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Despite the potential of photodynamic therapy (PDT), its comprehensive use in cancer treatment has not been achieved because of the nondegradable risks of photosensitizing drugs and limits of light penetration and instrumentation. Here, we present bioluminescence (BL)-induced proteinaceous PDT (BLiP-PDT), through the combination of luciferase and a reactive oxygen species (ROS)-generating protein (Luc-RGP), which is self-luminescent and degradable. After exposure to coelenterazine-h as a substrate for luciferase without external light irradiation, Luc-RGP fused with a small lead peptide-induced breast cancer cell death through the generation of BL-sensitive ROS in the plasma membrane. Even with extremely low light energy, BLiP-PDT exhibited targeted effects in primary breast cancer cells from patients and in in vivo tumor xenograft mouse models. These findings suggest that BLiP-PDT is immediately useful as a promising theranostic approach against various cancers.
The use of fluorescent proteins has revolutionized our understanding of biological processes. However, the requirement for external illumination precludes their universal application to the study of biological processes in all tissues. Although light can be created by chemiluminescence, light emission from existing chemiluminescent probes is too weak to use this imaging modality in situations when fluorescence cannot be used. Here we report the development of the brightest luminescent protein to date, Nano-lantern, which is a chimera of enhanced Renilla luciferase and Venus, a fluorescent protein with high bioluminescence resonance energy transfer efficiency. Nano-lantern allows real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence and sensitive tumour detection in freely moving unshaved mice. We also create functional indicators based on Nano-lantern that can image Ca(2+), cyclic adenosine monophosphate and adenosine 5'-triphosphate dynamics in environments where the use of fluorescent indicators is not feasible. These luminescent proteins allow visualization of biological phenomena at previously unseen single-cell, organ and whole-body level in animals and plants.
Nanoparticles have been recognized as promising tools for targeted drug-delivery and protein therapeutics. However, the mechanisms of protein-nanoparticle interaction and the dynamics underlying the binding process are poorly understood. Here, we present a general methodology for the characterization of protein-nanoparticle interaction on a molecular level. To this end we combined biophysical techniques including nuclear magnetic resonance (NMR), circular dichroism (CD), resonance energy transfer (RET) and surface plasmon resonance (SPR). Particularly, we analyzed molecular mechanisms and dynamics of the interaction of CaF2 nanoparticles with the prototypical calcium sensor calmodulin (CaM). We observed the transient formation of an intermediate encounter complex involving the structural region linking the two domains. Specific interaction of CaM with CaF2 NPs is driven by the N-terminal EF-hands, which seem to recognize Ca2+ on the surface of the nanoparticle. We conclude that CaF2 NP-CaM interaction is fully compatible with potential applications in nanomedicine. Overall, the methods presented in this work can be extended to other systems and may be useful to quantitatively characterize structural and dynamic features of protein-NP interactions with important implications for nanomedicine and nano-biotechnology.
Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage's ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics. Other techniques, such as classic adsorption assays, are required to define a phage's "adsorptive host range." The issue shared amongst all adsorption assays, however, is that they rely on the use of a complete bacteriophage and thus inherently describe when all adsorption-specific machinery is working together to facilitate bacterial surface adsorption. These techniques cannot be used to examine individual interactions between a singular set of a phage's adsorptive machinery (like long tail fibers, short tail fibers, tail spikes, etc.) and that protein's targeted bacterial surface receptor. To address this gap in knowledge we have developed a high-throughput, filtration-based, bacterial binding assay that can evaluate the adsorptive capability of an individual set of a phage's adsorption machinery. In this manuscript, we used a fusion protein comprised of an N-terminal bioluminescent tag translationally fused to T4's long tail fiber binding tip (gp37) to evaluate and quantify gp37's relative adsorptive strength against the Escherichia coli reference collection (ECOR) panel of 72 Escherichia coli isolates. Gp37 could adsorb to 61 of the 72 ECOR strains (85%) but coliphage T4 only formed plaques on 8 of the 72 strains (11%). Overlaying these two datasets, we were able to identify ECOR strains incompatible with T4 due to failed adsorption, and strains T4 can adsorb to but is thwarted in replication at a step post-adsorption. While this manuscript only demonstrates our assay's ability to characterize adsorptive capabilities of phage tail fibers, our assay could feasibly be modified to evaluate other adsorption-specific phage proteins.
Collagen has long been one of the top targets for biomimetic design due to its superior structural and functional properties. Significant progress has been achieved to construct self-assembling peptides to mimic the fibrous nanostructure of native collagen, while it is still very demanding to fabricate peptide assemblies that can recapitulate both structural and biofunctional features of collagen. Herein, collagen-like peptides have been synthesized to contain negatively charged amino acids as the binding groups of lanthanide ions and the integrin-binding motif GFOGER. The simultaneous inclusion of negatively charged amino acids in the middle as well as at both terminals drives the peptides to self-assemble to form well-ordered nanofibers with distinct periodic banding patterns specifically mediated by lanthanide ions. The aggregation tendency and the morphology of the final assembled materials for the peptides are modulated in a pH-cooperative manner, which well mimics the pH-dependent fibrillogenesis of Type I collagen. The utilization of lanthanide ions in the system not only offers a convenient external stimulus but also functionalizes assembled materials with excellent luminescent features. Most notably, the lanthanide-triggered peptide assembled nanomaterials possess good cell adhesion properties, which resemble the biological function of collagen. This peptide-Ln3+ system provides a facile and potent strategy to generate nanofibers that mimic both the structural and functional properties of natural collagen. These novel pH-responsive, luminescent, and biofunctional collagen mimetic nanofibers open fascinating opportunities in the development of improved functional biomaterials in tissue engineering, drug delivery, and medical diagnostics.
This paper reports on the synthesis and characterization of La2O3:Eu3+ luminescent aerogels fabricated by the sol-gel method and the supercritical drying technique. The % mol concentration of the Eu3+ ion was varied to study the effects on the luminescent properties of the aerogels. XRD and TEM analysis showed that the La2O3:Eu3+ aerogels exhibited a semi-crystalline behavior regardless of whether the concentration of europium was increased. SEM micrographs revealed a porous structure in the aerogels, which were composed of quasi-spherical nanoparticles that were interconnected and formed coral-shaped agglomerates. Photoluminescence spectroscopy characterization showed that the aerogels had an infrared emission located at λ = 793 nm, and the maximum photoluminescence emission intensity was observed for the aerogel with 50% Eu3+. The results demonstrate that, without heat treatment, it is possible to manufacture luminescent aerogels of rare-earth oxides that can be used in opto-electronic devices.
In this paper, we exploit the perspective of luminescent Si nanowires (NWs) in the growing field of commercial biosensing nanodevices for the selective recognition of proteins and pathogen genomes. We fabricated quantum confined fractal arrays of Si NWs with room temperature emission at 700 nm obtained by thin-film, metal-assisted, chemical etching with high production output at low cost. The fascinating optical features arising from multiple scattering and weak localization of light promote the use of Si NWs as optical biosensing platforms with high sensitivity and selectivity. In this work, label-free Si NW optical sensors are surface modified for the selective detection of C-reactive protein through antigen-gene interaction. In this case, we report the lowest limit of detection (LOD) of 1.6 fM, fostering the flexibility of different dynamic ranges for detection either in saliva or for serum analyses. By varying the NW surface functionalization with the specific antigen, the luminescence quenching of NW biosensors is used to measure the hepatitis B-virus pathogen genome without PCR-amplification, with an LOD of about 20 copies in real samples or blood matrix. The promising results show that NW optical biosensors can detect and isolate extracellular vesicles (EV) marked with CD81 protein with unprecedented sensitivity (LOD 2 × 105 sEV/mL), thus enabling their measurement even in a small amount of blastocoel fluid.
Two new neutral fac-[Re(CO)₃(phen)L] compounds (1,2), with phen = 1,10-phenanthroline and L = O₂C(CH₂)₅CH₃ or O₂C(CH₂)₄C≡CH, were synthetized in one-pot procedures from fac-[Re(CO)₃(phen)Cl] and the corresponding carboxylic acids, and were fully characterized by IR and UV-Vis absorption spectroscopy, ¹H- and 13C-NMR, mass spectrometry and X-ray crystallography. The compounds, which display orange luminescence, were used as probes for living cancer HeLa cell staining. Confocal microscopy revealed accumulation of both dyes in mitochondria. To investigate the mechanism of mitochondrial staining, a new non-emissive compound, fac-[Re(CO)₃(phen)L], with L = O₂C(CH₂)₃((C₅H₅)Fe(C₅H₄), i.e., containing a ferrocenyl moiety, was synthetized and characterized (3). 3 shows the same mitochondrial accumulation pattern as 1 and 2. Emission of 3 can only be possible when ferrocene-containing ligand dissociates from the metal center to produce a species containing the luminescent fac-[Re(CO)₃(phen)]⁺ core. The release of ligands from the Re center was verified in vitro through the conjugation with model proteins. These findings suggest that the mitochondria accumulation of compounds 1-3 is due to the formation of luminescent fac-[Re(CO)₃(phen)]⁺ products, which react with cellular matrix molecules giving secondary products and are uptaken into the negatively charged mitochondrial membranes. Thus, reported compounds feature a rare dissociation-driven mechanism of action with great potential for biological applications.
This article presents the results of research related to the development of cellulose man-made fibers with luminescent properties. The fibers were obtained from regenerated cellulose with the use of the N-Methylmorpholine-N-Oxide (NMMO) method for lyocell (Tencel) fiber formation. The method is named after the cellulose solvent (NMMO) used to obtain the spinning solution. Fibers are formed by the dry-wet spinning method. Due to the characteristic of the lyocell process, the fibers were easily modified to achieve luminescent properties with star-shaped organic compound poly((9-carbazolyl)methylthiirane) (KMT). Fibers were examined on their mechanical parameters with the use of Zwick Z2.5/TN1S tensile testing machine, and the results show the influence of the KMT concentration in the fiber matrix on mechanical parameters of the fibers. The study also attempted to determine the concentration of the modifier in the fibers with the use of UV-VIS Spectrofluorometer JASCO. The luminescent properties of fibers were estimated as well, using Jobin-Yvon spectrofluorometer FLUOROMAX-4, and the results are very promising as the fibers emit blue light in the range of visible light spectrum even for small concentrations of KMT (about 0.1 wt.%).
Efforts have been devoted to screening various prevalent diseases, such as severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19). Real-time polymerase chain reaction (RT-PCR), which is currently the most widely used, has high accuracy, but it requires several facilities and takes a relatively long time to check; so, new testing technology is necessary for a higher test efficiency. A chemiluminescence (CL) sensor is a relatively simple device and suitable as an alternative because it can detect very precise specimens. However, in measurements via CL, the quantitative formulation of reagents that cause color development is important. In the case of mixing using micropipettes, precise analysis is possible, but this technique is limited by uncontrollable errors or deviations in detection amounts. In addition, in using a microfluidic chip to increase field applicability, a syringe pump or other quantification injection tools are required, so problems must be overcome for practical use. Therefore, in this study, a microchip was designed and manufactured to supply a sample of a certain volume by simply blowing air and injecting a sample into the chamber. By utilizing the luminescence reaction of luminol, CuSO4 and H2O2 the performance of the prepared chip was confirmed, and the desired amount of the sample could be injected with a simple device with an error rate of 2% or less. For feasible applications, an experiment was performed to quantitatively analyze thrombin, a biomarker of heart disease. Results demonstrated that biomarkers could be more precisely detected using the proposed microchips than using micropipettes.
Multifunctional nanocomposites can combine multiple functions into a single nanosystem and thus have attracted extensive interest in various fields. The combination of magnetic and upconversion luminescent nanoparticles into one single nanoplatform, which have a good application in biomedical fields such as bio-magnetic separation, magnetic resonance imaging (MRI), and optical imaging, is highly desirable. Here we reported multifunctional nanocomposites by using hollow carbon sphere to integrate magnetic Fe3O4 and upconversion nanoparticles (UCNPs) into one nanosystem. The as-prepared UCNPs/Fe3O4@h-C have near-infrared (NIR) luminescence under 980 nm excitation and superparamagnetism. In addition, since the carbon layer can absorb NIR light and transfer it into heat with high efficiency, the nanocomposites can realize photo thermal (PT), upconversion luminescence (UCL) and MRI tri-mode imaging. The UCNPs/Fe3O4@h-C might be further utilized as a potential theranostic agent, including its in-depth monitoring through luminescent imaging and MRI diagnosis, as well as its direct use in tumors as a photothermal therapy (PTT) agent.
Bioluminescence is a powerful biological signal that scientists have repurposed as a reporter for gene expression in plants and animals. However, there are downsides associated with the need to provide a substrate to these reporters, including its high cost and non-uniform tissue penetration. In this work we reconstitute a fungal bioluminescence pathway (FBP) in planta using a composable toolbox of parts. We demonstrate that the FBP can create luminescence across various tissues in a broad range of plants without external substrate addition. We also show how our toolbox can be used to deploy the FBP in planta to build auto-luminescent reporters for the study of gene-expression and hormone fluxes. A low-cost imaging platform for gene expression profiling is also described. These experiments lay the groundwork for future construction of programmable auto-luminescent plant traits, such as light driven plant-pollinator interactions or light emitting plant-based sensors.
Nanoclusters (NCs) bridge the gap between atoms and nanomaterials in not only dimension but also physicochemical properties. Precise chemical and structural control, as well as clear understanding of formation mechanisms, have been important to fabricate NCs with high performance in optoelectronics, catalysis, nanoalloys, and energy conversion and harvesting. Herein, taking advantage of the close chemical properties of Ln3+ (Ln = Eu, Nd, Sm, Gd, etc.) and Gd3+-Eu3+ energy transfer ion-pair, we report a clickable LnF3 nanoparticle assembly strategy allowing reliable fabrication of diversely structured NCs, including single-component, dimeric, core-shelled/core-shell-shelled, and reversely core-shelled/core-shell-shelled, particularly with synergized optical functionalities. Moreover, the purposely-embedded dual luminescent probes offer great superiority for in situ and precise tracking of tiny structural variations and energy transfer pathways within complex nanoarchitectures.
Afterglow luminescence is an internal luminescence pathway that occurs after photo-excitation, holds great promise for non-background molecular imaging in vivo, but suffer from poor quantitative ability owing to luminescent attenuation over time. Moreover, the inert structure and insufficient reactive sites of current afterglow materials make it hard to design activatable afterglow probes for specific detection. Here, we report a ratiometric afterglow luminescent nanoplatform to customize various activatable afterglow probes for reliable quantification and molecular imaging of specific analytes, such as NO, ONOO- or pH. Notably, these afterglow probes can not only address the attenuation of afterglow intensity and eliminate the interference of factors (e.g., laser power, irradiation time, and exposure time), but also significantly improve the imaging reliability in vivo and signal-to-background ratios (~1200-fold), both of which enable more reliable quantitative analysis in biological systems. Moreover, as a proof-of-concept, we successfully design an NO-responsive ratiometric afterglow nanoprobe, RAN1. This nanoprobe can monitor the fluctuations of intratumoral NO, as a biomarker of macrophage polarization, making it possible to real-time dynamically evaluate the degree cancer immunotherapy, which provides a reliable parameter to predict the immunotherapeutic effect.
Herein we report on a novel inorganic peroxidase-mimicking nanocatalyst activated under blue LED photoirradiation. A novel flash-pyrolysis method has been developed for the generation of strong blue photoluminescence (PL) centers attributed to silicon and carbon-based sites within a mesoporous SBA-15 silica nanorod platform. The type of centers and their PL response can be controlled by varying the flash thermal treatment conditions. By tailoring the operating conditions the system can be driven towards the preferential generation of carbon-based luminescent centers, with or without the simultaneous generation of silicon-based centers. The properties and the nature of these luminescent centers within the mesoporous nanorods have been thoroughly corroborated by a battery of characterization techniques including fluorescence spectroscopy, X-ray photoelectron spectroscopy (XPS) and electron energy loss spectroscopy (EELS) at the local level of the structures combined with scanning transmission electron microscopy (STEM) imaging. In addition, these luminescent mesoporous nanorods have been successfully tested as robust photocatalysts able to display peroxidase-like activity and indirect glucose sensing in a wider range of pH conditions compared to the natural enzyme, especially when carbogenic dots and oxygen-deficient silica centers are simultaneously present in the structure.
In this study, we developed a highly stable polymeric vesicle using a nanosilica-armor membrane to achieve a sustainable colorimetric/luminescent response. The silica armor can be grown directly as ~ 5 nm spherical nanoparticles on the surface of the diacetylene (DA) vesicle with liposomal structure. This can be accomplished via the modified Stöber reaction in pure water on a layer of amine linkers deposited on the vesicles. Once formed, the structural stability of the DA vesicles dramatically increased and remained so even in a dried powder form that could be stored for a period of approximately 6 months. Then, redispersed in water, the armored vesicles did not agglomerate because of the electric charge of the silica armor. After polymerization, the polydiacetylene (PDA) vesicles maintained an average of 87.4% their sensing capabilities compared to unstored vesicles. Furthermore, the silica membrane thickness can be controlled by reiteration of the electrostatic layer-by-layer approach and the direct hydrolysis of silica. As the number of silica armor membranes increases, the passage of the stimuli passing through the membranes becomes longer. Consequently, three layers of silica armor gave the PDA vesicles size-selective recognition to filter out external stimuli. These discoveries are expected to have large-scale effects in the chemo- and biosensor fields by applying protective layers to organic nanomaterials.
NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies.
Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) can be used to separate proteins based mainly on their size such as in denaturing gels. Different staining methods have been reported to observe proteins in the gel matrix, where the most used dyes are generally anionic. Anionic dyes allow for interactions with protonated amino acids, retaining the dye in the proteins. Fluorescent staining is an alternative technique considered to be sensitive, safe, and versatile. Some anionic complexes based on d6 transition metals have been used for this purpose, where cationic dyes have been less explored in this context. In this work, we synthesized and characterized a new monocationic rhenium complex fac-[Re(CO)3(deeb)B2]+ (where deeb is 4,4'-bis(ethoxycarbonyl)-2,2'-bpy and B2 is 2,4-di-tert-butyl-6-(3H-imidazo[4,5-c]pyridine-2-yl)phenol). We carried out a structural characterization of this complex by MS+, FTIR, 1H NMR, D2O exchange, and HHCOSY. Moreover, we carried out UV-Vis, luminescence, and cyclic voltammetry experiments to understand the effect of ligands on the complex's electronic structure. We also performed relativistic theoretical calculations using the B3LYP/TZ2P level of theory and R-TDDFT within a dielectric continuum model (COSMO) to better understand electronic transitions and optical properties. We finally assessed the potential of fac-[Re(CO)3(deeb)B2]+ (as well as the precursor fac-Re(CO)3(deeb)Br and the free ligand B2) to stain proteins separated by SDS-PAGE. We found that only fac-[Re(CO)3(deeb)B2]+ proved viable to be directly used as a luminescent dye for proteins, presumably due to its interaction with negatively charged residues in proteins and by weak interactions provided by B2. In addition, fac-[Re(CO)3(deeb)B2]+ seems to interact preferentially with proteins and not with the gel matrix despite the presence of sodium dodecyl sulfate (SDS). In future applications, these alternative cationic complexes might be used alone or in combination with more traditional anionic compounds to generate counterion dye stains to improve the process.
The evolution of bioluminescence has prompted scientific attention to illuminate phylogenetic relationships of luminescent beetles. However, genomic resources are virtually lacking in rhagophthalmids (Rhagophthalmidae) and their related firefly beetles lampyrids (Lampyridae). Here, we employed the Illumina Hiseq 2000 platform and sequenced the whole-body transcriptomes of the four luminescent beetles: one rhagophthalmid (Rhagophthalmus sp.) and three fireflies (Asymmetricata circumdata, Aquatica ficta, and Pyrocoelia pectoralis). We obtained 55.4, 43.4, 38.6, and 36.7 million clean reads for the four species, respectively. All reads were assembled into contigs from which unigenes were derived. All unigenes were annotated by publicly available databases, and a total of 4325 orthologous genes were identified. Using multiple phylogenetic approaches, our transcriptome data confirmed the distinctiveness of Rhagophthalmidae from Lampyridae, which was also supported by our mitogenome analysis using three newly determined mitogenome sequences and 12 previously published ones. Together, this study is the first report of whole transcriptome sequencing data in Rhagophthalmidae and Lampyridae species, representing a valuable genomic resource for studying the origin and evolution of some remarkable traits in these beetles such as bioluminescence. Moreover, our transcriptome and mitogenome data provide useful phylogenetic information that could be of importance in future studies of phylogenetic inference.
We present a single-step, room-temperature synthesis of fluorescent organosilica nanobeads (FOS NBs). The FOS NBs were synthesized under aqueous conditions using (3-aminopropyl)triethoxysilane (APTES) as the silicon source in the presence of L-ascorbic acid (L-AA). In the APTES/L-AA/water ternary phase, the hydrolysis and condensation reaction of APTES occurred under acidic conditions to form spherical FOS NBs with an average diameter of 426.8 nm. FOS NBs exhibit excellent colloidal stability in aqueous media. The formation of FOS NBs was complete within a 10 min reaction time, which indicates potential for large-scale mass-production synthesis of luminescent colloidal NBs. The FOS NBs exhibited blue photoluminescence (PL) under UV excitation in the absence of an additional high temperature calcination process or with the incorporation of any fluorophores. This phenomenon is attributed to the presence of carbon-containing defects, which act as luminescent centers formed by the reaction between amino groups in the APTES and L-ascorbic acid reductant. Finally, the results of a cytotoxicity test and cellular uptake experiments revealed that the FOS NBs showed potential as optical contrast agents for bioimaging.
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