A Dual Purpose Non-Canonical Amino Acid for the Expanded Genetic Code: Combining Metal-Binding and Click Chemistry.

A rationally designed dual purpose non-canonical amino acid (Trz) has been synthesised and successfully incorporated into a protein scaffold via genetic code expansion. Trz contains a 5-pyridyl-1,2,4-triazine system, which allows for inverse electron-demand Diels-Alder (IEDDA) reactions to occur on the triazine ring and for metal ions to be chelated both before and after the click reaction. Trz was successfully incorporated into a protein scaffold and the IEDDA utility of Trz demonstrated through the site-specific labelling of the purified protein with a bicyclononyne. Additionally, Trz was shown to successfully coordinate a cyclometallated iridium(III) centre, providing access to a bioorthogonal luminogenic probe. The luminescent properties of the Ir(III)-bound protein blue-shift upon IEDDA click reaction with bicyclononyne, providing a unique method for monitoring the extent and location of the labelling reaction. In summary, Trz is a new dual purpose non-canonical amino acid, which has great potential for myriad bioapplications where metal-based functionality is required, for example in imaging, catalysis, or photo-dynamic therapy, in conjunction with a bioorthogonal reactive handle to impart additional functionalities, such as dual modality imaging or therapeutic payloads.

Factors Important in the Use of Fluorescent or Luminescent Probes and Other Chemical Reagents to Measure Oxidative and Radical Stress.

Numerous chemical probes have been used to measure or image oxidative, nitrosative and related stress induced by free radicals in biology and biochemistry. In many instances, the chemical pathways involved are reasonably well understood. However, the rate constants for key reactions involved are often not yet characterized, and thus it is difficult to ensure the measurements reflect the flux of oxidant/radical species and are not influenced by competing factors. Key questions frequently unanswered are whether the reagents are used under 'saturating' conditions, how specific probes are for particular radicals or oxidants and the extent of the involvement of competing reactions (e.g., with thiols, ascorbate and other antioxidants). The commonest-used probe for 'reactive oxygen species' in biology actually generates superoxide radicals in producing the measured product in aerobic systems. This review emphasizes the need to understand reaction pathways and in particular to quantify the kinetic parameters of key reactions, as well as measure the intracellular levels and localization of probes, if such reagents are to be used with confidence.

AIEgen-sensitized lanthanide nanocrystals as luminescent probes for intracellular Fe3+ monitoring.

The abnormal Fe3+ level is known to cause various diseases, such as heart failure, liver damage and neurodegeneration. In situ probing Fe3+ in living cells or organisms is highly desired for both biological research and medical diagnostics. Herein, hybrid nanocomposites NaEuF4@TCPP were constructed by the assembly of an aggregation-induced emission luminogen (AIEgen) TCPP and NaEuF4 nanocrystals (NCs). The anchored TCPP on the surface of NaEuF4 NCs can reduce rotational relaxation of the excited state and efficiently transfer the energy to the Eu3+ ions with minimized nonradiative energy loss. Consequently, the prepared NaEuF4@TCPP nanoparticles (NPs) exhibited an intense red emission with a 103-fold enhancement relative to that in NaEuF4 NCs under 365 nm excitation. A selectively quenching response to Fe3+ ions for the NaEuF4@TCPP NPs makes them luminescent probes for sensitive detection of Fe3+ ions with a low detection limit of 340 nM. Moreover, the luminescence of NaEuF4@TCPP NPs could be recovered by the addition of iron chelators. Benefiting from their good biocompatibility and stability in living cells, together with the characteristic of the reversible luminescence response, the lipo-coated NaEuF4@TCPP probes were successfully applied for real-time monitoring of Fe3+ ions in living HeLa cells. These results are expected to motivate the exploration of AIE-based lanthanide probes for sensing and biomedical applications.

Photoluminescence Probes in Data-Enabled Sensing.

This review summarizes the current status of development in photoluminescent probes, multidimensional photoluminescence detection, and multivariate data analysis methods. It then highlights reports featuring multivariate analysis of multidimensional measurements of photoluminescent probes published between June 2015 and June 2022, emphasizing work in the last 5 years. Important trends include the development of probe arrays, which provide fingerprint responses to the analyte(s) of interest and facilitate the analysis of complex samples; the application of neural networks and deep learning to pattern recognition and feature selection in photoluminescence images; and the application of multiway multivariate analysis to mining matrices, three-way arrays, and higher-order measurements, including hyperspectral intensity and lifetime images. These examples illustrate the increase in information extraction provided by the combination of multidimensional measurements and multivariate analysis.

Probing Tyrosine Nitration with a Small TbIII -Metallopeptide.

Tyrosine nitration, a post-translational modification (PTM) that takes place under nitrosative stress conditions, occurs through a non-enzymatic peroxynitrite-mediated reaction. Although protein nitration has long been considered an irreversible PTM involved in nitrosative stress-associated diseases, it has also been suggested to be a regulatory mechanism of signal transduction. Therefore, the development of tools that help to understand this protein modification is of great interest. Herein, we explore a TbIII -chelating metallopeptide to monitor tyrosine nitration. The luminescence of this probe decreases significantly between its non-nitrated and nitrated states, and this reduction in the luminescence intensity is directly related to the degree of tyrosine nitration after treatment with peroxynitrite. Remarkably, the luminescence intensity changes after nitration are not affected in the presence of complex biological media, which makes it a promising tool for understanding this protein modification.

Bifunctional Temperature and Oxygen Dual Probe Based on Anthracene and Europium Complex Luminescence.

In this work, we synthesized a polydimethylsiloxane membrane containing two emitter groups chemically attached to the membrane structure. For this, we attached the anthracene group and the [Eu(bzac)3] complex as blue and red emitters, respectively, in the matrix via hydrosilylation reactions. The synthesized membrane can be used as a bifunctional temperature and oxygen ratiometric optical probe by analyzing the effects that temperature changes and oxygen levels produce on the ratio of anthracene and europium(III) emission components. As a temperature probe, the system is operational in the 203-323 K range, with an observed maximum relative sensitivity of 2.06% K-1 at 290 K and temperature uncertainties below 0.1 K over all the operational range. As an oxygen probe, we evaluated the ratiometric response at 25, 30, 35, and 40 °C. These results show an interesting approach to obtaining bifunctional ratiometric optical probes and also suggest the presence of an anthracene → europium(III) energy transfer, even though there is no chemical bonding between species.

Molecular to Supramolecular Self-Assembled Luminogens for Tracking the Intracellular Organelle Dynamics.

Deciphering the dynamics of intracellular organelles has gained immense attention due to their subtle control over diverse, complex biological processes such as cellular metabolism, energy homeostasis, and autophagy. In this context, molecular materials, including small-organic fluorescent probes and their supramolecular self-assembled nano-/microarchitectures, have been employed to explore the diverse intracellular biological events. However, only a handful of fluorescent probes and self-assembled emissive structures have been successfully used to track different organelle's movements, circumventing the issues related to water solubility and long-term photostability. Thus, the water-soluble molecular fluorescent probes and the water-dispersible supramolecular self-assemblies have emerged as promising candidates to explore the trafficking of the organelles under diverse physiological conditions. In this review, we have delineated the recent progress of fluorescent probes and their supramolecular self-assemblies for the elucidation of the dynamics of diverse cellular organelles with a special emphasis on lysosomes, lipid droplets, and mitochondria. Recent advancement in fluorescence lifetime and super-resolution microscopy imaging has also been discussed to investigate the dynamics of organelles. In addition, the fabrication of the next-generation molecular to supramolecular self-assembled luminogens for probing the variation of microenvironments during the trafficking process has been outlined.

Hydroxypyridinone-Based Metal Chelators towards Ecotoxicity: Remediation and Biological Mechanisms.

Hydroxypyridinones (HPs) are recognized as excellent chemical tools for engineering a diversity of metal chelating agents, with high affinity for hard metal ions, exhibiting a broad range of activities and applications, namely in medical, biological and environmental contexts. They are easily made and functionalizable towards the tuning of their pharmacokinetic properties or the improving of their metal complex thermodynamic stabilities. In this review, an analysis of the recently published works on hydroxypyridinone-based ligands, that have been mostly addressed for environmental applications, namely for remediation of hard metal ion ecotoxicity in living beings and other biological matrices is carried out. In particular, herein the most recent developments in the design of new chelating systems, from bidentate mono-HP to polydentate multi-HP derivatives, with a structural diversity of soluble or solid-supported backbones are outlined. Along with the ligand design, an analysis of the relationship between their structures and activities is presented and discussed, namely associated with the metal affinity and the thermodynamic stability of the corresponding metal complexes.

Understanding the ancillary ligand effect on luminescent cyclometalated Ir(III) complex as a reporter for 2-acetylaminofluorene DNA(AAF-dG) adduct.

Mutagenic agents such as aromatic amines undergo metabolic activation and produce DNA adducts at C8 position of guanine bases. N-2-acetylaminofluorene (AAF) generates different mutational outcomes when placed at G1, G2, and G3 of a NarI sequence (-G1G2CG3CC/T-). These outcomes are dictated by the conformations adopted by these adducts. Detection of such lesions is of considerable interest owing to their hazardous effects. Here, we report the synthesis of three cyclometalated [Ir(L)2dppz]+ complexes (L = 2-phenylpyridine (ppy) 1; benzo[h]quinoline (bhq) 2; 2-phenylquinoline (pq) 3; dppz = dipyrido[3,2-a:2',3'-c]phenazine) and their interaction with AAF adducted NarI DNA. Remarkably, complexes 1 and 2 displayed dominant 3LC transition characteristic of polar environment despite binding to the adducted sites. On the other hand, complex 3 binds to NarI sequences and behaves as a luminescent reporter for AAF-modified DNA. The results reported here emphasize that molecular light switching phenomenon can be stimulated by switching ancillary ligands and might act as potential probes for covalent-DNA defects.

Ruthenium(II) Complex-Based G-quadruplex DNA Selective Luminescent 'Light-up' Probe for RNase H Activity Detection.

A bis-heteroleptic ruthenium(II) complex, 1[PF6 ]2 of benzothiazole amide substituted 2,2'-bipyridine ligand (bmbbipy) has been synthesized for the selective detection of G-quadruplex (GQ) DNA and luminescence-assay-based RNase H activity monitoring. Compound 1[PF6 ]2 exhibited aggregation-caused quenching (ACQ) in water. Aggregate formation was supported by DLS, UV-vis, and 1 H NMR spectroscopy results, and the morphology of aggregated particles was witnessed by SEM and TEM. 1[PF6 ]2 acted as an efficient GQ DNA-selective luminescent light-up probe over single-stranded and double-stranded DNA. The competency of 1[PF6 ]2 for selective GQ structure detection was established by PL and CD spectroscopy. For 1[PF6 ]2 , the PL light-up is exclusively due to the rigidification of the benzothiazole amide side arm in the presence of GQ-DNA. The interaction between the probe and GQ-DNA was analyzed by molecular docking analysis. The GQ structure detection capability of 1[PF6 ]2 was further applied in the luminescent 'off-on' RNase H activity detection. The assay utilized an RNA:DNA hybrid, obtained from 22AG2-RNA and 22AG2-DNA sequences. RNase H solely hydrolyzed the RNA of the RNA:DNA duplex and released G-rich 22AG2-DNA, which was detected via the PL enhancement of 1[PF6 ]2 . The selectivity of RNase H activity detection over various other restriction enzymes was also demonstrated.

Real-Time Imaging of Hepatic Inflammation Using Hydrogen Sulfide-Activatable Second Near-Infrared Luminescent Nanoprobes.

The sensing and visualized monitoring of hydrogen sulfide (H2S) in vivo is crucial to understand its physiological and pathological roles in human health and diseases. Common methods for H2S detection require the destruction of the biosamples and are not suitable to be applied in vivo. In this Communication, we report a "turn-on" second near-infrared (NIR-II) luminescent approach for sensitive, real-time, and in situ H2S detection, which is based on the absorption competition between the H2S-responsive chromophores (compound 1) and the NIR-II luminescent lanthanide nanoparticles. Specifically, the luminescence was suppressed by compound 1 due to the competitive absorption of the incident light. In the presence of H2S, the compound 1 was bleached to recover the luminescence. Thanks to the deep tissue penetration depth and the low absorbance/scattering on biological samples of the NIR-II nanoprobes, the monitoring of the endogenous H2S in lipopolysaccharide-induced liver inflammation was achieved, which is unattainable by the conventional histopathological and serological approaches.

Cell Temperature Measurement for Biometabolism Monitoring.

Temperature is an important factor in the process of life, as thermal energy transfer participates in all biological events in organisms. Due to technical limitations, there is still a lot more information to be explored regarding the correlation between life activities and temperature changes. In recent years, the emergence of a variety of new temperature measurement methods has facilitated further research in this field. Here, we introduce the latest advances in temperature sensors for biological detection and their related applications in metabolic research. Various technologies are discussed in terms of their advantages and shortcomings, and future prospects are presented.

Comparison of Aß (1-40, 1-28, 11-22, and 29-40) aggregation processes and inhibition of toxic species generated in early stages of aggregation by a water-soluble ruthenium complex.

Neurotoxicity of amyloid beta (Aß) species generated in early stages of aggregation has been associated with development of Alzheimer's disease (AD). Consequently, the field of action of compounds that can identify and inhibit the formation of these species has enlarged considerably. This study investigates the effect and influence of the luminescent, water soluble metal complex cis-[Ru(phen)2(3,4Apy)2]2+ (RuApy, 3,4Apy = 3,4-diaminopyridine, phen = 1,10-phenanthroline) on the aggregation process and toxicity of Aß1-40 and its Aß1-28, Aß11-22 and Aß29-40 fragments since their early stages. The absence of correlation between the conformations generated by Aß fragments and the full length 1-40 peptide during aggregation and the absence of toxicity of Aß fragments to PC12 cells in all stages of aggregation indicated that the aggregation pathway and toxicity found to the full-length Aß1-40 depends on specific interactions between the three fragments. The toxicity of Aß1-40 was dependent on the aggregation step investigated: species generated at the beginning (15 min) of aggregation were toxic, whereas mature (120 min) fibrils were not. The RuApy complex is not toxic to PC12 cells up to 60 µM, and does not interfere with the aggregation pathway of the Aß fragments, but interferes with the aggregation of Aß1-40 and protects the PC12 cells, maintaining 100% of cell viability against the toxicity of Aß1-40 species generated in early stages of aggregation.

Cd-Based Metal-Organic Framework for Selective Turn-On Fluorescent DMSO Residual Sensing.

Dimethyl sulfoxide (DMSO) is a universally used solvent in various synthetic reactions, and trace amounts of DMSO residual are often seen on the surface of chemical product. It is difficult to quickly determine whether the residual DMSO is washed completely. This work reports a CdII metal-organic framework (MOF) SXU-4 which can detect trace amounts of DMSO in various solvents. Fluorescence experiments reveal its turn-on fluorescence effect toward DMSO with high selectivity and sensitivity, indicating that it can be used as an effective luminescent probe for rapid chemical product purity detection by testing the washing solution. Crystallographically characterized DMSO loaded SXU-4 (DMSO@SXU-4), in combination with computational results uncover that the enhanced DMSO-MOF conjugation through multiple DMSO-MOF supramolecule interactions and charge rearrangement are the main causes of fluorescence intensification.

Measurement of Temperature Distribution at the Nanoscale with Luminescent Probes Based on Lanthanide Nanoparticles and Quantum Dots.

It is very challenging to probe the temperature in a nanoscale because of the lack of detection technique. Temperature-sensitive luminescent probes at a nanoscale provide the possibility to solve this problem. Herein, we fabricated a model, which combined two kinds of temperature sensitive nanoprobes and gold nanoparticle heater within mesoporous silica nanoparticles. Upconverting nanoparticles and quantum dots located at different positions inside 110 nm nanoparticles reported different temperatures when the gold nanoparticles generated heat by 532 nm laser irradiation. The temperature difference between two probes with an average distance of 55 nm can reach about 30 °C. Our results prove that the temperature distribution at a nanoscale can be measured, and it will be noteworthy if a nano-heater is applied.

Nonlinear Optical Pigments. Two-Photon Absorption in Crosslinked Conjugated Polymers and Prospects for Remote Nonlinear Optical Thermometry.

Nonlinear optical (NLO) pigments are compounds insoluble in solvents that exhibit phenomena related to nonlinear optical susceptibilities (χ(n) where n = 2,3,...), e.g., two-photon absorption (2PA) which is related to the imaginary part of χ(3). Determination of spectrally-resolved 2PA properties for NLO pigments of macromolecular nature, such as coordination polymers or crosslinked polymers, has long been a challenging issue due to their particulate form, precluding characterizations with standard techniques such as Z-scan. In this contribution, we investigate thus far unknown spectrally-resolved 2PA properties of a new subclass of NLO pigments-crosslinked conjugated polymers. The studied compounds are built up from electron-donating (triphenylamine) and electron-withdrawing (2,2'-bipyridine) structural fragments joined by vinylene (Pol1) or vinyl(4-ethynylphenyl) (Pol2) aromatic bridges. 2PA properties of these polymers have been characterized in broad spectral range by specially modified two-photon excited fluorescence (TPEF) techniques: solid state TPEF (SSTPEF) and internal standard TPEF (ISTPEF). The impact of self-aggregation of aromatic backbones on the 2PA properties of the polymers has been evaluated through extended comparisons of NLO parameters, i.e., 2PA cross sections (σ2) and molar-mass normalized 2PA merit factors (σ2/M) with those of small-molecular model compounds: Mod1 and Mod2. By doing this, we found that the 2PA response of Pol1 and Pol2 is improved 2-3 times versus respective model compounds in the solid state form. Further comparisons with 2PA results collected for diluted solutions of Mod1 and Mod2 supports the notion that self-aggregated structure contributes to the observed enhancement of 2PA response. On the other hand, it is clear that Pol1 and Pol2 suffer from aggregation-caused quenching phenomenon, well reflected in time-resolved fluorescence properties as well as in relatively low values of quantum yield of fluorescence. Accordingly, despite improved intrinsic 2PA response, the effective intensity of two-photon excited emission for Pol1 and Pol2 is slightly lower relative to Mod1 and Mod2. Finally, we explore temperature-resolved luminescence properties under one- (377 nm), two- (820 nm), and three-photon excitation (1020 nm) conditions of postsynthetically Eu3+-functionalized material, Pol1-Eu, and discuss its suitability for temperature sensing applications.

TADF Dye-Loaded Nanoparticles for Fluorescence Live-Cell Imaging.

Thermally activated delayed fluorescence (TADF) molecules offer nowadays a powerful tool in the development of novel organic light emitting diodes due to their capability of harvesting energy from non-emissive triplet states without using heavy-metal complexes. TADF emitters have very small energy difference between the singlet and triplet excited states, which makes thermally activated reverse intersystem crossing from the triplet states back to the singlet manifold viable. This mechanism generates a long-lived delayed fluorescence component which can be explored in the sensing of oxygen concentration, local temperature, or used in time-gated optical cell-imaging, to suppress interference from autofluorescence and scattering. Despite this strong potential, until recently the application of TADF outside lighting devices has been hindered due to the low biocompatibility, low aqueous solubility and poor performance in polar media shown by the vast majority of TADF emitters. To achieve TADF luminescence in biological media, careful selection or design of emitters is required. Unfortunately, most TADF molecules are not emissive in polar media, thus complexation with biomolecules or the formation of emissive aggregate states is required, in order to retain the delayed fluorescence that is characteristic of these compounds. Herein, we demonstrate a facile method with great generalization potential that maintains the photophysical properties of solvated dyes by combining luminescent molecules with polymeric nanoparticles. Using an established swelling procedure, two known TADF emitters are loaded onto polystyrene nanoparticles to prepare TADF emitting nanomaterials able to be used in live-cell imaging. The obtained particles were characterized by optical spectroscopy and exhibited the desired TADF emission in aqueous media, due to the polymeric matrix shielding the dye from solvent polarity effects. The prepared nanoparticles were incubated with live human cancer cells and showed very low cytotoxicity and good cellular uptake, thus making fluorescence microscopy imaging possible at low dye concentrations.

Fluorometric determination of the activity of the biomarker terminal deoxynucleotidyl transferase via the enhancement of the fluorescence of silver nanoclusters by in-situ grown DNA tails.

The activity of terminal deoxynucleotidyl transferase (TdTase) is a biomarker for routine diagnosis of acute leukemia. A method has been developed for the determination of TdTase activity. It is based on the use of silver nanoclusters (AgNCs) whose yellow fluorescence is enhanced by an in-situ grown DNA tail of TdTase-polymerized and guanine-rich DNA at the 3' end of a hairpin DNA. The fluorescence, best measured at excitation/emission peaks of 530/585 nm, increases linearly in the 1 to 35 mU mL-1 TdTase activity range. The detection limit is 0.8 mU mL-1. The method is cost-efficient, selective and convenient. It integrates enhancement of the fluorescence of AgNCs and target recognition into a single process. Graphical abstract Schematic presentation of a method for determination of TdTase activity. It is based on AgNCs fluorescence enhanced by in-situ grown TdTase-polymerized G-rich DNA tail. The method integrates AgNCs fluorescence enhancement and the target recognition into a single process.

Stable ZnI -Containing MOFs with Large [Zn70 ] Nanocages from Assembly of ZnII Ions and Aromatic [ZnI8 ] Clusters.

Two unique ZnI -containing MOFs {[ZnI8 ZnII3 (H2 O)x (HL)12 ](OH)2 ⋅13 H2 O}n (x=6, 1; x=2, 2) (HL=tetrazole monoanion) with high-nuclearity Zn-cages were prepared successfully. These Zn-cages are constructed from [ZnI8 ] clusters with multi-centered ZnI -ZnI bonds and ZnII ions. [ZnI8 ] clusters in 1 and 2 display Oh and D4h symmetry, respectively. Importantly, eight [ZnI8 ] clusters and six ZnII ions form a large [Zn70 ] nanocage in 1. To our knowledge, this is the first MOF based on polynuclear Zn-cages consisting of ZnI and ZnII ions. Compared with reported ZnI -species, 1 and 2 display high thermal and solvent stabilities. Theoretical investigations based on DFT calculations uncover that effective 4s-4s orbital overlap and electron delocalization through these a1g +t1u molecular orbitals on [ZnI8 ] clusters lead to considerable aromatic stabilization, which can explain well the intrinsic stability of 1 and 2. Interestingly, 1 can behave as a luminescent probe to detect the toxic CrVI ions in aqueous.

Quaternary Ammonium Polyamidoamine Dendrimer Modified Quantum Dots as Fluorescent Probes for p-Fluorophenoxyacetic Acid Detection in Aqueous Solution.

The wide use of pesticide p-fluorophenoxyacetic acid has caused the serious environmental contaminant. A novel fluorescent probe for sensitive detection of p-fluorophenoxyacetic acid in aqueous solutions based on 3.0G quaternary ammonium polyamidoamine (PAMAM) dendrimer modified quantum dots (QDs) (PAMAM@QDs) was reported. Through the solvent-evaporation method, quaternary ammonium PAMAM was employed to modify the QDs. Poloxamer 188 was used to improve the solubility and stability. The resultant PAMAM@QDs dispersed well in water. Fluorescence (FL) spectroscopic study showed that the FL intensity of the PAMAM@QDs was enhanced in the presence of p-fluorophenoxyacetic acid. Under optimal conditions, the enhanced FL intensity as a function of concentration matched very well in the range of 1 ~ 200 µg/mL of p-fluorophenoxyacetic acid, while the lower limits of detection were found to be 0.16 µg/mL. These results show that PAMAM@QDs is a promising luminescent probe for the detection of pesticides.