate with nitrogen-doped carbon quantum dots (NCDs). The ECL intensity of the nanowires showed a five-fold increase in ECL intensity compared with the iridium complex monomer under the same experimental conditions. A label-free ECL biosensing platform based on Ir-NCDs was established for Salmonella enteritidis (SE) detection. The ECL signal was quenched linearly in the range of 102-108 CFU/mL for SE with a detection limit of 102 CFU/mL. Moreover, the relative standard deviations (RSD) of the stability within and between batches were 0.98% and 3.9%, respectively. In addition, the proposed sensor showed high sensitivity, selectivity and stability towards SE in sheep feces samples with satisfactory results. In summary, the excellent ECL efficiency of Ir-NCDs demonstrates the prospects for Ir(III) complexes in bioanalytical applications.

Long-term spatiotemporal and highly specific imaging of the plasma membrane of diverse plant cells using a near-infrared AIE probe.

Fluorescent probes are valuable tools to visualize plasma membranes intuitively and clearly and their related physiological processes in a spatiotemporal manner. However, most existing probes have only realized the specific staining of the plasma membranes of animal/human cells within a very short time period, while almost no fluorescent probes have been developed for the long-term imaging of the plasma membranes of plant cells. Herein, we designed an AIE-active probe with NIR emission to achieve four-dimensional spatiotemporal imaging of the plasma membranes of plant cells based on a collaboration approach involving multiple strategies, demonstrated long-term real-time monitoring of morphological changes of plasma membranes for the first time, and further proved its wide applicability to plant cells of different types and diverse plant species. In the design concept, three effective strategies including the similarity and intermiscibility principle, antipermeability strategy and strong electrostatic interactions were combined to allow the probe to specifically target and anchor the plasma membrane for an ultralong amount of time on the premise of guaranteeing its sufficiently high aqueous solubility. The designed APMem-1 can quickly penetrate cell walls to specifically stain the plasma membranes of all plant cells in a very short time with advanced features (ultrafast staining, wash-free, and desirable biocompatibility) and the probe shows excellent plasma membrane specificity without staining other areas of the cell in comparison to commercial FM dyes. The longest imaging time of APMem-1 can be up to 10 h with comparable performance in both imaging contrast and imaging integrity. The validation experiments on different types of plant cells and diverse plants convincingly proved the universality of APMem-1. The development of plasma membrane probes with four-dimensional spatial and ultralong-term imaging ability provides a valuable tool to monitor the dynamic processes of plasma membrane-related events in an intuitive and real-time manner.

A novel fluorescence aptasensor based on PCN-223 as an efficient quencher for sensitive determination of prostate-specific antigen.

A novel fluorescence aptasensor based on PCN-223 as an efficient quencher was developed to sensitively detect prostate-specific antigen (PSA). The 5-carboxytetramethylrhodamine (TAMRA)-labeled PSA aptamer was adsorbed on PCN-223 by π-π stacking and hydrogen-bonding interactions, which contributed to fluorescence quenching because of the photoinduced electron transfer from TAMRA to PCN-223. In addition, the amount of quenched fluorescence of the PSA-binding aptamer complex-PCN-223 was lower than that of TAMRA aptamer-PCN-223 without PSA (at excitation/emission peaks of 545/582 nm), which can be explained by the fact that the PSA-binding aptamer complexes contributed to the separation of the aptamer from PCN-223. ∆F value of fluorescence intensities for TAMRA aptamer-PCN-223 with and without PSA showed a good linear relationship with PSA concentration over a range of 0.1 to 24 ng mL-1, with a detection limit of 0.05 ng mL-1. Compared with three metal-organic frameworks (MOFs) of UiO-66-NH2, ZIF-67, and Ni3(HITP)2 as quenchers, PCN-223 as a Zr-MOF exhibited the highest ∆F value for PSA detection. The advantage of PCN-223 could be attributed to its carboxyl, benzene, and porphyrin groups, the large specific surface area and good biocompatibility. This proposed aptasensor can be successfully used to detect PSA in sera of prostate cancer patients. The PSA detection results of this aptasensor were consistent with those which were obtained from hospital by Archtecti2000sr automatic chemiluminescence immunoanalyzer. The proposed aptasensor has potential clinical detection application.

Photochemically Enhanced Emission by Introducing Rational Photoactive Subunits into an Aggregation-Induced Emission Luminogen.

Herein, we propose a rational design strategy by introducing photoactive thienyl and pyridyl groups into an AIE-active tetraarylethene skeleton to achieve highly efficient photochemistry-activated fluorescence enhancement from dominantly photo-physical aggregation-induced emission behavior, and prove that such photoactivated fluorescence enhancement is perfectly suited for superstable photocontrollable dual-mode patterning applications in both solution and solid matrix. It is found that the photoactivated fluorescence of designed AIEgen is attributed to the irreversible cyclized-dehydrogenation reaction under UV irradiation, and the oxidation product has a brighter fluorescence in both solution and solid states owning to its rigid and planar structure. The overall transformation rate of the AIEgen from its opened form to dehydrogenated form is up to nearly 100 % in a short period of UV irradiation, and the fast transformation and the stable product of this photochemical reaction guarantees super stability of photocontrolled patterning, which can be applied in photoactivated dual-mode patterning and advanced anti-counterfeiting.

Dramatic emission enhancement of aggregation-induced emission luminogens by dynamic metal coordination bonds and the anti-heavy-atom effect.

Restriction of intramolecular motions of AIEgens is greatly intensified by introducing dynamic metal coordination bonds to achieve dramatic fluorescence enhancement, which provides a simple and effective way to dramatically improve the emission efficiency of AIEgens. AIEgen-based metal complexes have an abnormal anti-heavy-atom effect, which contributes to their high emission efficiencies without changing their emission nature.

Esterase-Activated Precipitating Strategy to Achieve Highly Specific Detection and Long-Term Imaging of Calcium Ions by Aggregation-Induced Phosphorescence Probe.

Spatial and temporal monitoring of bioactive targets such as calcium ions is vitally significant for their essential roles in physiological and biochemical functions. Herein, we proposed an esterase-activated precipitating strategy to achieve highly specific identification and long-term bioimaging of calcium ions via lighting up the calcium ions by precipitation using a water-soluble aggregation-induced phosphorescence (AIP) probe. The designed probe CaP2 has an AIP behavior and can be efficiently aggregated by calcium ions through the coupling coordination of carboxylic acid and cyanide groups, which enables it to light up Ca2+ by precipitating-triggered phosphorescence. Four hydrophilic groups of tetraethylene glycol were introduced to endow the resulting probe CaP3 with extraordinary water solubility as well as excellent cellular penetration. Only when the probe CaP3 penetrates inside the live cells the existing esterase in cells can activate the probe to be transformed active CaP2 probe selectively binding with calcium ion in the surroundings. The probe was used to further evaluate the imaging of intracellular calcium ions in model organisms. The excellent imaging performance of CaP3 in Arabidopsis thaliana seedling roots demonstrates that CaP3 has the excellent capability of monitoring calcium ions in live-cell imaging, and furthermore CaP3 exhibits much better photostability and thereby greater potential in long-term imaging. This work established a general esterase-activated precipitating strategy to achieve specific detection and bioimaging in situ triggered by esterase in live cells, and established a water-soluble aggregation-induced phosphorescence probe with high selectivity to achieve specific sensing and long-term imaging of calcium ions in live cells.

Novel Aggregation-Enhanced PEC Photosensitizer Based on Electrostatic Linkage of Ionic Liquid with Protoporphyrin IX for Ultrasensitive Detection of Molt-4 Cells.

Nowadays, aggregation quenching of most organic photosensitizers in aqueous media seriously restricts analytical and biomedical applications of photoelectrochemical (PEC) sensors. In this work, an aggregation-enhanced PEC photosensitizer was prepared by electrostatically bonding protoporphyrin IX (PPIX) with an ionic liquid of 1-butyl-3-methylimidazole tetrafluoroborate ([BMIm][BF4]), termed as PPIX-[BMIm] for clarity. The resultant PPIX-[BMIm] showed weak photocurrent in pure dimethyl sulfoxide (DMSO, good solvent), while the PEC signals displayed a 44.1-fold enhancement in a water (poor solvent)/DMSO binary solvent with a water fraction (fw) of 90%. Such PEC-enhanced mechanism was critically studied by electrochemistry and density functional theory (DFT) calculation in some detail. Afterward, a label-free PEC cytosensor was built for ultrasensitive bioassay of acute lymphoblastic leukemia (molt-4) cells by electrodepositing Au nanoparticles (Au NPs) on the PPIX-[BMIm] aggregates and sequential assembly of protein tyrosine kinase (PTK) aptamer DNA (aptDNA). The resultant cytosensor showed a wide linear range (300 to 3 × 105 cells mL-1) with a limit of detection (LOD) as low as 63 cells mL-1. The aggregation-enhanced PEC performance offers a valuable and practical pathway for synthesis of advanced organic photosensitizer to explore its PEC applications in early diagnosis of tumors.

Antipermeability Strategy to Achieve Extremely High Specificity and Ultralong Imaging of Diverse Cell Membranes Based on Restriction-Induced Emission of AIEgens.

Long-term in situ cell membrane-targeted bioimaging is of great significance for studying specific biological processes and functions, but currently developed membrane probes are rarely simultaneously used to image the plasma membrane of animal and plant cells, and these probes lack sufficiently high long-term targeting ability. Herein, we proposed an antipermeability strategy to achieve highly specific and long-term imaging of plasma membranes of both human and plant cells using the steric hindrance effect and restriction-induced emission of AIE-active probes based on an updated membrane model. A certain degree of rigidity of plasma membrane containing a large ratio of rigid cholesterol molecules in the updated membrane model provides a promising opportunity to design antipermeable probes by introducing a rigid steric hindrance group in the probe. The designed antipermeable probes can anchor inside plasma membrane for a long term relying on the combination of the steric hindrance effect and the electrostatic and hydrophobic interactions between the probe and the membrane, as well as light up the membrane via the restriction-induced emission mechanism. The excellent performance in imaging completeness and specificity for both human cells and plant cells clearly shows that these designed probes possess outstanding antipermeability to achieve long-term specific imaging of membrane. These probes also show some advanced features such as ultrafast staining, wash-free merit, favorable biocompatibility, good photostability, and effective resistance to viscosity and pH alteration. This work also provides a valuable design principle for membrane probes of plant cells that the designed probes require a suitable molecular size favoring the penetration of small pores of cell walls.

Achieving highly efficient aggregation-induced emission, reversible and irreversible photochromism by heavy halogen-regulated photophysics and D-A molecular pattern-controlled photochemistry of through-space conjugated luminogens.

It is extremely challenging but desirable to regulate the photophysical and photochemical processes of aggregation-induced emission luminogens (AIEgens) in distinct states in a controllable manner. Herein, we design two groups of AIEgens based on a triphenylacrylonitrile (TPAN) skeleton with through-space conjugation (TSC) property, demonstrate controlled regulation of photophysical emission efficiency/color and photochemical photochromic and photoactivatable fluorescence behaviours of these compounds, and further validate design principles to achieve highly efficient and emission-tuning AIEgens and to accomplish photo-dependent color switches and fluorescence changes. It is surprisingly found that the introduction of heavy halogens like bromine into a TPAN skeleton dramatically enhances the emission efficiency, and such an abnormal phenomenon against the heavy-atom effect is attributed to the specific through-space conjugation nature of the AIE-active skeleton, effective intermolecular halogen-bond-induced restriction of intramolecular motions, and heavy atom-induced vibration reduction. The incorporation of two electron-donating amino groups into the TPAN skeleton cause the luminogens to undergo a bathochromic shifted emission due to the formation of a D-A pattern. Apart from the regulation of photophysical processes in the solid state, the construction of the D-A pattern in luminogens also results in extremely different photochemical reactions accompanying reversible/irreversible photochromism and photoactivatable fluorescence phenomena in a dispersed state. It is revealed that photo-triggered cyclization and decyclization reactions dominantly contribute to reversible photochromism of the TPAN family, and the photo-induced cyclization-dehydrogenation reaction is responsible for the irreversible color changes and photoactivatable fluorescence behaviours of the NTPAN family. The demonstrations of multiple-mode signaling in photoswitchable patterning and information encryption highlight the importance of controlled regulation of photophysics and photochemistry of fused chromic and AIE-active luminogens in distinct states.

Nanoliposomal Ratiometric Fluorescent Probe toward ONOO- Flux.

Peroxynitrite (ONOO-), a powerful biological oxidant, is produced in the mitochondria and reacts with many biomolecular targets under various pathological conditions, leading to a range of disease states. In this work, we developed a nanoliposome-encapsulated ratiometrically fluorescent probe (NRF) based on a hemicyanine structure Cy-O obtained by facile synthesis. Upon reaction with ONOO-, the oxidation and hydrolysis of a π-conjugation system within the nanoliposome triggers a ratiometrically fluorescent response and a large-scale emission shift (238 nm), which provides a specific and sensitive means for the ONOO- detection. Moreover, we have performed DFT calculation at the 6-31+G(d,p) level using a suite of Gaussian 09 programs to obtain insights into the chemical structure optical properties of Cy-O. In addition, the practical applications of the nanoprobe to image exogenous and endogenous ONOO- were achieved further in live cells and animals triumphantly.

A phosphorescence "turn-on" probe for the detection and imaging of Al3+ based on aggregation-induced emission.

Aggregation-induced emission luminogens (AIEgens) have been widely used to design fluorescent probes for chemosensing and bioimaging. However, it is still challenging to design long-lived AIE-active probes due to the lack of aggregation-induced phosphorescence (AIP) luminogens. In this work, we design and synthesize a long-lived molecular probe with aggregation-induced phosphorescence property for aluminum ion-specific detection by introducing multiple carboxylic acid groups in a unique twisted molecular skeleton, and develop a first phosphorescent detection method for aluminum ion based on aggregation-induced emission mechanism. The introduction of six carboxylic acid groups into the probe not only significantly enhances the water-solubility but also provides specific recognition unit for aluminum ions via complexation. The probe shows a very sharp emission enhancement in the presence of aluminum ions via aluminum ion-triggered aggregation-induced emission. The cytotoxicity test of the probe shows its biocompatible nature, and further imaging results in live human cells and roots of live Arabidopsis thaliana demonstrates that the designed AIP-active probe is capable of monitoring aluminum ions in complex biological systems. This work proposes a general design strategy for AIP-active probes, and provides valuable use of these AIP-active probes in bioimaging.

Photochromism and Fluorescence Switch of Furan-Containing Tetraarylethene Luminogens with Aggregation-Induced Emission for Photocontrolled Interface-Involved Applications.

It is extremely challenging to design photocontrolled molecular switches with absorption and fluorescence dual-mode outputs that are suited for a solid surface and interface. Herein, we report a group of furan-containing tetraarylethene derivatives with unique photophysical behavior of aggregation-induced emission (AIE) and distinct photochemical reaction-triggered photochromic behaviors by combining a photoactive furan or benzofuran group and an AIE-active triphenylethene molecule. The introduction of a furyl or benzofuryl group into the AIE luminogen endows the molecules with significant reversible photochromism and solid-state fluorescence. The coloration and decoloration of these molecules can be switched by respective irradiation of UV and visible light in a reversible way, and the photochromic changes are accompanied by a switch-on and switch-off of the solid-state fluorescence. It is revealed that the photocontrolled cyclization and cycloreversion reactions are responsible for the reversible photochromism and fluorescence switching based on experimental data and theoretical analysis. Both the position and conjugation of the introduced photoactive units have significant influence on the color and strength of the photochromism, and the simultaneous occurrence of photoinduced fluorescence change in the solid state is perfectly suited for surface-involved applications. The demonstrations of dual-mode signaling in photoswitchable patterning on a filter paper and anti-counterfeiting of an anti-falsification paper strongly highlight the unique advantage of these photochromic molecules with an aggregation-induced emission characteristic in various practical applications. This work proposes a general strategy to design photochromic molecules with AIE activity by introducing photoactive functionals into an AIEgen and demonstrates incomparable advantage in dual-mode signaling and multifunctional applications of these molecules.

Clustering-Triggered Ultralong Room-Temperature Phosphorescence of Organic Crystals through Halogen-Mediated Molecular Assembly.

To achieve efficient room-temperature phosphorescence of organic materials with ultralong lifetime, it is imperative to resolve the dilemma that the introduction of heavy atoms simultaneously improves emission efficiencies and shortens the emission lifetimes. Herein, we report a new molecular design approach for halogenated luminogens with a methylene bridge to avoid the lifetime shortening induced by heavy halogens and propose a general molecular engineering strategy to realize efficient and ultralong room-temperature phosphorescence via halogen-mediated molecular clustering. The halogenated N-benzylcarbazole derivatives show distinct photophysical behaviors depending on different physical states, including single-molecule state and cluster state. Their crystals demonstrate the halogen-dependent emission duration of room-temperature phosphorescence upon excitation. Experimental data and theoretical analysis indicate that halogen-regulated molecular clustering in the crystal is responsible for the generation of efficient ultralong room-temperature phosphorescence, and halogen-dominated molecular engineering favors the promotion of the intersystem crossing process and the following triplet emissions.

Photophysical Switching between Aggregation-Induced Phosphorescence and Dual-State Emission by Isomeric Substitution.

It is attractive but highly challenging to achieve controllable regulation of photophysical properties of pure organic luminogens, due to distinct work mechanisms and molecular structures. Here, a strategy to regulate in a controllable way the emission behavior of luminogens is reported, according to which long-lived aggregation-induced emission (AIE) can be switched to short-lived dual-state emission (DSE) by an isomer-based substitution reaction. Three luminogens with sharply different photophysical behaviors, including aggregation-induced phosphorescence and dual-state fluorescence emission, were obtained through a substitution reaction with three isomers. Freely rotating structures are attributed to aggregation-induced phosphorescence behavior, whereas twisted rigidification of the molecule greatly contributes to its dual-state emission phenomenon. This work contributes to the controlled regulation of photophysical behaviors through simple reactions and provides a solid evidence to support the key role of the prohibition of intramolecular rotation in aggregation-induced emission process and molecular design of dual-state emitters.

Halogenated tetraphenylethene with enhanced aggregation-induced emission: an anomalous anti-heavy-atom effect and self-reversible mechanochromism.

Halogenated tetraphenylethene derivatives show a unique anti-heavy-atom effect where introducing heavy halogens like bromine greatly improves the fluorescence quantum yield upon aggregation, contrary to the classic heavy-atom effect. The unique self-reversible mechanochromism of brominated TPE is attributed to re-generation of halogen-halogen bonding after its breakage.

Rational Design of Dual-State Emission Luminogens with Solvatochromism by Combining a Partially Shared Donor-Acceptor Pattern and Twisted Structures.

We report a general design strategy for a new class of luminogens with dual-state emission (DSEgens) that are brightly emissive in both the solution and solid state, with solvatochromism properties, by constructing a partially shared donor-acceptor pattern based on a twisted molecule. The DSEgens with bright fluorescence emission in both the solid and solution state demonstrate a unique solvatochromism behaviour depending on solvent polarity and thus may have applications in anti-counterfeiting.