Fluorescent Cascade Research Articles

Fluorescence and colorimetric dual-mode multienzyme cascade nanoplatform based on CuNCs/FeMn-ZIF-8/PCN for detection of sarcosine.

It is critical to develop a highly efficient and sensitive method for detecting the biomarker sarcosine (SA) of prostate cancer due to its importance for men's health. In our work, a fluorescence (FL) and colorimetric dual-mode multienzyme cascade nanoplatform for SA detection was designed and constructed. CuNCs/FeMn-ZIF-8/PCN nanocomposites with high FL properties and peroxidase-like activity were successfully prepared by encapsulating copper nanoclusters (CuNCs) into FeMn-ZIF-8 and then loaded onto P-doped graphitic carbon nitride (PCN). Furthermore, the nanocomposites served as carriers for the immobilization of sarcosine oxidase (SOX) to construct a high-efficiency dual-mode multienzyme cascade nanoplatform CuNCs/SOX@FeMn-ZIF-8/PCN for the detection of SA. The intermediate H2O2 generated in the cascade caused the FL quenching of nanocomposites and the discoloration of 3,3',5,5'-tetramethylbenzidin. The linear ranges for SA detection in the dual-mode system were 1-100 μM (FL) and 1-200 μM (colorimetric), with detection limits of 0.34 and 0.59 μM, respectively. This nanoplatform exhibited notable repeatability, specificity, and stability, making it suitable for detecting sarcosine in real human urine samples. Therefore, this dual-mode multienzyme cascade nanoplatform would have a potential applicative prospect for detecting SA and other biomarkers in real clinical samples.

Reversible crowdedness of pH-responsive and host–guest active polymersomes: Mimicking µm-sized cell structures

The structure–function characteristics of isolated artificial organelles (AOs) in protocells are mainly known, but there are few reports on clustered or aggregated AOs. To imitate µm-sized complex and heterogeneous cell structures, approaches are needed that enable reversible changes in the aggregation state of colloidal structures in response to chemical, biological, and external stimuli. To construct adaptive organelle-like or cell-like reorganization characteristics, we present an advanced crosslinking strategy to fabricate clustered polymersomes as a platform based on host–guest interactions between azobenzene-containing polymersomes (Azo-Psomes) and a β-cyclodextrin-modified polymer (β-CD polymer) as a crosslinker. First, the reversible (dis)assembly of clustered Azo-Psomes is carried out by the alternating input of crosslinker and adamantane-PEG3000 as a decrosslinker. Moreover, cluster size dependence is demonstrated by environmental pH. These offer the controlled fabrication of various homogeneous and heterogeneous Azo-Psomes structures, including the size regulation and visualization of clustered AOs through a fluorescent enzymatic cascade reaction. Finally, a temperature-sensitive crosslinking agent with β-CD units can promote the coaggregation of Azo-Psomes mediated by temperature changes. Overall, these (co–)clustered Azo-Psomes and their successful transformation in AOs may provide new features for modelling biological systems for eukaryotic cells and systems biology.

The resonance fluorescence cascade of a laser-excited two-level atom

The cascade of fluorescence photons by a two-level atom excited by coherent laser light is reviewed. The discussion emphasizes the random nature of resonance fluorescence and uses the distribution of delays between two successively emitted photons as the primary characterization of the process. Other characterizations such as photon counting and photon correlation are deduced.

Triazine pyridinium derivative supramolecular cascade assembly extended FRET for two-photon NIR targeted cell imaging.

A triazine pyridinium derivative (TAZpy) was encapsulated into the cavity of a cucurbit[7]uril and further assembled with sulfonatocalix[4]-arene, hyaluronic acid and commercial dyes, which not only achieved fluorescence cascade enhancement and an effective FRET process based on macrocyclic confinement, but was also applied in two-photon NIR targeted cell imaging.

The Fluorescent Enzyme Cascade Detects Low Abundance Protein Modifications Suitable for the Assembly of Functionally Annotated Modificatome Databases.

Pathophysiological functions of proteins critically depend on both their chemical composition, including post-translational modifications, and their three-dimensional structure, commonly referred to as structure-activity relationship. Current analytical methods, like capillary electrophoresis or mass spectrometry, suffer from limitations, such as the detection of unexpected modifications at low abundance and their insensitivity to conformational changes. Building on previous enzyme-based analytical methods, we here introduce a fluorescence-based enzyme cascade (fEC), which can detect diverse chemical and conformational variations in protein samples and assemble them into digital databases. Together with complementary analytical methods an automated fEC analysis established unique modification-function relationships, which can be expanded to a proteome-wide scale, i. e. a functionally annotated modificatome. The fEC offers diverse applications, including hypersensitive biomarker detection in complex samples.

Monte Carlo method solution of the broadband fluorescent radiative transfer equation considering fluorescent cascade.

Altering wavelength via fluorescent particles is used in various applications. The solution of the broadband radiative transfer equation (RTE) for absorbing and anisotropically scattering a fluorescent medium is presented in this study considering fluorescent cascade, along with a Monte-Carlo-method-based solution of the equation. The path-length-based Monte Carlo method, the dual-stage method, and its modified version, the multi-stage method, which are used for solving the RTE in a fluorescent medium for biomedical and lighting applications, are not capable of accurately solving the broadband RTE with fluorescent cascade. Therefore, a collision-based Monte Carlo method is applied to overcome the limitations of these approaches. An accuracy comparison with the alternative methods is presented along with the flow chart and codes of the proposed method.

In Vivo Imaging of Hypoxia Associated with Inflammatory Bowel Disease by a Cytoplasmic Protein-Powered Fluorescence Cascade Amplifier.

Accurate and sensitive imaging of hypoxia associated with inflammatory bowel disease (IBD) is significant for the precise diagnosis and treatment of this disease, but it remains a challenge for traditional hypoxia-activatable fluorescence probes because of a more moderate hypoxic state during IBD than under other pathological conditions. To address this issue, herein, we designed a hypoxia-activatable and cytoplasmic protein-powered fluorescence cascade amplifier, named HCFA, to image hypoxia associated with IBD in vivo. In our design, a 4-aminobenzoic acid (azo)-modified mesoporous silica nanoparticle (MSN) was used as a container to load black hole quencher 2 (BHQ2) and cytoplasmic protein-binding squarylium dye (SQ); then, the β-cyclodextrin polymer (β-CDP) combined with azo through a host-guest interaction to form HCFA. Upon passive stagnation in the inflamed tissue of IBD, the azo band would be cleaved under a hypoxic microenvironment, and SQ was released to activate the fluorescence of HCFA. Moreover, the unconstrained SQ can bind with cytoplasmic protein to exhibit drastic fluorescence intensity enhancement, realizing the fluorescence signal amplification for imaging of hypoxia. When one takes advantage of the large load capacity of MSN and the unique property of SQ, HCFA can sense oxygen levels in the range of 0% to 10%. Meanwhile, the fluorescence imaging results demonstrate that HCFA can sensitively distinguish different levels of cellular hypoxia and monitor the variations of hypoxia in vivo, highlighting HCFA as a promising tool for the detection of hypoxia associated with IBD.

Engineering of ATP-Powered Photosensitizer for Targeted Recycling Activatable Imaging of MicroRNA and Controllable Cascade Amplification Photodynamic Therapy.

Owing to the low abundance of microRNAs (miRNAs) in living tumor cells, the development of intracellular cancer-relevant miRNA stimuli-activatable photosensitizers (PSs) for accurate imaging and efficient photodynamic therapy (PDT) of tumors in vivo is extremely challenging. Herein, we engineered a tumor targeting and intracellular trace miRNA-activatable nanophotosensitizer Y-motif/FA@HyNP on the basis of an endogenous ATP-powered strand-displacement cascade amplification strategy, which was prepared by assembly of a quencher BHQ2-labeled Y-motif DNA structure (containing ATP-binding aptamer and target miRNA-binding complementary sequence) on the surface of folate (FA) and amine-functionalized hybrid micellar nanoparticles. We showed that the fluorescence emissions at both 555 and 627 nm were effectively inhibited due to BHQ2 in Y-motif/FA@HyNPs, leading to negligible PDT efficacy. Once Y-motif/FA@HyNPs were selectively internalized into tumor cells via FA-receptor-mediated endocytosis, the intracellular trace target miRNA initiated the dissociation of the BHQ2-terminated sequences from Y-motif/FA@HyNPs by means of abundant endogenous ATP-powered strand-displacement reactions, causing remarkable fluorescence enhancement and cascade amplification PDT. The activated dual-color fluorescence emissions at 555 and 627 nm were feasible to achieve real-time, highly sensitive, and specific imaging of trace target miRNA in living tumor cells. With the guidance of excellent imaging in living mice, Y-motif/FA@HyNPs exhibited the precise and efficient PDT of tumors as well as insignificant side effects in vivo. This work revealed the great potential of using an integration of receptor-mediated cell uptake and target-triggered recycling cascade amplification strategy to design early cancer-relevant stimuli-activatable PSs for both fluorescence imaging and PDT ablation of tumors in vivo, which could effectively facilitate the timeliness and precision of early cancer diagnosis and therapy.

A fluorescent cascade amplification method for sensitive detection of Salmonella based on magnetic Fe3O4 nanoparticles and hybridization chain reaction

We developed a fluorescence sensor based on hybridization chain reaction (HCR) and Fe3O4 nanoparticles for the detection of Salmonella, common pathogen related to numerous food poisoning outbreaks. In this study, a pair of specific primers based on invA of Salmonella was designed and used in asymmetric polymerase chain reaction (aPCR) to produce a long target single-stranded DNA (ssDNA) that was captured by biotin-probe labeled magnetic beads (MBs) forming the MBs-ssDNA. The target ssDNA triggered HCR amplification with the two carboxyfluorescein (FAM) labeled DNA hairpin reporter probes (H1-FAM, H2-FAM) that enabled the measurements of the fluorescence intensities from the resulting ssDNA∼(H1-FAM ∼H2-FAM)n. The Fe3O4 nanoparticles were used to isolate the target ssDNA which hybridized with the reporter probes and led to decreased background signal that resulted in a detection limit of 7.4 × 101 CFU/mL in buffer and 6.9 × 102 CFU/g in spiked lettuce. The method developed also showed excellent selectivity over several common foodborne pathogens. The Fe3O4 nanoparticles capture coupled with fluorescence detection sensor showed promise for the detection of Salmonella.

Entangled photons from single atoms and molecules

The first two-photon entanglement experiment performed 50 years ago by Kocher and Commins (KC) provided isolated pairs of entangled photons from an atomic three-state fluorescence cascade. In view of questioning of Bell’s theorem, data from these experiments are re-analyzed and shown sufficiently precise to confirm quantum mechanical and dismiss semi-classical theory without need for Bell’s inequalities. Polarization photon correlation anisotropy (A) is useful: A is near unity as predicted quantum mechanically and well above the semi-classic range, 0⩽A⩽1/2. Although yet to be found, one may envisage a three-state molecule emitting entangled photon pairs, in analogy with the KC atomic system. Antibunching in fluorescence from single molecules in matrix and entangled photons from quantum dots promise it be possible. Molecules can have advantages to parametric down-conversion as the latter photon distribution is Poissonian and unsuitable for producing isolated pairs of entangled photons. Analytical molecular applications of entangled light are also envisaged.

3D visualization of additive occlusion and tunable full-spectrum fluorescence in calcite.

From biomineralization to synthesis, organic additives provide an effective means of controlling crystallization processes. There is growing evidence that these additives are often occluded within the crystal lattice. This promises an elegant means of creating nanocomposites and tuning physical properties. Here we use the incorporation of sulfonated fluorescent dyes to gain new understanding of additive occlusion in calcite (CaCO3), and to link morphological changes to occlusion mechanisms. We demonstrate that these additives are incorporated within specific zones, as defined by the growth conditions, and show how occlusion can govern changes in crystal shape. Fluorescence spectroscopy and lifetime imaging microscopy also show that the dyes experience unique local environments within different zones. Our strategy is then extended to simultaneously incorporate mixtures of dyes, whose fluorescence cascade creates calcite nanoparticles that fluoresce white. This offers a simple strategy for generating biocompatible and stable fluorescent nanoparticles whose output can be tuned as required.

Ligand-centred fluorescence and electronic relaxation cascade at vibrational time scales in transition-metal complexes.

Using femtosecond-resolved photoluminescence up-conversion, we report the observation of the fluorescence of the high-lying ligand-centered (LC) electronic state upon 266 nm excitation of an iridium complex, Ir(ppy)3, with a lifetime of 70 ± 10 fs. It is accompanied by a simultaneous emission of all lower-lying electronic states, except the lowest triplet metal-to-ligand charge-transfer ((3)MLCT) state that shows a rise on the same time scale. Thus, we observe the departure, the intermediate steps, and the arrival of the relaxation cascade spanning ∼1.6 eV from the (1)LC state to the lowest (3)MLCT state, which then yields the long-lived luminescence of the molecule. This represents the first measurement of the total relaxation time over an entire cascade of electronic states in a polyatomic molecule. We find that the relaxation cascade proceeds in ≤10 fs, which is faster than some of the highest-frequency modes of the system. We invoke the participation of the latter modes in conical intersections and their overdamping to low-frequency intramolecular modes. On the basis of literature, we also conclude that this behavior is not specific to transition-metal complexes but also applies to organic molecules.

Solving the radiative transfer equation with a mathematical particle method.

We solve the radiative transfer equation (RTE) using a recently proposed mathematical particle method, originally developed for solving general functional equations. We show that, in the case of the RTE, it gives several advantages, such as handling arbitrary boundary conditions and phase functions and avoiding numerical instability in strongly forward-scattering media. We also solve the RTE, including fluorescence, and an example is shown with a fluorescence cascade where light is absorbed and emitted in several steps. We show that the evaluated particle method is straightforward to implement, which is in contrast with many traditional RTE solvers, but a potential drawback is the tuning of the method parameters.

Control of emission peaks of X-ray phosphor using dye-silica nanoparticles

Simple method yielding new emission peaks to the X-ray phosphor and its mechanism were investigated using concentrated-dye-molecule-doped silica nanoparticles (dye-silica nanoparticles) and X-ray phosphors. The dye-silica nanoparticles were coated on the sheet of the X-ray phosphor using 20 wt% polyvinyl alcohol solution. The fluorescence of the dye-silica nanoparticle coated onto X-ray phosphor was successfully observed by X-ray irradiation. The fluorescent cascade from the X-ray-irradiated phosphor could be used in the excitation of the dye-silica nanoparticles by coating on the calcium tungstate (X-ray phosphor). The observed new fluorescence was based on the extent of the overlapping of wavelengths between the emission of the X-ray phosphor and the excitation of the dye-silica nanoparticles. The fluorescent peak of the calcium tungstate as the X-ray phosphor shifted from 434 to 425 nm. The dye-silica particle–calcium tungstate hybrid material showed new emission peaks from 543 to 601 nm due to the addition of fluorescein, rhodamine red-X, or cy5. The new emission peaks changed by the content of dye and the size of the silica nanoparticles. The fluorescence intensity ratio of the new fluorescence peaks at 543 nm against that of the X-ray phosphor depended on the fluorescent cascade from the X-ray-irradiated X-ray phosphor. It can closely match the overall emission of the phosphorous intensity screens to the sensitivity of the film allowing lower dose X-rays to obtain the same image clarity.

Fluorescent Cascade and Direct Assays for Characterization of RAF Signaling Pathway Inhibitors

RAF kinases are part of a conserved signaling pathway that impacts cell growth, differentiation, and survival, and RAF pathway dysregulation is an attractive target for therapeutic intervention. We describe two homogeneous fluorescent formats that distinguish RAF pathway inhibitors from direct RAF kinase inhibitors, using B-RAF, B-RAF V599E, and C-RAF. A Förster-resonance energy transfer (FRET) based method was used to develop RAF and MEK cascade assays as well as a direct ERK kinase assay. This method uses a peptide substrate, that is terminally labeled with a FRET-pair of fluorophores, and that is more sensitive to proteolysis relative to the phosphorylated peptide. A second time-resolved FRET-based assay using fluorescently labeled MEK substrate was used to detect direct inhibitors of RAF kinase activity. The cascade assays detect compounds that interact with activated and unactivated kinases within the recapitulated RAF pathway, and the direct assays isolate the point of action for an inhibitor.

Design and Synthesis of Alkyne-Substituted Boron in Dipyrromethene Frameworks

4,4-Difluoro-3a,4a-diaza-4-bora-s-indacenes, commonly named boron dipyrromethene dyes (F-Bodipy), were utilized as precusors to 4,4-diethynyl-3a,4a-diaza-4-bora-s-indacenes. Symmetrically substituted 4,4-diethynyl-3a,4a-diaza-4-bora-s-inda­cenes were synthesized using substituted ethynyllithium reagents, while the use of a 1:1 mixture of two different ethynyl Grignard reagents gave dissymmetric products. Using (trialkylsilyl)ethynyllithiums (alkyl = Me, Et) gave 4,4-bis[(trialkylsilyl)ethynyl]-3a,4a-diaza-4-bora-s-indacenes, that in the case of the trimethylsilyl derivative could be deprotected to give the mono- or dideprotected product. The dideprotected 4,4-diethynyl-3a,4a-diaza-4-bora-s-indacene was coupled with bromopyrene, -anthracene, or -terpyridine to give the corresponding 4,4-bis(arylethynyl)-3a,4a-diaza-4-bora-s-indacenes. The monodeprotected 4-ethynyl-4-[(trimethylsil­yl)ethynyl]-3a,4a-diaza-4-bora-s-indacenes were either coupled with bromopyrene and then deprotected and oxidatively homocoupled or directly oxidatively homocoupled to give the 1,4-bis(3a,4a-diaza-4-bora-s-indacen-4-yl)butadiyne products. The synthesis of disymmetric 3a,4a-diaza-4-bora-s-indacenes allows the design of fluorescent dyes bearing various functionalities including an oxazol­ine ring. All the dyes synthesized are strongly fluorescent and redox active and cascade energy transfer is evidenced when excited in the pyrene fragment.

Branching ratios in the radiative decay of helium doubly excited states

The doubly excited singlet states of He below the $N=2$ threshold may decay by autoionization or fluorescence. In the fluorescence decay channel, most decay cascades consist of emission of three photons, of which the first is a VUV photon, the second is in or near the visible range, and the last is another VUV photon. We have studied the fluorescence channel decay dynamics of the $(2,{0}_{\mathrm{n}})$, $(2,{1}_{\mathrm{n}})$ and $(2,\ensuremath{-}{1}_{\mathrm{n}})$ $^{1}\mathrm{P}$, $n=3--7$, states by wavelength dispersed photon-induced fluorescence spectroscopy. We have detected the photons in the second step of the cascade and determined the branching ratios for the strongest lines in this step. From these data we are able to calculate the branching ratios of the first step in the cascade. The results are in good agreement with calculations of the main decay channels of the higher resonances, but about 20--30 % lower, and so we are able to describe quantitatively the whole fluorescence cascade of the above-mentioned doubly excited states.

Fluorescence of low-lying doubly photoexcited states in helium

In this work we investigate fluorescence decay of low-lying $(n<~8)$ resonances with odd parity and total angular momentum $J=1$ below the $N=2$ threshold of helium. After discussing the photoabsorption cross section for creation of such states, we estimate the probability for the primary fluorescence transition to $1snl$ states below the first ionization threshold. The subsequent fluorescence cascade decay brings helium into the ground state or into one of the two long lived $1s2s{}^{1,3}S$ states. We discuss the time scale of the cascade and combine all the calculated transition probabilities to determine branching ratios for singlet and triplet metastable production. Finally, we estimate the angular distribution of the emitted uv photons and perform a comparison with the recent experimental data of Penent et al. [Phys. Rev. Lett. 86, 2758 (2001)].

Spatial density profiles of the N2 metastable stateA 3Σu+, the B 3Πg and C 3Πustates in an asymmetric rf discharge

A laser-induced fluorescence technique, in combination withoptical emission spectroscopy, was applied to investigate the spatialdistribution of the number densities of neutral species in an asymmetriclow-pressure 27.12 MHz discharge in pure nitrogen. The primary targets ofthis investigation were the three nitrogen triplet states C 3Πu, B 3Πg and the metastable A 3Σu+ each intheir lowest vibrational levels v = 0,1,2 and additionally v = 8 ofA 3Σu+ which can be populated very efficiently in theplasma sheath. The absolute number densities of all these states weredetermined in a Rayleigh scattering experiment, the results of which havebeen published elsewhere (Krames B, Glenewinkel-Meyer T and Meichsner J2001 J. Appl. Phys. 89 3115). The spatial profiles as afunction of the discharge parameters were studied by varying the gaspressure between 10 and 100 Pa and the effective rf voltage up to 260 V.These profiles of the different molecular states vary markedly in theplasma bulk as well as in the sheath near the powered electrode. A model,which includes the production, diffusion and quenching rates ofcontributing species and also fluorescence cascade processes, explainsthese varieties. A comparison with the experimental data at 50 Pa confirmsthe results of the model calculations.

Transdermal macromolecular delivery: real-time visualization of iontophoretic and chemically enhanced transport using two-photon excitation microscopy.

To investigate the transdermal delivery of a model macromolecule by passive and iontophoretic means following pretreatment with C12-penetration enhancers and to visualise transport across human stratum corneum (SC) in real time. Transport studies of dextran, labelled with fluorescent Cascade Blue (D-CB: M(R) = 3 kDa) across human stratum corneum, were conducted during passive and iontophoretic modes of delivery following pretreatment with either dodecyltrimethylammonium bromide (DTAB), sodium dodecyl sulphate (SDS) or Azone. Size-exclusion chromatography was used to assess maintenance of dextran structural integrity throughout experimental lifetime. Two-photon excitation microscopy was employed to visualise real-time dextran transport during current application. The positively charged C12-enhancer DTAB elevated passive D-CB steady-state flux (J(ss)) and was the only enhancer to do so above control during iontophoresis. The negatively charged SDS had the least effect during both stages. On-line macromolecular transport was visualised, indicating both inter- and intra-cellular pathways across SC during current application. No transport was visible across untreated SC during passive transport. Use of a positively charged enhancer may improve J(ss) of anionic macromolecular penetrants during passive and iontophoretic delivery. On-line visualisation of iontophoresis across SC was possible and can provide mechanistic insight into SC transport pathways.