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While an array of taxa are capable of producing fluorescent pigments, fluorescence in mammals is a novel and poorly understood phenomenon. A first step towards understanding the potential adaptive functions of fluorescence in mammals is to develop an understanding of fluorescent compounds, or fluorophores, that are present in fluorescent tissue. Here we use Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) of flying squirrel fur known to fluoresce under ultraviolet (UV) light to identify potentially fluorescent compounds in squirrel fur. All of the potentially fluorescent compounds we identified were either present in non-fluorescent fur or were not present in all species of fluorescent flying squirrel. Therefore, we suggest that the compounds responsible for fluorescence in flying squirrels may also be present in non-fluorescent mammal fur. Some currently unexplained factor likely leads to excitation of fluorophores in flying squirrel fur. A recently suggested hypothesis that fluorescence in mammals is widely caused by porphyrins is consistent with our findings., Competing Interests: The authors have declared that no competing interest exist.

A single-step and rapid chromatographic method-based purification of Gracilaria corticata (J. Agardh) R-phycoerythrin (R-PE) was attained using polyacrylamide gel electrophoresis (PAGE) technique without affecting structural integrity. The purified R-PE had a characteristic UV-Vis spectrum with three absorbance maxima at 496, 535, and 565 nm, and fluorescence at 575 nm. R-PE was obtained with a purity index of 4.2 and a recovery yield of 44.3%. SDS-PAGE analysis exhibited three sub-units i.e., 18, 21, and 31 kDa, which corresponds to α, β, and γ, respectively. This report's purification process was considered less time-consuming and could be efficiently applied to purify phycobiliproteins. The purified R-PE showed optimal stability up to 6 h at pH 7.0 when exposed to light (3000 lx), while the temperature at which the maximum stability was retained was at 20 °C. The cellular imaging property of R-PE was effectively implemented to evaluate its credentials without affecting the cell proliferation of Vero and Hep-2 cell lines with the higher IC50 concentrations in vitro. Under fluorescence microscopy and flow cytometry analysis, purified R-PE displayed the characteristic affinity towards cell imaging functions in preliminary in vitro studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sorption ( i.e. , adsorption and absorption) of small-molecule compounds to polydimethylsiloxane (PDMS) is a widely acknowledged phenomenon. However, studies to date have largely been conducted under atypical conditions for microfluidic applications (lack of perfusion, lack of biological fluids, etc.), especially considering biological studies such as organs-on-chips where small-molecule sorption poses the largest concern. Here, we present an in-depth study of small-molecule sorption under relevant conditions for microphysiological systems, focusing on a standard geometry for biological barrier studies that find application in pharmacokinetics. We specifically assess the sorption of a broad compound panel including 15 neuropsychopharmaca at in vivo concentration levels. We consider devices constructed from PDMS as well as two material alternatives (off-stoichiometry thiol-ene-epoxy, or tape/polycarbonate laminates). Moreover, we study the much neglected impact of peristaltic pump tubing, an essential component of the recirculating systems required to achieve in vivo -like perfusion shear stresses. We find that the choice of the device material does not have a significant impact on the sorption behavior in our barrier-on-chip-type system. Our PDMS observations in particular suggest that excessive compound sorption observed in prior studies is not sufficiently described by compound hydrophobicity or other suggested predictors. Critically, we show that sorption by peristaltic tubing, including the commonly utilized PharMed BPT, dominates over device sorption even on an area-normalized basis, let alone at the typically much larger tubing surface areas. Our findings highlight the importance of validating compound dosages in organ-on-chip studies, as well as the need for considering tubing materials with equal or higher care than device materials.

The field of extracellular vesicles (EVs) is relatively new and the methods for EV isolation and quantification are still maturing. For example, there is no consensus on how to separate free stain from labelled EVs. Here we report a comparison of the recovery of labelled EVs following separation from free stain using ultracentrifugation, diafiltration with different devices and a charged size exclusion chromatography column. Of the methods we tested, the charged size exclusion column provided the greatest recovery of labelled EVs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Due to the redox properties closely related to numerous physiological and pathological processes, biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), have received considerable attention in biological science. On account of the important physiological roles of these biothiols, it is of profound significance to develop sensitive and selective detection of biothiols to understand their biological profiles. In this work, we reported an efficient fluorescent probe, PHPQ-SH , for detecting biothiols in vitro and vivo, based on the phenothiazine-HPQ skeleton, with DNBS (2,4-dinitrobenzenesulfonate) as the response unit. Probe PHPQ-SH exhibited brilliant sensing performances toward thiols, including a large Stokes shift (138 nm), excellent sensitivity (for GSH, LOD = 18.3 nM), remarkable fluorescence enhancement (163-fold), low cytotoxicity, rapid response (8 min), and extraordinary selectivity. Finally, the probe PHPQ-SH illustrated herein was capable of responding and visualizing biothiols in MCF-7 cells and zebrafish.

Sensors that can quickly and accurately diagnose and monitor human health are currently at the forefront of medical research. Single walled carbon nanotube (SWNT) based optical biosensors are a growing area of research due to the high spatiotemporal resolution of their near infrared fluorescence leading to high tissue transparency and unparalleled sensitivity to analytes of interest. Unfortunately, due to the functionalization requirements of SWNT-based sensors, there are concerns surrounding accumulation and persistence when applied in vivo. In this study, we developed protocols to extract and quantify SWNT from complex solutions and show an 89% sensor retention by hydrogel platforms when implanted in vivo. Animal tissues of interest were also extracted and probed for SWNT content showing no accumulation (0.03 mg l -1 SWNT detection limit). The methods developed in this paper demonstrated one avenue for applying SWNT sensors in vivo without concern for accumulation.

Zinc (Zn 2+ ) is an essential micronutrient that is required for a wide variety of cellular processes. Tools and methods have been instrumental in revealing the myriad roles of Zn 2+ in cells. This review highlights recent developments fluorescent sensors to measure the labile Zn 2+ pool, chelators to manipulate Zn 2+ availability, and fluorescent tools and proteomics approaches for monitoring Zn 2+ -binding proteins in cells. Finally, we close with some highlights on the role of Zn 2+ in regulating cell function and in cell signaling., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Rhizoclosmatium globosum genome encodes three rhodopsin-guanylyl cyclases (RGCs), which are predicted to facilitate visual orientation of the fungal zoospores. Here, we show that RGC1 and RGC2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR). RGC1/2 utilize conventional green or blue-light-sensitive rhodopsins (λ max  = 550 and 480 nm, respectively), with short-lived signaling states, responsible for light-activation of the enzyme. The bistable NeoR is photoswitchable between a near-infrared-sensitive (NIR, λ max  = 690 nm) highly fluorescent state (Q F  = 0.2) and a UV-sensitive non-fluorescent state, thereby modulating the activity by NIR pre-illumination. No other rhodopsin has been reported so far to be functional as a heterooligomer, or as having such a long wavelength absorption or high fluorescence yield. Site-specific mutagenesis and hybrid quantum mechanics/molecular mechanics simulations support the idea that the unusual photochemical properties result from the rigidity of the retinal chromophore and a unique counterion triad composed of two glutamic and one aspartic acids. These findings substantially expand our understanding of the natural potential and limitations of spectral tuning in rhodopsin photoreceptors.

A novel combination of CE-based separation techniques was used for the precise fractionation of ionic compounds from impurities. The combination of on-capillary concentration and separation using transient isotachophoresis, with multiple injections and a two-point detection system provided higher efficiency, and accuracy at a microliter-scale injection volume, than when CE was individually used for purification. In this paper, we present successful applications of the CE fractionation techniques for the purification of fluorescein, fluorescein-4-isothiocyanate, two fluorescent metal ion probes, and a fluorescein-modified DNA aptamer. The purity of the isolated fluorescent probes ranged from 95 to 99%. Such high purity could not be achieved using chromatographic purification techniques. With relatively low dilution factors of 6-9, the purified probe solutions were practical for use as purified stock solutions. In addition, the fluorescein-modified DNA aptamer purified by our method was successfully used in a thrombin binding assay. The method developed was useful for the purification of anionic fluorescent reagents to be of ultratrace analytical grade for use with CE-LIF., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fluorescent dyes are of immense interest due to their nontrivial applications in cell imaging to observe different micro organelles and their functions. We have discovered organic Beet-root extracted water soluble fluorescent (BREWSF) dye as an efficient pigment for effective cell imaging. The extraction of this pigment from beet root and its preservation is very easy and cost effective. The BREWSF dye is characterized by IR, UV-Vis, NMR, mass, fluorescence spectroscopy and flowcytometry. By applying this dye on different types of human cells we obtained very good results in fluorescence microscopy, flowcytometry and confocal cell imaging. We also have noticed that this dye takes very less internalization time to produce very good fluorescence image and flowcytometry results of cells and also remains stable at different pH levels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

We designed and synthesized a water-soluble near-infrared (NIR) fluorescent probe with the recognition unit of the cyanine-like structure and acrylate group. Through an aromatic ring nucleophilic substitution reaction based on sulfhydryl moiety, an off-on fluorescence response toward cysteine (Cys) was realized. The probe exhibited excellent spectral performance with an emission wavelength of 720nm and a detection limit of 0.20μM. The spectral properties, selectivity and anti-interference performance of the probe were systematically investigated. Density functional theory (DFT) calculations were conducted to clarify the luminescence mechanism of the probe. Furthermore, the probe was successfully applied to the detection of free Cys in human serum and the NIR imaging of endogenous Cys in living cells. Thus, the probe has a promising application prospect in clinical diagnosis and fluorescence imaging., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We have 3D printed and fabricated micro free-flow electrophoresis (µFFE) devices in acrylonitrile butadiene styrene (ABS) that exhibit minimal surface adsorption without requiring additional surface coatings or specialized buffer additives. 2D, nano LC-micro free flow electrophoresis (2D nLC × µFFE) separations were used to assess both spatial and temporal broadening as peaks eluted through the separation channel. Minimal broadening due to wall adsorption was observed in either the spatial or temporal dimensions during separations of rhodamine 110, rhodamine 123, and fluorescein. Surface adsorption was observed in separations of Chromeo P503 labeled myoglobin and cytochrome c but was significantly reduced compared to previously reported glass devices. Peak widths of < 30 s were observed for both proteins. For comparison, Chromeo P503 labeled myoglobin and cytochrome c adsorb strongly to the surface of glass µFFE devices resulting in peak widths >20 min. A 2D nLC × µFFE separation of a Chromeo P503 labeled tryptic digest of BSA was performed to demonstrate the high peak capacity possible due to the low surface adsorption in the 3D printed ABS devices, even in the absence of surface coatings or buffer additives., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A molecular imaging probe to fluorescently image the β-site of the amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) and cathepsin D (CatD) enzymes associated with Alzheimer's disease (AD) was designed and synthesized. This imaging probe was built upon iron oxide nanoparticles (cross-linked dextran iron oxide nanoparticles, or CLIO). Peptide substrates containing a terminal near-infrared fluorochrome (fluorophore emitting at 775 nm for CatD or fluorophore emitting at 669 nm for BACE1) were conjugated to the CLIO nanoparticles. The CatD substrate contained a phenylalanine-phenylalanine cleavage site more specific to CatD than BACE1. The BACE1 substrate contained the sequence surrounding the leucine-asparagine cleavage site of the BACE1 found in the Swedish mutation of APP, which is more specific to BACE1 than CatD. These fluorescently-labeled peptide substrates were then conjugated to the nanoparticle. The nanoparticle probes were purified by gel filtration, and their fluorescence intensities were determined using a fluorescence plate reader. The CatD peptide substrate demonstrated a 15.5-fold increase in fluorescence when incubated with purified CatD enzyme, and the BACE1 substrate exhibited a 31.5-fold increase in fluorescence when incubated with purified BACE1 enzyme. Probe specificity was also demonstrated in the human H4 neuroglioma cells and the H4 cells stably transfected with BACE1 in which the probe monitored enzymatic cleavage. In the H4 and H4-BACE1 cells, BACE1 and active CatD activity increased, an occurrence that was reflected in enzyme expression levels as determined by immunoblotting. These results demonstrate the applicability of this probe for detecting potential Alzheimer's enzyme biomarkers.

A novel AIE probe with ESIPT characteristics, myricetin, has been easily purified from vine tea for reversible, selective and sensitive targeting of O2˙- in turn-on mode. Its AIE nanocrystals exhibit large Stokes shift, high photostability, excellent biocompatibility, and low cytotoxicity for endogenous O2˙- detection and imaging in vitro and in vivo.

The effect of the counteranion of hexadecyltrimethylammonium salts on the physico-chemical properties of organoclays was investigated, using a selected natural clay mineral with a cation exchange capacity of 95 meq/100 g. The uptake amount of C 16 cations was dependent on the hexadecyltrimethylammonium (C 16 ) salt solution used, the organoclay prepared from C 16 Br salt solution exhibited a value of 1. 05 mmole/g higher than those prepared from C 16 Cl and C 16 OH salt solutions. The basal spacing of these organoclays was in the range of 1.81 nm to 2.10 nm, indicating a similar orientation of the intercalated surfactants, and could indicated that the excess amount of surfactants, above the cation exchange capacity of 0.95 meq/g could be adsorbed on the external surface of the clay mineral sheets. These organoclays were found to be stable in neutral, acidic, and basic media. The thermal stability of these organoclays was carried out using thermogravimetric analysis and in-situ X-ray diffraction (XRD) techniques. The decomposition of the surfactant occurred at a maximum temperature of 240 °C, accompanied with a decrease of the basal spacing value close to 1.42 nm. The application of these organoclays was investigated to remove an acidic dye, eosin. The removal amount was related to the initial used concentrations, the amount of the surfactants contents, and to the preheated temperatures of the organoclays. The removal was found to be endothermic process with a maximum amount of 55 mg of eosin/g of organoclay. The value decreased to 25 mg/g, when the intercalated surfactants were decomposed. The reuse of these organoclays was limited to four regeneration recycles with a reduction of 20 to 30%. However, noticeable reduction between 35% to 50% of the initial efficiency, was achieved after the fifth cycle, depending of the used organoclays.

In this initial research on feasibility, removal of unconjugated fluorescein isothiocyanate (FITC) after fluorescent labeling of human serum albumin (HSA) with electromembrane extraction (EME) was investigated for the first time. A 100 μL solution of 0.1 mg/mL HSA was fluorescently labeled with 0.01 mg/mL FITC in a molar ratio of 10:1 in an Eppendorf tube for 30 min under agitation and absence of light. Then the labeled solution was transferred to a 96-well EME with 3 μL 0.1% (w/w) Aliquat 336 in 1-octanol as the supported liquid membrane (SLM) and 200 μL 10 mM NaOH as waste solution. EME was performed for 10 min with a voltage of 50 V, with the anode in the waste solution and at 900 rpm agitation. Negatively charged and unconjugated FITC was extracted electrokinetically into the SLM and to the waste solution. Analysis of purified samples, by Taylor dispersion analysis (TDA), showed a 92% removal of unconjugated FITC (FITC clearance: 92%, RSD: 3%), while 79% of the HSA/FITC complex remained in the sample (protein retention: 79%, RSD: 18%). Conserved functionality of the HSA/FITC complex after EME was proven by a binding affinity study with anti-HSA using flow induced dispersion analysis (FIDA). In this real sample, the dissociation constant (K d ) and hydrodynamic radius of the complex were determined to be 0.8 μM and 5.87 nm, respectively, which was in concordance with previously reported values.

Canthin-6-one alkaloids, which are present in plants of the genus Simaba, are natural compounds that are capable of acting as fluorescent probes. However, the chemical composition and fluorescent properties of most species of this genus have not been analyzed. The objective of this study was to characterize the fluorescent properties of an extract of S. bahiensis and identify the chemical entities responsible for these properties. In addition, the cell-labeling properties of the fluorescent dye from A and of the isolated compounds were characterized by confocal fluorescence microscopy and flow cytometry. One quassinoid and three fluorescent alkaloids were isolated from S. bahiensis, all compounds were identified by using NMR spectroscopy and high-resolution mass spectrometry. Staining experiments and HPLC-FL analysis shown that canthin-6-one alkaloids are the main green fluorescent compounds in the analyzed dyes. All compounds evaluated showed a cytoplasmic marker with a residence time of 24 h. The present study is the first to describe the presence of canthin-6-one alkaloids in S. bahiensis, in addition to demonstrating promising cell-labeling properties of fluorescent compounds from S. bahiensis with broad emission wavelengths., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fluorescent small molecules are important tools in many aspects of modern biology. A two-stage evaluation process involving fluorescence screening and live-cell imaging was developed to facilitate the identification of new fluorescent probes from extracts housed within the NCI Natural Products Repository. To this end, over 2000 extracts and prefractionated samples were examined, including an extract from the marine crinoid Pterometra venusta. An optically guided evaluation involving stepwise fluorescence screening and live-cell imaging was developed to enable the isolation of fluorescent natural products. These efforts resulted in the isolation of six hydroxyanthraquinone compounds, three of which are new natural products. These purified metabolites were examined for their potential as cellular imaging probes, and they demonstrate that natural product libraries can be a good source of new fluorescent agents.

Small-molecule fluorescent probes are powerful and ubiquitous tools for measuring the concentration and distribution of analytes in living cells. However, accurate characterization of these analytes requires rigorous evaluation of cell-to-cell heterogeneity in fluorescence intensities and intracellular distribution of probes. In this study, we perform a parallel and systematic comparison of two small-molecule fluorescent vesicular Zn 2+ probes, FluoZin-3 AM and SpiroZin2, to evaluate each probe for measurement of vesicular Zn 2+ pools. Our results reveal that SpiroZin2 is a specific lysosomal vesicular Zn 2+ probe and affords uniform measurement of resting Zn 2+ levels at the single cell level with proper calibration. In contrast, FluoZin-3 AM produces highly variable fluorescence intensities and non-specifically localizes in the cytosol and multiple vesicular compartments. We further applied SpiroZin2 to lactating mouse mammary epithelial cells and detected a transient increase of lysosomal free Zn 2+ at 24-hour after lactation hormone treatment, which implies that lysosomes play a role in the regulation of Zn 2+ homeostasis during lactation. This study demonstrates the need for critical characterization of small-molecule fluorescent probes to define the concentration and localization of analytes in different cell populations, and reveals SpiroZin2 to be capable of reporting diverse perturbations to lysosomal Zn 2+ .

Extracellular vesicles (EVs) are nanosized vesicular structures released by cells to communicate with one another. The growing interest in the (patho)physiological function and potential pharmaceutical application of these vesicles is accompanied by a vast number of new research groups entering this research field and a plethora of different protocols to separate EVs from non-vesicular components. This lack of uniformity often generates conflicting or difficult-to-compare results. Here we provide a comparative analysis of different EV isolation strategies, discussing the purity of the final isolate and highlighting the importance of purity on downstream experimental readouts. First, we show that ultracentrifugation (UC) of B16F10 melanoma cell-derived conditioned medium co-purifies proteins or protein complexes with nuclease activity. Such contaminants should be taken into account when aiming to apply EVs as delivery carriers for exogenous nucleic acids. Second, three commonly used purification strategies (i.e. precipitation, UC and density-gradient centrifugation) were evaluated for their ability to remove non-incorporated fluorescent dye (i.e. the lipophilic PKH67 dye), important when probing EV interactions with cells. For both types of impurities, endogenous and exogenous, density gradient purification outperforms the other evaluated methods. Overall, these results demonstrate that the implementation of stringent purification protocols and adequate controls is of pivotal importance to draw reliable conclusions from downstream experiments performed with EV isolates., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Molecularly imprinted covalent organic polymers were constructed by an imine-linking reaction between 1,3,5-triformylphloroglucinol and 2,6-diaminopyridine and used for the selective solid-phase extraction of benzoxazole fluorescent whitening agents from food samples. Binding experiments showed that imprinting sites on molecularly imprinted polymers had higher selectivity for targets compared with those of the corresponding non-imprinted polymers. Parameters affecting the solid-phase extraction procedure were examined. Under optimal conditions, actual samples were treated and the eluent was analyzed with high-performance liquid chromatography with diode-array detection. The results showed that the established method has a wide linearity, satisfactory detection limits and quantification limits, and acceptable recoveries. Thus, this developed method possesses the practical potential for the selective determination of benzoxazole fluorescent whitening agents in complex food samples., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

With an increased use of color dye in textile industries and elevated fabrics output, more scientific studies and technology developments are needed to effectively treat wastewater containing dyes. However, better understanding of the interactions between dyes and suspended solids is a necessity to advance such developments. In this study the interactions between rhodamine 6G (R6G), a cationic dye, and different types of clays minerals, commonly found in the wastewater sludge, were elucidated. The uptake of R6G on the clay minerals was attributed to cation exchange on the external surfaces of non-swelling clays, and at both external and interlayer spaces for swelling clays. In the interlayer of montmorillonite, the R6G molecules form monolayer and bilayer configurations under low and high uptake levels. The significant amounts of R6G uptake indicate that clay minerals are good sorbents for the removal of cationic dyes from water. And the R6G could be readily removed in wastewater treatment by adding small amount of clays and flocculated the clays out., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Magnetic chicken bone biochar (MCB) was fabricated and characterised. The specific surface area, magnetisation value and pH pzc of the MCB were found to be 328 m 2  g -1 , 64.7 emu/g and 8.3 respectively. The adsorptive performance of MCB for rhodamine B dye (RB) and tetracycline (TC) removal in a single and two-stage stirred adsorber (TSA) was evaluated. The TSA reduced the pressure drops, mass transfer resistances, and fouling of the adsorbent. 63.0 g MCB is required to remove 75% of RB and TC in a single-stage system within 12 h. However, the optimised TSA confirmed that 33.2 g of MCB is needed to achieve 96% removal of TC and 22.2 g for RB within 180 min of 100 mgL -1 effluent solutions. The sorption was suitably described by the Freundlich mechanism. Based on the comparative performance, the MCB is considered a viable efficient and magnetically separable alternative adsorbent., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Previously, we reported a method for facile purification of oligonucleotides labeled with hydrophobic dyes, based on the solubility difference between the hydrophilic DNA and unreacted dye. Here, we present a new purification method applicable to any dye regardless of its hydrophobicity. We exploited the population shift of a fluorescent dye in a low-pH aqueous solution from its anionic form toward its neutral form. When the pH of an aqueous solution containing dye-labeled DNA and unreacted free dye is lowered, and the solution is mixed with a hydrophobic organic solvent (butanol), the neutral free dye is preferentially dissolved in the organic phase, leaving behind the hydrophilic dye-labeled DNA in the aqueous phase. We experimentally verified that our new method results in high yields of dye-labeled oligonucleotides and the efficient removal of free dye.

The synthesis of a small collection of novel bile acid-bisphosphonate (BA-BP) conjugates as potential drug candidates is reported. The disclosed methodology relied on the installation of azide and thiol functionalities at the head and tail positions, respectively, of the BA scaffold and its subsequent decoration by orthogonal click reactions (copper-catalyzed azide-alkyne cycloaddition, thiol-ene or thiol-yne coupling) to introduce BP units and a fluorophore. Because of the troublesome isolation of the target conjugates by standard procedures, the methodology culminated with the functionalization of the BA scaffold with a light fluorous tag to rapidly and efficiently purify intermediates and final products by fluorous solid-phase extraction.

The fluorescence quenchers BHQ1 and BHQ2 can be modified by trace amounts of ammonium persulfate, used for initiating gel polymerization, in electrophoretic purification of TaqMan probes using a denaturing polyacrylamide gel. The case study of BHQ1 quencher has demonstrated that a Boyland-Sims reaction proceeds in the presence of ammonium persulfate to give the corresponding sulfate. The absorption maximum of the resulting quencher shifts to the short-wavelength region relative to the absorption maximum of the initial BHQ1. The TaqMan probe containing such a quencher is less efficient as compared with the probe carrying an unmodified BHQ1. The presence of fluorescein in TaqMan probe plays decisive role in this transformation: the quencher modification proceeds at a considerably lower rate when the fluorescein is absent or replaced with a rhodamine dye (for example, R6G). It is assumed that the observed reaction can take place in two ways-both in darkness and in the reaction of the quencher in an excited state due to energy transfer from the fluorophore irradiated by light.

The emission spectrum of widely used CyPet is pH-sensitive. In order to synthesize a pH-insensitive cyan fluorescent protein by rational design, we solved the crystal structures of CyPet under different pH conditions. The indole group of the CyPet chromophore adopts a cis-coplanar conformation in acidic and neutral conditions, while it converts to trans-coplanar under basic conditions. His148 and Glu222 play a vital role in this isomerization. The pH-sensitive chromophore isomerization and change in the emission spectrum can be explained by the coexistence of several different fluorescent states. We trap the chromophore in the trans conformation by A167I mutation (CyPet2), which also prevents the multiconformation of the seventh β-strand. CyPet2 exhibits an unchanged emission spectral shape as a function of pH., (© 2017 Federation of European Biochemical Societies.)

Fluorescent carbon dots, zero-dimensional nanomaterials with surface ligands, have been studied extensively over the past few years in biolabelling or fluorescence-based live cell assays. In the past, synthetic organic dyes have been used as cell tracking materials, but they have severe limitations; fluorescent carbon dots may pave the way to biolabelling and cell imaging. In this work, green fluorescent carbon dots have been synthesized from a green source, gram, without any sort of covalent or ionic modifications. These gram-derived carbon dots are unique with respect to synthetic commercial cell-tracking dyes as they are non-toxic, cell internalization occurs quickly, and they have excellent bioconjugation with bacterial cells. Our aim is to establish these carbon dots in a biolabelling assay with its other physicochemical features like the tunable luminescence property, high degree of water solubility and low toxicity, towards various environments (wide range of pH, high ionic strength). Our study introduces a new perspective on the commercialization of carbon dots as a potential alternative to synthetic organic dyes for fluorescence-based cell-labelling assays.

Simplified lab-on-a-chip techniques are desirable for quick and efficient detection of analytes of interest in the field. The following work involves the use of deterministic pillar arrays on the micro-scale as a platform to separate compounds, and the use of Ag colloid within the arrays as a source of increased signal via surface enhanced Raman spectroscopy (SERS). One problem traditionally seen with SERS surfaces containing Ag colloid is oxidation; however, our platforms are superhydrophobic, reducing the amount of oxidation taking place on the surface of the Ag colloid. This work includes the successful separation and SERS detection of a fluorescent dye compounds (resorufin and sulforhodamine 640), fluorescent anti-tumor drugs (Adriamycin and Daunomycin), and purine and pyrimidine bases (adenine, cytosine, guanine, hypoxanthine, and thymine)., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Herein, we report a convenient approach to purify water-soluble dihydrolipoic acid (DHLA)-capped Ag nanoclusters (Ag NCs) by pH-induced precipitation under acidic conditions. The fluorescence of Ag NCs could be completely recovered by re-dispersing the precipitate into a basic solution using DHLA and NaBH4 as stabilizing ligands and etching reagent. DHLA-Ag NCs-doped agarose hydrogels have been prepared to monitor pH with a wide range from 8.0 to 4.0. When pH decreased, the fluorescence of the hydrogels under a UV lamp decreased and completely disappeared after pH 5. The DHLA-Ag NCs-doped agarose hydrogels biosensor showed low cytotoxicity and long stability. Accordingly, a fluorescent pH sensor for bacterial monitoring has been employed based on the "OFF-ON" signal switch of the Ag NCs-agarose hydrogel., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A novel ultra high performance liquid chromatography with diode array detection method, based on the dispersive solid-phase extraction by using polymer weak anion exchange as the sorbent, was established for the simultaneous determination of fluorescent whitening agents 85, 28, 351, and 71 in facial mask. The amounts of polymer weak anion exchange, adsorption time, and volume of elution solvent in the dispersive solid-phase extraction technology were optimized, and the developed method was validated in terms of the method limit of detection, method limit of quantitation, linear range, recovery, accuracy, and precision. Results indicated that the standard curves were linear over the selected concentration ranges of 0.05-100 mg/L for four target analytes, with determination coefficients greater than 0.999. The method limits of quantitation of the target analytes were in the range of 0.6-2.8 mg/kg. Recoveries were calculated at the concentrations of 1.0-30 mg/kg spiked in negative samples and the values were between 89.3 and 102% with an RSD of 2.5-5.1% for intraday precision and 3.8-5.0% for interday precision. The method was successfully applied to analyze 20 facial mask samples and fluorescent whitening agent 85 was detected in one sample with the concentration of 4.7 mg/kg., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The packed nanofiber solid-phase extraction of crude extracts of a mass viscous sample is challenging because the interference and recalcitrant particulates in the sample may attach to the nanofiber and block the column, which leads to insufficient sample extraction. A novel concentric layered nanofiber solid-phase extraction (SPE) column using polystyrene-based electrospun nanofiber as the stationary phase has been employed for the pretreatment of mass viscous crude extracts. The layered column was fabricated by using untouched nanofiber with its natural morphology rather than hand-packing of spoiled fiber to the control packing density of the column. In the novel column, the SPE packed bed was divided into a multi-layer structure to provide uniform radial and axial packing and to part the mobile phase stream by the isolated layer with great superiority in aspects such as lower column pressure and faster elution speed. The feasibility and efficiency of the LFSPE column were then evaluated via determination of rhodamine B (RB) from spiked chili samples. Based on the LFSPE column, a linear spiked calibration curve in the range of 0.02-5 mg/kg was obtained. The limit of detection (LOD) and limit of quantification (LOQ) of the method were 0.001 and 0.004 mg/kg, respectively; recoveries at 0.1, 1, and 2 mg/kg (n = 3) were all up to 95 %; and the RSD values of inter-day and intra-day were all below 5 %. This novel LFSPE column overcame heterogeneous packing and exploited the wall effect in subtle ways, and exhibited great superiority by comparison with some existing methods. Graphical Abstract ᅟ.

In this paper, an efficient method for determination of total pyrene concentration in the biological samples including plasma, liver, spleen, lung and kidney of Sprague-Dawley rats were investigated and established using steady-state fluorescence method. Equilibrium dialysis method was applied to determine plasma protein binding rate of pyrene. The results illustrated that the protein binding rate depends on the concentration of pyrene in plasma. Extraction of pyrene in plasma was studied by using biomedical nanopartical which was prepared from synthesized associating polymer poly(ethylene glycol) end-capped by hexadecane. The Critical Micelle Concentration (CMC) of the polymeric micelle in aqueous solution was determined to equal 0.0063 mg/mL using 1-pyrenemethanol as a fluorescent probe. The distribution of free pyrene and pyrene loaded nanoparticals in blood were determined. The results showed that over 95% of the free pyrene was distributed into the erythrocyte, and the pyrene-loaded nanoparticles were less distributed in to the erythrocyte than free pyrene, but it was higher than 60%. This study provides an efficient method to detect pyrene in different tissues as well as an extraction method at the molecular level, which might contribute to the development of modern molecular diagnosis and identification in vivo.

Herein, we present a water dispersable, magnetic nanoparticle supported "click and release" system. The cleavable linker has been synthesized by using a strain-promoted copper-free "click" reagent to establish the specific link and a fluoride cleavable silane moiety for mild cleavage. Small organic molecules, azide-bearing dyes and functionalized enzymes have been bound to the magnetic particle and released in a bioorthogonal way.

The ion concentration polarization (ICP) phenomenon at micronanofluidic interfaces has been extensively utilized to preconcentrate low-abundance biological samples. Although preconcentration by ICP is robust, its multiphysics phenomenon does not permit a clear prediction of the preconcentration conditions and sites. Here, we present a new method for spatiotemporally defining preconcentration, which can generate target-condensed plugs in a very specific region (<100 μm) regardless of the operating conditions (time, applied voltage, ionic strength, and pH). In contrast to previous devices that use only ion depletion, this device uses merged ICP zones with opposite polarity, i.e., ion depletion and ion enrichment. In this regard, ICP is initiated between two line-patterned cation exchange membranes. When voltage is applied across two membranes, an ion depletion (enrichment) zone occurs on the anodic (cathodic) side of the membranes. Two ICP zones are then merged and confined between the membranes. Consequently, the preconcentration action is also confined between the membranes. We demonstrate that fluorescent dyes are always preconcentrated at the designated location at all lengths of operating time and at broad voltage (0.5-100 V), ionic strength (1-100 mM KCl), and pH (3.7-10.3) ranges. This device successfully condenses proteins up to 10000-fold in a specific region of the channel (100 × 50 × 10 μm(3)) in 10 min. This work not only characterizes the unique scientific phenomenon of ICP overlapping but also opens the possibility of integrating ICP preconcentrators into commercial analysis equipment, which requires a known, stationary preconcentration site.

Several types of dyes or fluorophores are used for the detection of interactions between drug carriers and cells, within biomedicine field. However, many of them have a certain level of toxicity and instability affecting their biological properties. Different studies have demonstrated that nanoparticles (NPs) have interesting properties that could be used to stabilize diverse biomolecules, including dyes. Here, we report the synthesis of a novel nanosystem by the functionalization of silica NPs using biocompounds extracted from Mexican tree "Palo azul" (Eysenhardtia polystachya) and APTES as a coupling agent. Particle size, electrical properties, and morphology of the novel nanosystem were analyzed. The extracted biocompounds presented fluorescence which prevails over time, even after nanosystem formation and apparent cellular internalization. These were detected using MCF-7 cells visualized by confocal laser-scanning microscopy (CLSM), finding that the nanosystem was able to internalize into cells and act as a fluorescent biomarker. By this method, our novel nanosystem opens the possibilities to obtain sensitive data in a noninvasive manner for biological applications, such as early-stage cancer diagnosis, drug delivery, and pathogen detection., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Glutathione (GSH) is a major endogenous antioxidant that has a central role in cellular defense against toxins and free radicals. This protocol describes the preparation of CPDSA, a cyanine-based near-infrared (NIR) fluorescent probe for the detection of GSH in cells and in vivo. CPDSA is prepared with high yield through a simple two-step process. The first step is to react commercially available IR-780 iodide with excess anhydrous piperazine in anhydrous N,N-dimethyl formamide at 85 °C to form cyanine-piperazine (CP). The second step is the sulfonylation of CP with dansyl chloride in anhydrous dichloromethane. CPDSA selectively detects GSH in cells, and it has been shown to not react with other biothiols such as cysteine (Cys) and homocysteine (Hcy). This probe can also be used to monitor the GSH level of mouse bone marrow-derived neutrophils (BMDNs). The preparation of probe CPDSA takes 2 d, and experiments in cells and mice take 12-13 d.

Ultrasmall polymer nanoparticles are rapidly gaining importance as nanocarriers for drugs and contrast agents. Here, a straightforward modular approach to efficiently loaded and stable sub-20-nm polymer particles is developed. In order to obtain ultrasmall polymer nanoparticles, we investigated the influence of one to two charged groups per polymer chain on the size of particles obtained by nanoprecipitation. Negatively charged carboxylate and sulfonate or positively charged trimethylammonium groups were introduced into the polymers poly(d,l-lactide-co-glycolide) (PLGA), polycaprolactone (PCL), and poly(methyl methacrylate) (PMMA). According to dynamic light scattering, atomic force and electron microscopy, the presence of one to two charged groups per polymer chain can strongly reduce the size of polymer nanoparticles made by nanoprecipitation. The particle size can be further decreased to less than 15 nm by decreasing the concentration of polymer in the solvent used for nanoprecipitation. We then show that even very small nanocarriers of 15 nm size preserve the capacity to encapsulate large amounts of ionic dyes with bulky counterions at efficiencies >90%, which generates polymer nanoparticles 10-fold brighter than quantum dots of the same size. Postmodification of their surface with the PEG containing amphiphiles Tween 80 and pluronic F-127 led to particles that were stable under physiological conditions and in the presence of 10% fetal bovine serum. This modular route could become a general method for the preparation of ultrasmall polymer nanoparticles as nanocarriers of contrast agents and drugs.

Ag3PO4/TiO2 nanotube (TNT) heterojunctions were fabricated via a facile in situ growth method. Hemispherical Ag3PO4 nanocrystals were uniformly grown on the TNT surface, and their size was confined to 5-10 nm. A joint area was distinctly observed between the Ag3PO4 nanocrystals and TNT, indicating the formation of a Ag3PO4/TNT heterojunction. Compared with pure Ag3PO4, the Ag3PO4/TNT heterojunction possesses more active sites, less bulk defects, more efficient electron-hole separation, as well as better dye adsorption properties, and thus exhibits a significantly elevated photocatalytic activity for Rhodamine B (RhB) degradation. The study of the reactive species demonstrates that the photocatalytic degradation of RhB over the Ag3PO4/TNT heterojunction is primarily driven by both photogenerated h(+) and ˙OH radicals. This easily-fabricated Ag3PO4/TNT heterojunction with promising photocatalytic activity may find potential applications in energy and environmental related areas.

Self-assembly of gold nanoparticles into one-dimensional (1D) nanostructures with finite primary units was achieved by introducing a thin salt (NaCl) solution layer into density gradient before centrifugation. The electrostatic interactions between Au nanoparticles would be affected and cause 1D assembly upon passing through the salt layer. A negatively charged polymer such as poly(acrylic acid) was used as an encapsulation/stabilization layer to help the formation of 1D Au assemblies, which were subsequently sorted according to unit numbers at succeeding separation zones. A centrifugal field was introduced as the external field to overcome the random Brownian motion of NPs and benefit the assembly effect. Such a facile "one-tube synthesis" approach couples assembly and separation in one centrifuge tube by centrifuging once. The method can be tuned by changing the concentration of interference salt layer, encapsulation layer, and centrifugation rate. Furthermore, positively charged fluorescent polymers such as perylenediimide-poly(N,N-diethylaminoethyl methacrylate) could encapsulate the assemblies to give tunable fluorescence properties., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

High-throughput, quantitative, and rapid microfluidic-based separations has been a long-sought goal for applications in proteomics, genomics, biomarker discovery, and clinical diagnostics. Using droplet-interfaced microchip electrophoresis (MCE) techniques, we have developed a novel parallel MCE platform, based on the concept of combining the Slipchip principle with a newly developed "Gelchip". The platform consists of two plastic plates, with droplet wells on one plate and separation channels with preloaded/cured gel in the other. A single relative movement of one plate enables generation and then loading of multiple sample droplets in parallel into the separation channels, allowing electrophoretic separation of biomolecules in the droplets in parallel and with high-throughput. As proof of concept, we demonstrated the separation of 30 sub-nL sample droplets containing fluorescent dyes or DNA fragments.

A novel visible light active and magnetically separable nanophotocatalyst, Ni0.5Zn0.5Fe2O4/Zn0.95Ni0.05O (denoted as NZF@Z), with varying amount of Ni0.5Zn0.5Fe2O4, has been synthesized by egg albumen assisted sol gel technique. The structural, optical, magnetic, and photocatalytic properties have been studied by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), fourier transform infrared spectroscopy (FTIR), UV-visible (UV-Vis) spectroscopy, and vibrating sample magnetometry (VSM) techniques. Powder XRD, TEM, FTIR and energy dispersive spectroscopic (EDS) analyses confirm coexistence of Ni0.5Zn0.5Fe2O4 and Zn0.95Ni0.05O phases in the catalyst. Crystallite sizes of Ni0.5Zn0.5Fe2O4 and Zn0.95Ni0.05O in pure phases and nanocomposites, estimated from Debye-Scherrer equation, are found to be around 15-25 nm. The estimated particle sizes from TEM and FESEM data are ∼(22±6) nm. The calculated energy band gaps, obtained by Tauc relation from UV-Vis absorption spectra, of Zn0.95Ni0.05O, 15%NZF@Z, 40%NZF@Z and 60%NZF@Z are 2.95, 2.72, 2.64, and 2.54 eV respectively. Magnetic measurements (field (H) dependent magnetization (M)) show all samples to be super-paramagnetic in nature and saturation magnetizations (Ms) decrease with decreasing ferrite content in the nanocomposites. These novel nanocomposites show excellent photocatalytic activities on Rhodamin Dye., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Low-cost paper-based assays are emerging as the platform for diagnostics worldwide. Paper does not, however, readily enable advanced functionality required for complex diagnostics, such as analyte concentration and controlled analyte transport. That is, after the initial wetting, no further analyte manipulation is possible. Here, we demonstrate active concentration and transport of analytes in fully wet paper-based assays by leveraging nanoporous material (mean pore diameter ≈ 4 nm) and ion concentration polarization. Two classes of devices are developed, an external stamp-like device with the nanoporous material separate from the paper-based assay, and an in-paper device patterned with the nanoporous material. Experimental results demonstrate up to 40-fold concentration of a fluorescent tracer in fully wet paper, and directional transport of the tracer over centimeters with efficiencies up to 96%. In-paper devices are applied to concentrate protein and colored dye, extending their limits of detection from ∼10 to ∼2 pmol/mL and from ∼40 to ∼10 μM, respectively. This approach is demonstrated in nitrocellulose membrane as well as paper, and the added cost of the nanoporous material is very low at ∼0.015 USD per device. The result is a major advance in analyte concentration and manipulation for the growing field of low-cost paper-based assays.

In this study, the efficiency of a novel copper containing ionic liquid based nanoporous organosilica (Cu@IL-ONO) and palladium nanoparticles loaded on activated carbon (Pd-NP-AC) for the removal of Eosin Y from aqueous solution was investigated. The Cu@IL-ONO was prepared by hydrolysis and co-condensation of tetramethoxysilane (TMOS) and 1,3-bis (trimethoxysilylpropyl) imidazolium chloride in the presence of surfactant template following immobilization of copper chloride dihydrate. These materials were characterized by nitrogen adsorption-desorption analysis and scanning electron microscopy (SEM) and subsequently used for the successful removal of Eosin Yellow (EY) from aqueous solution. The effects of pH, contact time, amount of adsorbents, initial dye concentration was optimized and set as following: 0.005g/50mL Cu@IL-ONO and 0.015g/50mL Ag-NP-AC at pH=2 for Cu@IL-ONO and pH=3 for Ag-NP-AC and contact time less than 14min. The experimental removal percentage data at various situations was fitted by conventional isotherm models like Langmuir, Freundlich, Tempkin and Dubinin-Radushkevich (D-R). Judgment based on linear regression coefficient (R(2)) and error analysis show high usability of the Langmuir isotherm for best explanation of experimental data with maximum monolayer adsorption capacities 286 and 250mgg(-1) at room temperatures for Cu@IL-ONO and Ag-NP-AC, respectively. Fitting the corresponding data of removal percentage at various experimental conditions shows the suitability of second order and interparticle diffusion model for interpretation of real data., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Polyelectrolyte complexes of the chitosan-chondroitin sulfate and chitosan-hyaluronate polycation-polyanion pairs were synthesized and characterized as potential dye adsorbents at different pH levels. Equilibrium isotherm analysis was applied to investigate the efficiency and the mechanism of the adsorption of fluorone dyes (fluorescein, eosin Y, erythrosin B) on the synthesized complexes. The inefficiency of the fluorescein adsorption was proved by two different quantitative spectroscopic methods. The adsorption isotherm for both eosin Y and erythrosin B was adequately described in terms of the Langmuir-Freundlich model. The observed room-temperature phosphorescence of the adsorbed erythrosin B was attributed to the surface inhomogeneity of the synthesized complexes. The revealed variation in the adsorption properties of fluorone dyes was related to the differences in their ionic forms as well as in their polarity and hydrophobicity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Using fluorescent and non-fluorescent recombinant proteins has proved to be a very successful technique for following postfertilization events, in both male and female pronuclei during the first cell cycle of sea urchin in vivo. Proteins and dyes are introduced by microinjection into the unfertilized egg, and their function can be monitored by fluorescence or confocal/two-photon (2P) and transmitted light microscopy after insemination. Here, we describe expression and purification of GFP/RFP-tagged proteins involved in regulation of DNA replication. We also explain the techniques used to introduce recombinant proteins and fluorescent tubulin into sea urchin eggs and embryos.

The most common method to determine the EOF in CE is to measure the migration time for a neutral marker. In this study, 12 compounds (three novel and some previously used) were investigated as EOF markers in aqueous and nonaqueous BGEs. In the aqueous buffer systems (ammonium acetate, sodium phosphate, and sodium borate) the evaluation included a wide pH range (2-12). Two BGEs contained chiral selectors (sulphated-β-CD, (-)-diketogulonic acid) and one that contained a micellar agent (SDS) were included in the study. The majority of the evaluated compounds were found to migrate with the EOF in the water-based BGEs and are thus useful as EOF markers. However, in the SDS-based BGE only four of the compounds (acetone, acrylamide, DMSO, and ethanol) were found to be applicable. In the nonaqueous BGEs 11 markers (acetone, acetophenone, acrylamide, anthracene, benzene, 4-(4-methoxybenzylamino)-7-nitro-2,1,3-benzoxadiazole, benzyl alcohol, 2,5-diphenyloxazole, ethanol, flavone, and mesityl oxide) seemed to be functional as EOF markers. Even though several of the evaluated compounds can be used as EOF markers in the investigated BGEs, the authors would recommend the use of acrylamide as a general marker for UV detection. Furthermore, the four fluorescent markers (of which three were novel) gave RSD values equal to the other markers and can be used for the determination of the EOF in CE or microchip CE with fluorescence detection., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Semiconductor quantum dots (QDs) are fluorescent nanoparticles that can be used for biological imaging. Because of their brightness and photostability, which is far superior to those of organic dyes and fluorescent proteins, they can be detected at the single-particle level over long periods of time using standard fluorescence microscopy techniques. QDs can be conjugated to biomolecules and then used to track the motion of these molecules. Commercial, soluble QDs are available either unconjugated or functionalized with specific biomolecules. In the latter case, biomolecules such as streptavidin, Protein A, or antibodies are attached to the QD surface. Free biomolecules are often present in the QD solution, and these can be detrimental for live-cell imaging or other fluorescence assays. It is thus desirable to purify the functionalized QDs from these contaminating free biomolecules using size-exclusion chromatography. This article describes a simple procedure for purifying functionalized QDs using MicroSpin SR-400 columns.

Particle separation is a crucial step in sample preparation processes. The preparation of low volume samples is especially important for clinical diagnosis and chemical analysis. The advantages of microfluidic techniques have lead them to become potential candidates for particle separation. However, existing microfluidic devices require external pumping sources and extensive geometric patterns to attain high separation efficiency, which is disadvantageous when handling low volume samples. This paper presents a low sample volume particle separation microfluidic device with low voltage electrokinetic pumping based on circular travelling-wave electroosmosis (TWEO). Computational numerical software was utilized to simulate two electrokinetic mechanisms: circular TWEO and dielectrophoresis (DEP). The circular TWEO shear flow generates a velocity gradient in the radial direction which causes a shear stress-induced force to drag particles into the center region of the device. In contrast, the non-parallel electrodes induce negative DEP forces which push polystyrene beads towards the peripheral regions; the magnitude of the DEP forces are dependent on the sizes of the polystyrene beads. We used particles of various sizes to experimentally prove the concept of particle separation. Our experiments show that 15 μm beads are dragged into the center region due to the shear stress-induced force, and 1 μm beads move towards the outer region because of the large negative DEP force. The results show a separation purity of 94.4% and 80.0% for 15 μm and 1 μm beads respectively. We further demonstrated particle isolation from a sample of containing a small proportion of 6 μm beads mixed with 1 μm beads at a concentration ratio of 1 : 300. Therefore, the innovative device developed in this paper provides a promising solution to allow particle separation in sample volumes as low as 50 nL.