Unprecedented Sensitivity in a Probe for Monitoring Cathepsin B: Chemiluminescence Microscopy Cell‐Imaging of a Natively Expressed Enzyme

Until recently, chemiluminescence cell images could only be obtained using luciferase‐activated probes. Moreover, chemiluminescence microscopy cell‐imaging has not been demonstrated for natively expressed enzymes like cathepsin B. Herein, we describe the design, synthesis, and evaluation of the first chemiluminescence probe for the detection and imaging of cathepsin B. The probe activation mechanism relies on the release of a dioxetane intermediate, which undergoes chemiexcitation to emit green light with high efficiency under physiological conditions. Using the probe, we obtained clear images of cancerous leukemia and colon cells. This is the first demonstration of chemiluminescence cell images obtained by a probe for a natively expressed endogenous enzyme. We anticipate that the concept presented in this study will be broadly used to develop analogous probes for other important proteases relevant to biomolecular processes.     

Ultrasensitive Imaging of Cells and Sub-Cellular Entities by Electrochemiluminescence

Here we report on a label-free electrochemiluminescence (ECL) microscopy using exceptionally low concentrations of the [Ru(bpy)₃]²⁺ luminophore. This work addresses the central point of the minimal concentration of the ECL luminophore required to image single entities. We demonstrate the possibility to record ECL images of cells and mitochondria at concentrations down to nM and pM. This is 7 orders of magnitude lower than classically-used concentrations and corresponds to a few hundreds of luminophores diffusing around the biological entities. Yet, it produces remarkably sharp negative optical contrast ECL images, as demonstrated by structural similarity index metric analyses and supported by predictions of the ECL image covering time. Finally, we show that the reported approach is a simple, fast, and highly sensitive method, which opens new avenues for ultrasensitive ECL imaging and ECL reactivity at the single molecule level.     

Iridium(III) complexes as polymer bound oxygen sensors

New luminescent oxygen sensors have been prepared by covalent attachment of iridium complex luminophores to a silicone polymer. The oxygen sensor properties of these novel materials were compared to related sensors in which the luminophore is dispersed within the polymer matrix. Covalently bound luminophore materials showed increased sensitivity to oxygen over dispersions in pure silicone polymer as well as in blends with polystyrene, which was added to improve the mechanical properties of the material.     

The comparison of radiation and photochemical stability of luminophores in polystyrene scintillators

The kinetics of photoluminescence and secondary luminophore consumption was studied during γ radiolysis or photolysis of polystyrene film scintillators in air. The stability of secondary luminophores was considered as a substantial factor that has an effect on the overall stability of scintillators. Radiation chemical and quantum yields of luminophore consumption were estimated. The influence of primary luminophores on the secondary luminophore stability as well as on the overall stability of scintillators was analysed. It was shown that radiation stability of scintillators in a number of cases is practically determined by secondary processes that occur with the participation of excited states of the polymer and luminophores.     

Surface confinement and its effects on the luminescence quenching of a ruthenium-containing metallopolymer

Luminescence quenching of the metallopolymers [Ru(bpy)(2)(PVP)(10)](2+) and [Ru(bpy)(2)(PVP)(10)Os(bpy)(2)](4+), both in solution and as thin films, is reported, where bpy is 2,2'-bipyridyl and PVP is poly(4-vinylpyridine). When the metallopolymer is dissolved in ethanol, quenching of the ruthenium excited state, Ru(2+*), within [Ru(bpy)(2)(PVP)(10)](2+) by [Os(bpy)(3)](2+) proceeds by a dynamic quenching mechanism and the rate constant is (1.1 +/- 0.1) x 10(11) M(-1) s(-1). This quenching rate is nearly two orders of magnitude larger than that found for quenching of monomeric [Ru(bpy)(3)](2+) under the same conditions. This observation is interpreted in terms of an energy transfer quenching mechanism in which the high local concentration of ruthenium luminophores leads to a single [Os(bpy)(3)](2+) centre quenching the emission of several ruthenium luminophores. Amplifications of this kind will lead to the development of more sensitive sensors based on emission quenching. Quenching by both [Os(bpy)(3)](2+) and molecular oxygen is significantly reduced within a thin film of the metallopolymer. Significantly, in both optically driven emission and electrogenerated chemiluminescence, emission is observed from both ruthenium and osmium centres within [Ru(bpy)(2)(PVP)(10)Os(bpy)(2)](4+) films, i.e. the ruthenium emission is not quenched by the coordinated [Os(bpy)(2)](2+) units. This observation opens up new possibilities in multi-analyte sensing since each luminophore can be used to detect separate analytes, e.g. guanine and oxoguanine.     

A Glowing Trajectory between Bio‐ and Chemiluminescence: From Luciferin‐Based Probes to Triggerable Dioxetanes

Bioluminescent and chemiluminescent probes are widely used for noninvasive imaging applications because of their high sensitivity and the simplicity of the equipment required to perform the measurement. Synthetic luciferin‐analogue probes with in vivo imaging performance better than that of luciferin are now available. In addition, caged luciferin‐based bioluminogenic probes have been emerged as a general tool for the visualization of different enzymes and analytes in vivo. Recent discoveries have led to development of highly efficient chemiluminescent probes that are extremely bright under physiological conditions. As discussed in this Minireview, chemiluminescence is ready to realize its potential as a valuable tool for imaging in living systems.     

A Chemiluminescent Metal–Organic Framework

The synthesis and characterization of a chemiluminescent metal–organic framework with high porosity is reported. It consists of Zr₆O₆(OH)₄ nodes connected by 4,4′-(anthracene-9,10-diyl)dibenzoate as the linker and luminophore. It shows the topology known for UiO-66 and is therefore denoted PAP-UiO. The MOF was not only obtained as bulk material but also as a thin film. Exposure of PAP-UiO as bulk or film to a mixture of bis-(2,4,6-trichlorophenyl) oxalate, hydrogen peroxide, and sodium salicylate in a mixture of dimethyl and dibutyl phthalate evoked strong and long lasting chemiluminescence of the PAP-UiO crystals. Time dependent fluorescence spectroscopy on bulk PAP-UiO and, for comparison, on dimethyl 4,4′-(anthracene-9,10-diyl)dibenzoate provided evidence that the chemiluminescence originates from luminophores being part of the PAP-UiO, including the luminophores inside the crystals.     

Integration of the 1,2,3‐Triazole “Click” Motif as a Potent Signalling Element in Metal Ion Responsive Fluorescent Probes

In a systematic approach we synthesized a new series of fluorescent probes incorporating donor–acceptor (D‐A) substituted 1,2,3‐triazoles as conjugative π‐linkers between the alkali metal ion receptor N‐phenylaza‐[18]crown‐6 and different fluorophoric groups with different electron‐acceptor properties (4‐naphthalimide, meso‐phenyl‐BODIPY and 9‐anthracene) and investigated their performance in organic and aqueous environments (physiological conditions). In the charge‐transfer (CT) type probes 1 , 2 and 7 , the fluorescence is almost completely quenched by intramolecular CT (ICT) processes involving charge‐separated states. In the presence of Na⁺ and K⁺ ICT is interrupted, which resulted in a lighting‐up of the fluorescence in acetonitrile. Among the investigated fluoroionophores, compound 7 , which contains a 9‐anthracenyl moiety as the electron‐accepting fluorophore, is the only probe which retains light‐up features in water and works as a highly K⁺/Na⁺‐selective probe under simulated physiological conditions. Virtually decoupled BODIPY‐based 6 and photoinduced electron transfer (PET) type probes 3 – 5 , where the 10‐substituted anthracen‐9‐yl fluorophores are connected to the 1,2,3‐triazole through a methylene spacer, show strong ion‐induced fluorescence enhancement in acetonitrile, but not under physiological conditions. Electrochemical studies and theoretical calculations were used to assess and support the underlying mechanisms for the new ICT and PET 1,2,3‐triazole fluoroionophores.     

Immobilized 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole as solid phase luminophore in peroxyoxalate chemiluminescence

A new luminophore for application in peroxyoxalate chemiluminescence is presented. An analogue of the well-known chemiluminescence compound lophine, i.e. 2-(4-hydrazinocarbonylphenyl)-4,5-diphenylimidazole (HCPI), has been covalently immobilized to controlled pore glass and a porous methacrylate resin. By using this reagent in a solid phase detection reactor, sensitive determinations of hydrogen peroxide have been demonstrated. In homogeneous solution HCPI emits poorly as a result of 1,1-oxalyldiimidazole excitation, but when immobilized its efficiency is almost comparable to highly efficient luminophores such as 3-aminofluoranthene. Linearity extends in the single stream flow system over several orders of magnitude with both materials. The limit of detection was 1 nmol/l (10 fmole injected), when using the porous methacrylate support.     

No-wash protein labeling with designed fluorogenic probes and application to real-time pulse-chase analysis

Small molecule labeling techniques for cellular proteins under physiological conditions are very promising for revealing new biological functions. We developed a no-wash fluorogenic labeling system by exploiting fluorescence resonance energy transfer (FRET)-based fluorescein-cephalosporin-azopyridinium probes and a mutant β-lactamase tag. Fast quencher elimination, hydrophilicity, and high resistance against autodegradation were achieved by rational refinement of the structure. By applying the probe to real-time pulse-chase analysis, the trafficking of epidermal growth factor receptors between cell surface and intracellular region was imaged. In addition, membrane-permeable derivatization of the probe enabled no-wash fluorogenic labeling of intracellular proteins.     

Synthesis and photo- and electroluminescent properties of copolyfluorenes with nile red fragments in side chains

New copolyfluorenes containing units of 4,7-dibromo-2,1,3-benzothiadiazole (green luminophore) and 3,6- or 2,7-dibromocarbazole derivatives with side-chain fragments of green (4-pyrrolidinyl-1,8-naphthalimide) and red (Nile red) luminophores and additional carbazole or diphenyloxadiazole groups are synthesized via the Suzuki copolycondensation reaction. The structure of the polymers is modified via insertion of triphenylamine, aryloxadiazole, and quinoxaline units in the backbone of copolyfluorenes and via introduction of triphenylamine, fluorene, and benzene terminal groups. The molecular-weight characteristics of the polycondensation products and the specific features of the transfer of polyfluorene emission energy to the indicated luminophores in solutions and films are studied. Coating the emissive copolyfluorene layer with electron-conducting and hole-blocking layers of poly[9,9-bis(6′-diethoxyphosphorylhexyl)fluorene] makes it possible to increase the brightness of light-emitting diodes (to 2380–3900 cd/m²) and their current and luminance efficiencies.     

Hot electron-induced electrogenerated chemiluminescence of 1-aminonaphthalene-4-sulphonate at oxide-covered aluminium electrodes in aqueous solution

Electrogenerated chemiluminescence of 1-aminonaphthalene-4-sulphonate (ANS) provides a sensitive means for the detection of the ANS in aqueous solution when oxide-covered aluminium electrodes are used as tunnel emission electrodes and cathodic pulse polarisation is used for the excitation of the luminophore. During the pulse polarisation of insulating oxide film-coated aluminium cathodes, hot electrons are tunnel emitted from the aluminium to the aqueous electrolyte solution by direct field-assisted tunnelling (in the case of oxide films of thickness 2-6 nm) or by Fowler-Nordheim (FN) tunnelling in the cases of thicker oxide films. As a result of direct tunnel emission of these energetic electrons, the generation of hydrated electrons (e_aq⁻) becomes possible. These electrochemically generated, extremely strong reductants (dry hot or hydrated electrons) make the efficient excitation of various types of luminophores at thin insulating film-covered electrodes possible and provide a means for sensitive immunoassays and DNA-probing assays when these luminophores are used as label molecules.     

Ultrasensitive Detection of Salmonella and Listeria monocytogenes by Small‐Molecule Chemiluminescence Probes

Detection of Salmonella and L. monocytogenes in food samples by current diagnostic methods requires relatively long time to results (2–6 days). Furthermore, the ability to perform environmental monitoring at the factory site for these pathogens is limited due to the need for laboratory facilities. Herein, we report new chemiluminescence probes for the ultrasensitive direct detection of viable pathogenic bacteria. The probes are composed of a bright phenoxy‐dioxetane luminophore masked by triggering group, which is activated by a specific bacterial enzyme, and could detect their corresponding bacteria with an LOD value of about 600‐fold lower than that of fluorescent probes. Moreover, we were able to detect a minimum of 10 Salmonella cells within 6 h incubation. The assay allows for bacterial enrichment and detection in one test tube without further sample preparation. We anticipate that this design strategy will be used to prepare analogous chemiluminescence probes for other enzymes relevant to specific bacteria detection and point‐of‐care diagnostics.     

Fluorescence resonance energy transfer inhibition assay for α-fetoprotein excreted during cancer cell growth using functionalized persistent luminescence nanoparticles

Persistent-luminescence nanoparticles (PLNPs) are promising as a new generation of photoluminescent probes for detection of biomolecules and bioimaging. Here we report a fluorescence resonance energy transfer (FRET) inhibition assay for α-fetoprotein (AFP) excreted during cancer cell growth using water-soluble functionalized PLNPs based on Eu2+- and Dy3+-doped Ca1.86Mg0.14ZnSi2O7. Polyethyleneimine-coated PLNPs were conjugated with AFP-antibody-coated gold nanoparticles as a sensitive and specific persistent photoluminescence probe for detection of AFP in serum samples and imaging of AFP excreted during cancer cell growth. Such PLNPs do not contain toxic heavy metals. Their long-lasting afterglow nature allows detection and imaging without external illumination, thereby eliminating the autofluorescence and scattering light from biological matrixes encountered under in situ excitation.     

A Highly Efficient Chemiluminescence Probe for the Detection of Singlet Oxygen in Living Cells

Singlet oxygen is among the reactive oxygen species (ROS) with the shortest life‐times in aqueous media because of its extremely high reactivity. Therefore, designing sensors for detection of ¹O₂ is perhaps one of the most challenging tasks in the field of molecular probes. Herein, we report a highly selective and sensitive chemiluminescence probe ( SOCL‐CPP ) for the detection of ¹O₂ in living cells. The probe reacts with ¹O₂ to form a dioxetane that spontaneously decomposes under physiological conditions through a chemiexcitation pathway to emit green light with extraordinary intensity. SOCL‐CPP demonstrated promising ability to detect and image intracellular ¹O₂ produced by a photosensitizer in HeLa cells during photodynamic therapy (PDT) mode of action. Our findings make SOCL‐CPP the most effective known chemiluminescence probe for the detection of ¹O₂. We anticipate that our chemiluminescence probe for ¹O₂ imaging would be useful in PDT‐related applications and for monitoring ¹O₂ endogenously generated by cells in response to different stimuli.     

Near-Infrared Photodynamic Chemiluminescent Probes for Cancer Therapy and Metastasis Detection

There is growing interest in the development of chemiluminescence (CL) probes for phototheranostics because of their minimized tissue autofluorescence. However, due to a lack of near-infrared (NIR)-absorbing chemiluminophores, current probes for NIR CL-guided phototherapy are based on nanoparticles made up of multiple components. We report bright unimolecular chemiluminophores with NIR absorptions and emissions, long CL half-lives and ideal photodynamic efficiency. One luminophore is modified into an activatable probe, DBP_OL, with a turn-on CL signal and photodynamic activity that are specific to a cancer biomarker. The highly sensitive DBP_OL allows CL-guided photodynamic therapy which completely inhibits tumor growth and lung metastasis in mouse models, and can be applied for noninvasive monitoring of lung metastasis. We provide molecular guidelines for NIR-absorbing CL probes for imaging-guided phototherapy.     

新型荧光/EPR双功能次氯酸探针的构建及性能

构建了一种新型香豆素-萘酰亚胺荧光/电子顺磁共振双功能探针CNNOH,并结合荧光光谱、电子顺磁共振(EPR)波谱和紫外-可见吸收光谱对其性能进行了研究.结果表明,该探针可结合荧光光谱的灵敏性和EPR波谱的特异性进行次氯酸的检测;由于香豆素与萘酰亚胺之间存在荧光共振能量转移(FRET)效应,探针分子具有较大的Stokes位移(135 nm),可有效避免由激发光导致的杂散光对检测的干扰.该双功能探针具有检出限低(0.214μmol/L)、反应速度快(~10 s)、检测范围宽(0~5 mmol/L)、选择性好及在生理条件下稳定的特点,预期在活体细胞检测方面有良好的应用前景.     

Charge Transfer Through Dithieno[2,3‐a:3′,2′‐c]phenazine: Effect of Substitution Pattern on the Optoelectronic Properties of Regioisomeric Luminophores

Two series of regioisomeric luminophores that contained a dithieno[2,3‐a:3′,2′‐c]phenazine (DTP) unit as an electron acceptor have been designed and synthesized. To investigate the effect of substitution pattern on the optoelectronic properties of these luminophores, electron donors (N,N‐dihexylaniline or N,N‐dihexyl‐4‐vinylaniline) were incorporated at the 2,5‐, 8,11‐, and 9,10‐positions of the DTP unit. We found that the optoelectronic properties of the regioisomeric luminophores were greatly affected by the substitution pattern: functionalization at the 8,11‐positions of the DTP unit was superior to the other two substitution patterns in extending the effective π‐conjugation and strengthening the intramolecular charge‐transfer interactions. Moreover, the insertion of vinyl groups between the DTP and N,N‐dihexylaniline units narrowed the energy band‐gap for isomers 4 and 5 . However, hypsochromically shifted absorption and photoluminescence maxima were observed for isomeric luminophore 6 , in which electron donors were substituted at the 2,5‐positions of the DTP unit. These results should facilitate greater understanding of the structure–property relationships in regioisomeric semiconductors and present a new way to design optoelectronic materials with effective substitution patterns.     

Electrogenerated Chemiluminescence and Electroluminescence of N-Doped Graphene Quantum Dots Fabricated from an Electrochemical Exfoliation Process in Nitrogen-Containing Electrolytes

Artificial lighting sources are one of the most important technological developments for our modern lives; the search for cost-effective and efficient luminophores is therefore crucial to a sustainable future. Graphene quantum dots (GQDs) are carbon-based nanomaterials that exhibit exceptional optical and electronic properties, making them a prime candidate for a luminophore in a light-emitting device. Nitrogen-doped GQDs fabricated from a facile top-down electrochemical exfoliation process with a nitrogen-containing electrolyte in this report showed strong photoluminescent emission at 450 nm, and electrogenerated chemiluminescence at 660 nm in the presence of benzoyl peroxide as a coreactant. When introduced into solid-state light-emitting electrochemical cells, for the first time, the GQDs displayed a broad white emission centered at 610 nm, corresponding to Commision Internationale de l'eclairage (CIE) colour coordinates of (0.38, 0.36).     

A General Strategy for Through-Bond Energy Transfer Fluorescence Probes Combining Intramolecular Charge Transfer: A Silyl Ether System for Endogenous Peroxynitrite Sensing

A general strategy is reported for developing through-bond energy transfer (TBET) fluorescence probes by combining intramolecular charge transfer (ICT). The strategy uses a coplanar donor-π-bridge-acceptor system (SiOPh-PyOH) without spirolactam. The off-on switch of TBET and ICT is controlled by coplanar structure changes in the sensing process instead of spirolactam ring-opening in traditional TBET probes. DFT calculations showed that the energy and charge transfers from SiOPh to PyOH are prohibited. Since the SiOPh has no fluorescence, the probe SiOPh-PyOH shows fluorescence properties similar to that of pyrene. After sensing ONOO⁻, the silyl ether is removed and the probe changes into ⁻OPh-PyO⁻. Electron-donating ICT from ⁻OPh to PyO⁻ induces a large redshift of emission to 594 nm (179 nm shift). TBET from ⁻OPh to PyO⁻ ensures the probe exhibits a large pseudo-Stokes shift of 213 nm. Furthermore, the probe was successfully used in endogenous ONOO⁻ detection. This study offers a new strategy for the construction of TBET probes emitting in the red region without spirolactam ring-opening, a new ONOO⁻ sensing system using silyl ether as a reaction site, and a method for the deprotection of silyl ethers with ONOOH under mild conditions.