A Ratiometric Luminescence Probe for Highly Reactive Oxygen Species Based on Lanthanide Complexes

Reactive oxygen species (ROS) are important mediators in a variety of pathological events, but the oxidative stress owing to excessive generation of ROS is implicated in many human diseases. In this work, we designed and synthesized a novel dual-functional chelating ligand, [4′-(<i>p</i>-aminophenoxy)­methylene-2,2′:6′,2″-terpyridine-6,6″-diyl]­bis­(methylenenitrilo)­tetrakis­(acetic acid) (AMTTA), that can strongly coordinate with both Eu³⁺ and Tb³⁺ in aqueous solutions for the recognition and time-gated luminescence detection of highly ROS (hROS), hydroxyl radical (^•OH), and hypochlorite (ClO^–). The complexes AMTTA-Ln³⁺ (Ln = Eu and Tb) are almost nonluminescent because of the photoinduced electron transfer from the electron-rich aminophenyl group to the terpyridine-Ln³⁺ moiety but can rapidly react with hROS to afford highly luminescent complexes (4′-hydroxymethyl-2,2′:6′,2″-terpyridine-6,6″-diyl)­bis­(methylenenitrilo)­tetrakis­(acetate)-Ln³⁺ (HTTA-Ln³⁺). Interestingly, when the AMTTA-Eu³⁺/Tb³⁺ mixture (AMTTA/Eu³⁺/Tb³⁺ = 2/1/1) was reacted with hROS, the intensity ratio of its Tb³⁺ emission at 540 nm to its Eu³⁺ emission at 610 nm, <i>I</i>₅₄₀/<i>I</i>₆₁₀, showed a ratiometric response toward hROS, and the dose-dependent increase of the ratio displayed a double-exponential correlation to the concentration of hROS. This unique luminescence response allowed the AMTTA-Eu³⁺/Tb³⁺ mixture to be used as a ratiometric probe for the time-gated luminescence detection of hROS

New Class of Tetradentate β-Diketonate-Europium Complexes That Can Be Covalently Bound to Proteins for Time-Gated Fluorometric Application

Luminescent lanthanide complexes that can be covalently bound to proteins have shown great utility as biolabels for highly sensitive time-gated luminescence bioassays in clinical diagnostics and biotechnology discoveries. In this work, three new tetradentate β-diketonate–europium complexes that can be covalently bound to proteins to display strong and long-lived Eu³⁺ luminescence, 1,2-bis­[4′-(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-benzyl]-4-chlorosulfobenzene-Eu³⁺ (BHHBCB-Eu³⁺), 1,2-bis­[4′-(1″,1″,1″,2″,2″-pentafluoro-3″,5″-pentanedion-5″-yl)-benzyl]-4-chlorosulfobenzene-Eu³⁺ (BPPBCB-Eu³⁺), and 1,2-bis­[4′-(1″,1″,1″-trifluoro-2″,4″-butanedion-4″-yl)-benzyl]-4-chlorosulfobenzene-Eu³⁺ (BTBBCB-Eu³⁺), have been designed and synthesized as biolabels for time-gated luminescence bioassay applications. The luminescence spectroscopy characterizations of the aqueous solutions of three complex-bound bovine serum albumin reveal that BHHBCB-Eu³⁺ has the strongest luminescence with the largest quantum yield (40%) and longest luminescence lifetime (0.52 ms) among the complexes, which is superior to the other currently available europium biolabels. The BHHBCB-Eu³⁺-labeled streptavidin was prepared and used for both the time-gated luminescence immunoassay of human prostate specific antigen and the time-gated luminescence microscopy imaging of a pathogenic microorganism Cryptosporidium muris. The results demonstrated the practical utility of the new Eu³⁺ complex-based biolabel for time-gated luminescence bioassay applications

A Lanthanide Complex-Based Ratiometric Luminescence Probe for Time-Gated Luminescence Detection of Intracellular Thiols

A lanthanide complex-based ratiometric luminescence probe, [4′-(2,4-dinitrobenzenesulfonyloxy)-2,2′:6′,2′′-terpyridine-6,6′′-diyl] bis­(methylenenitrilo) tetrakis­(acetate)-Eu³⁺/Tb³⁺ (NSTTA-Eu³⁺/Tb³⁺), has been designed and synthesized for the specific recognition and time-gated luminescence detection of biothiols in physiological pH aqueous media. The probe itself is almost nonluminescent due to the presence of photoinduced electron transfer (PET) from the terpyridine-Ln³⁺ moiety to the 2,4-dinitrobenzenesulfonyl (DNBS) moiety. In the presence of biothiols, the reaction of NSTTA-Eu³⁺/Tb³⁺ with biothiols results in the cleavage of DNBS to afford the deprotonated (4′-hydroxy-2,2′:6′,2′′-terpyridine-6,6′′-diyl) bis­(methylenenitrilo) tetrakis­(acetate)-Eu³⁺/Tb³⁺ (HTTA-Eu³⁺/Tb³⁺), which terminates the PET process. After the reaction, the intensity of Eu³⁺ emission at 610 nm is unchanged, while that of Tb³⁺ emission at 540 nm is remarkably increased, which provides a ∼36-fold enhanced intensity ratio of Tb³⁺ emission to Eu³⁺ emission (<i>I</i>₅₄₀/<i>I</i>₆₁₀). This unique luminescence response allows NSTTA-Eu³⁺/Tb³⁺ to be used as a ratiometric probe for the time-gated luminescence detection of biothiols, using the intensity ratio of <i>I</i>₅₄₀/<i>I</i>₆₁₀ as a signal. Thus, based on the probe NSTTA-Eu³⁺/Tb³⁺, a ratiometric time-gated luminescence detection method for biothiols was established and successfully used for the quantitative detection of the total biothiols in several living cell samples

Placental Barrier-on-a-Chip: Modeling Placental Inflammatory Responses to Bacterial Infection

Placental inflammation, as a recognized cause of preterm birth and neonatal mortality, displays extensive placental involvement or damage with the presence of organisms. The inflammatory processes are complicated and tightly associated with increased inflammatory cytokine levels and innate immune activation. However, the deep study of the underlying mechanisms was limited by conventional cell and animal models because of great variations in the architecture and function of placenta. Here, we established a microengineered model of human placental barrier on the chip and investigated the associated inflammatory responses to bacterial infection. The multilayered design of the microdevice mimicked the microscopic structure in the fetal-maternal interfaces of human placenta, and the flow resembled the dynamic environment in the mother’s body. <i>Escherichia coli</i> (<i>E. coli</i>), one of the predominant organisms found in fetal organs, were applied to the maternal side, modeling acute placental inflammation. The data demonstrated the complex responses including the increased secretion of inflammatory cytokines by trophoblasts and the adhesion of maternal macrophages following bacterial infection. Particularly, transplacental communication was observed between two placental cells, and implied the potential role of trophoblast in fetal inflammatory response syndrome in clinic. These complex responses are of potential significance to placental dysfunctions, even abnormal fetal development and preterm birth. Collectively, placental barrier-on-a-chip microdevice presents a simple platform to explore the complicated inflammatory responses in human placenta, and might help our understanding of the mechanisms underlying reproductive diseases

Ratiometric Time-Gated Luminescence Probe for Nitric Oxide Based on an Apoferritin-Assembled Lanthanide Complex-Rhodamine Luminescence Resonance Energy Transfer System

Using apoferritin (AFt) as a carrier, a novel ratiometric luminescence probe based on luminescence resonance energy transfer (LRET) between a Tb³⁺ complex (PTTA-Tb³⁺) and a rhodamine derivative (Rh-NO), PTTA-Tb³⁺@AFt-Rh-NO, has been designed and prepared for the specific recognition and time-gated luminescence detection of nitric oxide (NO) in living samples. In this LRET probe, PTTA-Tb³⁺ encapsulated in the core of AFt is the energy donor, and Rh-NO, a NO-responsive rhodamine derivative, bound on the surface of AFt is the energy acceptor. The probe only emits strong Tb³⁺ luminescence because the emission of rhodamine is switched off in the absence of NO. Upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from Tb³⁺ complex to rhodamine occurs, which results in the remarkable increase and decrease of the long-lived emissions of rhodamine and PTTA-Tb³⁺, respectively. After the reaction, the intensity ratio of rhodamine emission to Tb³⁺ emission, <i>I</i>₅₆₅/<i>I</i>₅₃₉, is ∼24.5-fold increased, and the dose-dependent enhancement of <i>I</i>₅₆₅/<i>I</i>₅₃₉ shows a good linearity in a wide concentration range of NO. This unique luminescence response allowed PTTA-Tb³⁺@AFt-Rh-NO to be conveniently used as a ratiometric probe for the time-gated luminescence detection of NO with <i>I</i>₅₆₅/<i>I</i>₅₃₉ as a signal. Taking advantages of high specificity and sensitivity of the probe as well as its good water-solubility, biocompatibility, and cell membrane permeability, PTTA-Tb³⁺@AFt-Rh-NO was successfully used for the luminescent imaging of NO in living cells and <i>Daphnia magna</i>. The results demonstrated the efficacy of the probe and highlighted it’s advantages for the ratiometric time-gated luminescence bioimaging application

Ratiometric Time-Gated Luminescence Probe for Hydrogen Sulfide Based on Lanthanide Complexes

Developments of ratiometric bioprobes are highly appealing due to the superiority of their self-calibration capability for the quantitative biotracking. In this work, we designed and synthesized a novel lanthanide complex-based ratiometric luminescence probe, [4′-(2,4-dinitrophenyloxy)-2,2′:6′,2″-terpyridine-6,6″-diyl]­bis­(methylenenitrilo) tetrakis­(acetate)-Eu³⁺/Tb³⁺ (NPTTA-Eu³⁺/Tb³⁺), for the specific recognition and quantitative time-gated luminescence detection of hydrogen sulfide (H₂S) in aqueous and living cell samples. Due to the presence of the photoinduced electron transfer (PET) process from the terpyridine-Eu³⁺/Tb³⁺ moiety to 2,4-dinitrophenyl (DNP), the probe itself is weakly luminescent. In physiological pH aqueous media, the reaction of NPTTA-Eu³⁺/Tb³⁺ with H₂S leads to the cleavage of DNP moiety from the probe molecule, which affords the deprotonated (4′-hydroxy-2,2′:6′,2″-terpyridine-6,6″-diyl)­bis­(methylenenitrilo) tetrakis­(acetate)-Eu³⁺/Tb³⁺ and terminates the PET process. Meanwhile, the intensity of Tb³⁺ emission at 540 nm is remarkably increased, while that of the Eu³⁺ emission at 610 nm is slightly decreased. After the reaction, the intensity ratio of Tb³⁺ emission to Eu³⁺ emission, <i>I</i>₅₄₀/<i>I</i>₆₁₀, was ∼220-fold increased, and the dose-dependent enhancement of <i>I</i>₅₄₀/<i>I</i>₆₁₀ showed a good linearity upon the increase of H₂S concentration with a detection limit of 3.5 nM. This unique luminescence response allowed NPTTA-Eu³⁺/Tb³⁺ to be conveniently used as a ratiometric probe for the time-gated luminescence detection of H₂S with <i>I</i>₅₄₀/<i>I</i>₆₁₀ as a signal. In addition, the applicability of the probe for the quantitative time-gated luminescence imaging of intracellular H₂S in living cells was investigated. The results demonstrated the efficacy and advantage of the new probe for the time-gated luminescence cell imaging application

Lanthanide Complex-Based Luminescent Probes for Highly Sensitive Time-Gated Luminescence Detection of Hypochlorous Acid

Two novel lanthanide complex-based luminescent probes, ANMTTA-Eu³⁺ and ANMTTA-Tb³⁺ {ANMTTA, [4′-(4-amino-3-nitrophenoxy)­methylene-2,2′:6′,2″-terpyridine-6,6″-diyl] bis­(methylenenitrilo) tetrakis­(acetic acid)}, have been designed and synthesized for the highly sensitive and selective time-gated luminescence detection of hypochlorous acid (HOCl) in aqueous media. The probes are almost nonluminescent due to the photoinduced electron transfer (PET) process from the 4-amino-3-nitrophenyl moiety to the terpyridine-Ln³⁺ moiety, which quenches the lanthanide luminescence effectively. Upon reaction with HOCl, the 4-amino-3-nitrophenyl moiety is rapidly cleaved from the probe complexes, which affords strongly luminescent lanthanide complexes HTTA-Eu³⁺ and HTTA-Tb³⁺ {HTTA, (4′-hydroxymethyl-2,2′:6′,2″-terpyridine-6,6″-diyl) bis­(methylenenitrilo) tetrakis­(acetic acid)}, accompanied by the remarkable luminescence enhancements. The dose-dependent luminescence enhancements show good linearity with detection limits of 1.3 nM and 0.64 nM for HOCl with ANMTTA-Eu³⁺ and ANMTTA-Tb³⁺, respectively. In addition, the luminescence responses of ANMTTA-Eu³⁺ and ANMTTA-Tb³⁺ to HOCl are pH-independent with excellent selectivity to distinguish HOCl from other reactive oxygen/nitrogen species (ROS/RNS). The ANMTTA-Ln³⁺-loaded HeLa and RAW 264.7 macrophage cells were prepared, and then the exogenous HOCl in HeLa cells and endogenous HOCl in macrophage cells were successfully imaged with time-gated luminescence mode. The results demonstrated the practical applicability of the probes for the cell imaging application

Development of a Ruthenium(II) Complex-Based Luminescent Probe for Hypochlorous Acid in Living Cells

A novel Ru­(II) complex, [Ru­(bpy)₂(DNPS-bpy)]­(PF₆)₂ (bpy: 2,2′-bipyridine, DNPS-bpy: 4-(2,4-dinitrophenylthio)­methylene-4′-methyl-2,2′-bipyridine), has been designed and synthesized as a highly sensitive and selective luminescence probe for the recognition and detection of hypochlorous acid (HOCl) in living cells by exploiting a “signaling moiety-recognition linker-quencher” sandwich approach. The complex possesses large stokes shift (170 nm), long emission wavelength (626 nm), and low cytotoxicity. Owing to the effective photoinduced electron transfer (PET) from Ru­(II) center to the electron acceptor, 2,4-dinitrophenyl (DNP), the red-emission of bipyridine-Ru­(II) complex was completely withheld. In aqueous media, HOCl can trigger an oxidation reaction to cleave the DNP moiety from the Ru­(II) complex, which results in the formation of a highly luminescent bipyridine-Ru­(II) complex derivative, [Ru­(bpy)₂(COOH-bpy)]­(PF₆)₂ (COOH-bpy: 4′-methyl-2,2′-bipyridyl-4-carboxylic acid), accompanied by a 190-fold luminescence enhancement. Cell imaging experimental results demonstrated that [Ru­(bpy)₂(DNPS-bpy)]­(PF₆)₂ is membrane permeable, and can be applied for capturing and visualizing the exogenous/endogenous HOCl molecules in living cell samples. The development of this Ru­(II) complex probe not only provides a useful tool for monitoring HOCl in living systems, but also strengthens the application of transition metal complex-based luminescent probes for bioimaging

Developing Red-Emissive Ruthenium(II) Complex-Based Luminescent Probes for Cellular Imaging

Ruthenium­(II) complexes have rich photophysical attributes, which enable novel design of responsive luminescence probes to selectively quantify biochemical analytes. In this work, we developed a systematic series of Ru­(II)-bipyrindine complex derivatives, [Ru­(bpy)_3‑<i>n</i>(DNP-bpy)_<i>n</i>]­(PF₆)₂ (<i>n</i> = 1, 2, 3; bpy, 2,2′-bipyridine; DNP-bpy, 4-(4-(2,4-dinitrophenoxy)­phenyl)-2,2′-bipyridine), as luminescent probes for highly selective and sensitive detection of thiophenol in aqueous solutions. The specific reaction between the probes and thiophenol triggers the cleavage of the electron acceptor group, 2,4-dinitrophenyl, eliminating the photoinduced electron transfer (PET) process, so that the luminescence of on-state complexes, [Ru­(bpy)_3‑<i>n</i>(HP-bpy)_<i>n</i>]²⁺ (<i>n</i> = 1, 2, 3; HP-bpy, 4-(4-hydroxyphenyl)-2,2′-bipyridine), is turned on. We found that the complex [Ru­(bpy)­(DNP-bpy)₂]²⁺ remarkably enhanced the on-to-off contrast ratio compared to the other two (37.8 compared to 21 and 18.7). This reveals a new strategy to obtain the best Ru­(II) complex luminescence probe via the most asymmetric structure. Moreover, we demonstrated the practical utility of the complex as a cell-membrane permeable probe for quantitative luminescence imaging of the dynamic intracellular process of thiophenol in living cells. The results suggest that the new probe could be a very useful tool for luminescence imaging analysis of the toxic thiophenol in intact cells

Resolving Low-Expression Cell Surface Antigens by Time-Gated Orthogonal Scanning Automated Microscopy

We report a highly sensitive method for rapid identification and quantification of rare-event cells carrying low-abundance surface biomarkers. The method applies lanthanide bioprobes and time-gated detection to effectively eliminate both nontarget organisms and background noise and utilizes the europium containing nanoparticles to further amplify the signal strength by a factor of ∼20. Of interest is that these nanoparticles did not correspondingly enhance the intensity of nonspecific binding. Thus, the dramatically improved signal-to-background ratio enables the low-expression surface antigens on single cells to be quantified. Furthermore, we applied an orthogonal scanning automated microscopy (OSAM) technique to rapidly process a large population of target-only cells on microscopy slides, leading to quantitative statistical data with high certainty. Thus, the techniques together resolved nearly all false-negative events from the interfering crowd including many false-positive events