Intracellular flow cytometric lipid analysis - a multiparametric system to assess distinct lipid classes in live cells.

The lipid content of mammalian cells varies greatly between cell type. Current methods for analysing lipid components of cells are technically challenging and destructive. Here, we report a facile, inexpensive method to identify lipid content - intracellular flow cytometric lipid analysis (IFCLA). Distinct lipid classes can be distinguished by Nile Blue fluorescence, Nile Red fluorescence or violet autofluorescence. Nile Blue is fluorescent in the presence of unsaturated fatty acids with a carbon chain length greater than 16. Cis-configured fatty acids induce greater Nile Blue fluorescence than their trans-configured counterparts. In contrast, Nile Red exhibits greatest fluorescence in the presence of cholesterol, cholesteryl esters, some triglycerides and phospholipids. Multiparametric spanning-tree progression analysis for density-normalized events (SPADE) analysis of hepatic cellular lipid distribution, including vitamin A autofluorescence, is presented. This flow cytometric system allows for the rapid, inexpensive and non-destructive identification of lipid content, and highlights the differences in lipid biology between cell types by imaging and flow cytometry.

Novel Benzo[a]phenoxazinium Chlorides Functionalized with Sulfonamide Groups as NIR Fluorescent Probes for Vacuole, Endoplasmic Reticulum, and Plasma Membrane Staining.

The demand for new fluorophores for different biological target imaging is increasing. Benzo[a]phenoxazine derivatives are fluorochromophores that show promising optical properties for bioimaging, namely fluorescent emission at the NIR of the visible region, where biological samples have minimal fluorescence emission. In this study, six new benzo[a]phenoxazinium chlorides possessing sulfonamide groups at 5-amino-positions were synthesized and their optical and biological properties were tested. Compared with previous probes evaluated using fluorescence microscopy, using different S. cerevisiae strains, these probes, with sulfonamide groups, stained the vacuole membrane and/or the perinuclear membrane of the endoplasmic reticulum with great specificity, with some fluorochromophores capable of even staining the plasma membrane. Thus, the addition of a sulfonamide group to the benzo[a]phenoxazinium core increases their specificity and attributes for the fluorescent labeling of cell applications and fractions, highlighting them as quite valid alternatives to commercially available dyes.

Targeting Lysosomes in Colorectal Cancer: Exploring the Anticancer Activity of a New Benzo[a]phenoxazine Derivative.

Colorectal cancer (CRC) has been ranked as one of the cancer types with a higher incidence and one of the most mortal. There are limited therapies available for CRC, which urges the finding of intracellular targets and the discovery of new drugs for innovative therapeutic approaches. In addition to the limited number of effective anticancer agents approved for use in humans, CRC resistance and secondary effects stemming from classical chemotherapy remain a major clinical problem, reinforcing the need for the development of novel drugs. In the recent years, the phenoxazines derivatives, Nile Blue analogues, have been shown to possess anticancer activity, which has created interest in exploring the potential of these compounds as anticancer drugs. In this context, we have synthetized and evaluated the anticancer activity of different benzo[a]phenoxazine derivatives for CRC therapy. Our results revealed that one particular compound, BaP1, displayed promising anticancer activity against CRC cells. We found that BaP1 is selective for CRC cells and reduces cell proliferation, cell survival, and cell migration. We observed that the compound is associated with reactive oxygen species (ROS) generation, accumulates in the lysosomes, and leads to lysosomal membrane permeabilization, cytosolic acidification, and apoptotic cell death. In vivo results using a chicken embryo choriollantoic membrane (CAM) assay showed that BaP1 inhibits tumor growth, angiogenesis, and tumor proliferation. These observations highlight that BaP1 as a very interesting agent to disturb and counteract the important roles of lysosomes in cancer and suggests BaP1 as a promising candidate to be exploited as new anticancer lysosomal-targeted agent, which uses lysosome membrane permeabilization (LMP) as a therapeutic approach in CRC.

An easy and rapid staining method for confocal microscopic observation and reconstruction of three-dimensional images of echinoderm larvae and juveniles.

The morphologies of the internal organs of echinoderm larvae and juveniles are difficult to study using conventional optical microscopes because of their structural complexity and opaqueness. This paper describes an easy and rapid protocol involving Nile blue staining followed by benzyl alcohol/benzyl benzoate (BABB) clearing to overcome this limitation. This method was developed for a three-dimensional (3D) analysis of the internal structures of advanced larvae and juveniles of echinoderms (the sea lily Metacrinus rotundus, the sea urchin Hemicentrotus pulcherrimus, and the sand dollar Scaphechinus mirabilis) and is suitable for obtaining serial optical images by confocal microscopy without the use of specific antibodies or special reagents for labeling. Nile blue is an easy-to-use stain that offers several advantages for confocal microscopy such as it can stain various tissues with strong fluorescent signals without substantial bleaching during observation. We found that the strong fluorescence signal of Nile blue quickly yielded clear high-resolution optical section images for 3D reconstruction. BABB clearing rendered opaque larvae highly transparent. The clearing procedure was also easy and quick. During the process, agarose embedding prior to staining and clearing was found to be critical for handling the samples of less than 500-µm length and stabilizing their orientations. To conclude, the protocol described is useful for performing a rapid and accurate 3D morphological analysis of echinoderm larvae and juveniles.

Histological validation of adipogenic differentiation potential of ASC on collagen-based 2D scaffolds.

Determination of the adipogenic potential and behavior of adipose-derived mesenchymal stem/stromal cells (ASCs) is particularly relevant for their potential clinical application in regenerative medicine, especially when regeneration is supported by biomaterials or scaffolds. Scaffolds need to be able to induce tissue repair and limit undesired adipogenic differentiation. Depending on the scaffold employed, determination of cell behavior may be hindered by material interference with staining, which will limit either cells identification or dye quantification. Collagen is a promising biomaterial in regenerative medicine, however, histological analysis of cells cultured on collagen-based scaffolds is challenging. Here we describe a new histological method based on iron hematoxylin combined with Oil red O (ORO) staining, for the determination of the adipogenic differentiation of ASCs cultivated on a collagen-based 2D scaffold. ASCs were seeded on collagen films or plastic, differentiated into adipocytes for 14 days, and then stained with either ORO or iron hematoxylin and ORO combined. The collagen films avidly absorbed the ORO dye; conventional staining and quantification by dye extraction failed to discriminate between differentiated and undifferentiated cells on the films. On the contrary, the iron hematoxylin-ORO combination provided a quantitative and more reliable determination of adipocytes based on single cell count. This method is particularly recommended for determining the adipogenic differentiation potential of ASCs and other cell types grown on highly absorptive materials that need to be validated for their potential use in bioengineering and regenerative medicine.

Benzo[a]phenoxazinium chlorides: Synthesis, antifungal activity, in silico studies and evaluation as fluorescent probes.

Four new benzo[a]phenoxazinium chlorides with combinations of chloride, ethyl ester and methyl as terminals of the amino substituents were synthesized. These compounds were characterized and their optical properties were studied in absolute dry ethanol and water. Their antiproliferative activity was tested against Saccharomyces cerevisiae in a broth microdilution assay, along with an array of 36 other benzo[a]phenoxazinium chlorides. Minimum Inhibitory Concentration (MIC) values between 1.56 and >200 µM were observed. Fluorescence microscopy studies, used to assess the intracellular distribution of the dyes, showed that these benzo[a]phenoxazinium chlorides function as efficient and site specific probes for the detection of the vacuole membrane. The added advantage of some of the compounds, that displayed the lower MIC values, was the simultaneous staining of both the vacuole membrane and the perinuclear membrane of endoplasmic reticulum (ER). Molecular docking studies were performed on the human membrane protein oxidosqualene cyclase (OSC), using the crystal structure available on PDB (code 1W6K). The results showed that these most active compounds accommodated better in the active sites of ER enzyme OSC suggesting this enzyme as a potential target. As a whole, the results demonstrate that the benzo[a]phenoxazinium chlorides are interesting alternatives to the available commercial dyes. Changes in the substituents of these compounds can tailor both their staining specificity and antimicrobial activity.

Real-Time In†Vivo Detection of Cellular Senescence through the Controlled Release of the NIR Fluorescent Dye Nile Blue.

In vivo detection of cellular senescence is accomplished by using mesoporous silica nanoparticles loaded with the NIR-FDA approved Nile blue (NB) dye and capped with a galactohexasaccharide (S3). NB emission at 672 nm is highly quenched inside S3, yet a remarkable emission enhancement is observed upon cap hydrolysis in the presence of ß-galactosidase and dye release. The efficacy of the probe to detect cellular senescence is tested in vitro in melanoma SK-Mel-103 and breast cancer 4T1 cells and in vivo in palbociclib-treated BALB/cByJ mice bearing breast cancer tumor.

Biogenic synthesis of copper oxide nanoparticles using plant extract and its prodigious potential for photocatalytic degradation of dyes.

The development of benign and efficient approaches for treating industrial grade toxic organic dyes is an ongoing challenge. To this end, copper oxide nanoparticles (CuO NPs) were prepared by a simple, environment friendly, and economical green synthesis procedure by using Psidium guajava leaf extract as reducing agent (i.e., for the reduction of metal salt) as well as capping agent and copper acetate monohydrate as metal salt. The formation of mono-dispersed and spherical (average size 2-6 nm with BET surface area 52.6 m2/g) CuO NPs was confirmed by various spectroscopic and microscopic techniques. The CuO NPs exhibited excellent degradation efficiency for the industrial dyes, i.e., Nile blue (NB) (93% removal in 120 min) and reactive yellow 160 (RY160) (81% removal in 120 min) with apparent rate constants of 0.023 and 0.014 min-1, respectively. The CuO catalyst was found to be reusable for photocatalytic dye degradation even after five consecutive cycles. The limit of detection (LOD) values for NB and RY160 were 4 and 9 mg/L, respectively. In light of their high reusability and photocatalytic efficiency along with adaptability to green synthesis, the use of biogenic CuO NPs is a promising option for the purification of water resources contaminated with industrial dye.

A vertical flow microarray chip based on SERS nanotags for rapid and ultrasensitive quantification of α-fetoprotein and carcinoembryonic antigen.

A vertical flow microarray chip is described that uses core-shell SERS nanotags as tags for ultrasensitive quantification of the tumor markers α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) by detecting the intensity of the specific Raman bands at 592 cm-1. The nanotags warrant high sensitivity, and the use of porous nitrocellulose warrants a high surface-to-volume ratio. The linear dynamic ranges are 0.1 ng mL-1 - 10 µg mL-1 for both AFP and CEA, and the limits of detection) are 0.27 pg mL-1 and 0.96 pg mL-1, respectively. Quantification is rapid and can be performed without preconcentration. Graphical abstract Schematic representation of a vertical flow microarray chip using AuNBA@Ag SERS nanotags (where NBA stands for Nile blue A) as labels for rapid, ultrasensitive, and simultaneous detection of tumor biomarkers CEA and AFP.

The monoamine oxidase inhibition properties of selected structural analogues of methylene blue.

The thionine dye, methylene blue (MB), is a potent inhibitor of monoamine oxidase (MAO) A, a property that may, at least in part, mediate its antidepressant effects in humans and animals. The central inhibition of MAO-A by MB has also been linked to serotonin toxicity (ST) which may arise when MB is used in combination with serotonergic drugs. Structural analogues and the principal metabolite of MB, azure B, have also been reported to inhibit the MAO enzymes, with all compounds exhibiting specificity for the MAO-A isoform. To expand on the structure-activity relationships (SARs) of MAO inhibition by MB analogues, the present study investigates the human MAO inhibition properties of five MB analogues: neutral red, Nile blue, new methylene blue, cresyl violet and 1,9-dimethyl methylene blue. Similar to MB, these analogues also are specific MAO-A inhibitors with cresyl violet (IC50=0.0037µM), Nile blue (IC50=0.0077µM) and 1,9-dimethyl methylene blue (IC50=0.018µM) exhibiting higher potency inhibition compared to MB (IC50=0.07µM). Nile blue also represents a potent MAO-B inhibitor with an IC50 value of 0.012µM. From the results it may be concluded that non-thionine MB analogues (e.g. cresyl violet and Nile blue) also may exhibit potent MAO inhibition, a property which should be considered when using these compounds in pharmacological studies. Benzophenoxazines such as cresyl violet and Nile blue are, similar to phenothiazines (e.g. MB), representative of high potency MAO-A inhibitors with a potential risk of ST.

Reversible Absorption and Emission Responses of Nile Blue and Azure A Derivatives in Extreme Acidic and Basic Conditions.

Oxazinium derivatives have recently played an important role in bioanalysis attributing to the distinguished properties, thus a detailed study of the structure-property relationship is especially significant. Herein, pH-sensitive optical properties of Nile Blue (1a), N-monoalkyl-Nile Blue (1b) and Azure A (1c) have been carried out in extreme acid and base conditions. Dyes 1a and 1c showed colorimetric changes by the protonation of nitrogen atom in strong acidic condition (pH < 2.0), and dyes 1a - c exhibited colorimetric changes by equilibrium between amino and imide groups in very strong basic case (pH > 7.6). Besides, their fluorescent properties were closed to ON - OFF and OFF - ON emissions at 640-820 nm under strong acidic and basic conditions. Moreover, the absorption and emission properties were reversible, and there were no remarkable optical intensity changes of dyes 1a - c under subacidic and neutral solutions (pH = 3.0-7.0). The (TD) DFT calculations were used to optimize the most stable structures of their corresponding protonated and deprotonated forms, and their absorption and emission properties were also explained. Their fluorescent properties nearly ON-OFF and OFF - ON in strong acidic and basic conditions at near-infrared region will give the possible application in pH detection for extreme conditions. Graphical abstract ᅟ.

Nile Red and Nile Blue: Applications and Syntheses of Structural Analogues.

Nile red and Nile blue are highly fluorescent and photostable organic dyes from the benzo[a]phenoxazine family. They have been used as histological stains for imaging lysosomes and lipids in vitro. The dyes' high quantum yields and solvent-dependent optical properties make them ideal scaffolds for the development of pH probes and local polarity indicators. Reviews of the literature in this area are scarce with only one review ever published in 2006. It has been 10 years since and the field has evolved. This review aims to expand upon topics covered by the previous reviewers and to report on recent advances in the literature. As authors, we hope to convey a sense of scope and to spark renewed interest in this useful niche of dye chemistry.

Targosomes: Anti-HER2 PLGA nanocarriers for bioimaging, chemotherapy and local photothermal treatment of tumors and remote metastases.

Developing combined cancer therapy strategies is of utmost importance as it can enhance treatment efficacy, overcome drug resistance, and ultimately improve patient outcomes by targeting multiple pathways and mechanisms involved in cancer growth and progression. Specifically, the potential of developing a combination chemo&photothermal therapy using targeted polymer nanoparticles as nanocarriers offers a promising approach for synergistic cancer treatment by combining the benefits of both therapies, such as targeted drug delivery and localized hyperthermia. Here, we report the first targeted anti-HER2 PLGA nanocarriers, called targosomes, that simultaneously possess photothermal, chemotherapeutic and diagnostic properties using only molecular payloads. Biocompatible poly(lactic-co-glycolic acid), PLGA, nanoparticles were loaded with photosensitizer phthalocyanine, diagnostic dye Nile Blue, and chemotherapeutic drug irinotecan, which was chosen as a result of screening a panel of theragnostic nanoparticles. The targeted delivery to cell surface oncomarker HER2 was ensured by nanoparticle modification with the anti-HER2 monoclonal antibody, trastuzumab, using the one-pot synthesis method without chemical conjugation. The irradiation tests revealed prominent photothermal properties of nanoparticles, namely heating by 35 °C in 10 min. Nanoparticles exhibited a 7-fold increase in binding and nearly an 18-fold increase in cytotoxicity for HER2-overexpressing cells compared to cells lacking HER2 expression. This enhancement of cytotoxicity was further amplified by >20-fold under NIR light irradiation. In vivo studies proved the efficacy of nanoparticles for bioimaging of primary tumor and metastasis sites and demonstrated 93% tumor growth inhibition, making these nanoparticles excellent candidates for translation into theragnostic applications.

Anticancer Activity of Benzo[a]phenoxazine Compounds Promoting Lysosomal Dysfunction.

Specific cancer therapy remains a problem to be solved. Breast and colorectal cancer are among the cancers with the highest prevalence and mortality rates. Although there are some therapeutic options, there are still few effective agents for those cancers, which constitutes a clinical problem that requires further research efforts. Lysosomes play an important role in cancer cells' survival, and targeting lysosomes has gained increased interest. In recent years, our team has been synthetizing and testing novel benzo[a]phenoxazine derivatives, as they have been shown to possess potent pharmacological activities. Here, we investigated the anticancer activity of three of the most potent derivatives from our library, C9, A36, and A42, on colorectal- and breast-cancer-derived cell lines, and compared this with the effect on non-neoplastic cell lines. We observed that the three compounds were selective for the cancer cells, namely the RKO colorectal cancer cell line and the MCF7 breast cancer cell line. In both models, the compounds reduced cell proliferation, cell survival, and cell migration, accumulated on the lysosome, and induced cell death accompanied by lysosomal membrane permeabilization (LMP), increasing the intracellular pH and ROS accumulation. Our results demonstrated that these compounds specifically target lysosomes from cancer cells, making them promising candidates as LMP inducers for cancer therapy.

Antileukemic potential of Nile blue-mediated photodynamic therapy on HL60 human myeloid leukemia cells.

Background/aim: Photodynamic therapy (PDT) has received great attention over the past decade in the treatment of diseases such as leukemia which is a cancer of the blood and bone marrow cells that causes a significant number of deaths worldwide. In this study, it was aimed to investigate the effects of Nile blue-mediated PDT (NB-mediated PDT) on HL60 cells. Materials and methods: The effect of NB-mediated PDT on cell proliferation was evaluated with cell volume analysis using flow cytometry at 24 h. Cell apoptosis, ROS production, mitochondrial membrane potential, and cell cycle analysis were evaluated using annexin V-FITC, H2DCFDA, JC-1, and PI staining, respectively, by flow cytometry and fluorescence microscopy. The morphological and ultrastructural analyses were examined by Giemsa staining and SEM. CD11b staining is used to determine the differentiation of leukemia cells. Results: NB-mediated PDT induced an apoptotic response at 12.5 µM in HL60 cells. When Giemsa staining and SEM images were evaluated, apoptotic bodies, holes, and occasional folds were detected on the surfaces of cells in the NB-mediated PDT group. Conclusion: The NB-mediated PDT had no effect on the differentiation of leukemia cells, but this therapy affects the growth of HL60 cells in vitro, which may provide a new idea for removing leukemic cells from bone marrow intended for autologous transplant.

Nile blue as reporter dye in salt aggregation based-colorimetric aptasensors for peptide, small molecule and metal ion detection.

Herein, we report a novel approach for the design of a colorimetric aptasensor, relying on a Dye Salt Aggregation-based Colorimetric Oligonucleotide assay (DYSACO assay). This method is based on the use of an intercalating agent, Nile Blue (NB), whose aggregation capacities (and thus modification of its absorption spectrum) are drastically amplified by adding salts to the working solution. The presence of an aptamer could protect NB from such aggregation process due to its intercalation into double-stranded DNA and/or interaction with nucleobases. In response to the addition of the specific ligand, the competition between NB and the target for binding to the aptamer occurs, resulting in an increase in the dye salt aggregation and then in the blue-to-blank color change of the solution. The proof-of-principle was demonstrated by employing the anti-l-tyrosinamide aptamer and the assay was successfully applied to the trace enantiomer detection, allowing the detection of an enantiomeric impurity down to approximately 2% in a non-racemic sample. Through a reversed mechanism based on the increased capture of NB by DNA upon analyte binding, the sensing platform was further demonstrated for the Hg(II) detection. Water samples of different origin were spiked with Hg(II) analyte at final range concentrations comprised between (0.5-15 µM). An excellent overall recovery of 122 ± 14%; 105 ± 14%; 99 ± 9%; was respectively obtained from river, tap and mineral water, suggesting that the sensor can be used under real sample conditions. The assay was also shown to work for sensing the ochratoxin A and d-arginine vasopressin compounds, revealing its simplicity and generalizability potentialities.

A novel ADA-coated UCNPs@NB sensing platform combined with nucleic acid amplification for rapid detection of Escherichia coli.

In this study, we reported a novel sensing platform based on fluorescence quenching composed of alendronic acid (ADA) coated upconversion nanoparticles (UCNPs) and Nile Blue (NB) combined with polymerase chain reaction (PCR) for rapid, sensitive, and specific detection of Escherichia coli (E. coli). As a fluorescence acceptor, NB has a broad absorption band and can quench upconversion fluorescence intensity at 544 nm and 658 nm based on IFE. PCR is a double-stranded DNA (dsDNA) amplification technique with high specificity. The NB-dsDNA complex can be formed by intercalation of NB between base pairs and groove of dsDNA, leading to upconversion fluorescence recovery. The ADA-coated UCNPs@NB sensing platform achieved to detect E. coli in 1.5 h, with a lower limit of detection (33 CFU mL-1). In addition, the sensitivity of the ADA@UCNPs-NB fluorescence sensor under different PCR cycle numbers was discussed. The results showed that the proposed sensor could effectively shorten the assay time (1.0 h) while maintaining excellent sensitivity. This study demonstrated a rapid and sensitive analytical method for detecting E. coli in chicken, providing a reference for constructing PCR fluorescence sensors.

Targeted Two-Step Delivery of Oncotheranostic Nano-PLGA for HER2-Positive Tumor Imaging and Therapy In Vivo: Improved Effectiveness Compared to One-Step Strategy.

Therapy for aggressive metastatic breast cancer remains a great challenge for modern biomedicine. Biocompatible polymer nanoparticles have been successfully used in clinic and are seen as a potential solution. Specifically, researchers are exploring the development of chemotherapeutic nanoagents targeting the membrane-associated receptors of cancer cells, such as HER2. However, there are no targeting nanomedications that have been approved for human cancer therapy. Novel strategies are being developed to alter the architecture of agents and optimize their systemic administration. Here, we describe a combination of these approaches, namely, the design of a targeted polymer nanocarrier and a method for its systemic delivery to the tumor site. Namely, PLGA nanocapsules loaded with a diagnostic dye, Nile Blue, and a chemotherapeutic compound, doxorubicin, are used for two-step targeted delivery using the concept of tumor pre-targeting through the barnase/barstar protein "bacterial superglue". The first pre-targeting component consists of an anti-HER2 scaffold protein, DARPin9_29 fused with barstar, Bs-DARPin9_29, and the second component comprises chemotherapeutic PLGA nanocapsules conjugated to barnase, PLGA-Bn. The efficacy of this system was evaluated in vivo. To this aim, we developed an immunocompetent BALB/c mouse tumor model with a stable expression of human HER2 oncomarkers to test the potential of two-step delivery of oncotheranostic nano-PLGA. In vitro and ex vivo studies confirmed HER2 receptor stable expression in the tumor, making it a feasible tool for HER2-targeted drug evaluation. We demonstrated that two-step delivery was more effective than one-step delivery for both imaging and tumor therapy: two-step delivery had higher imaging capabilities than one-step and a tumor growth inhibition of 94.9% in comparison to 68.4% for the one-step strategy. The barnase*barstar protein pair has been proven to possess excellent biocompatibility, as evidenced by the successful completion of biosafety tests assessing immunogenicity and hemotoxicity. This renders the protein pair a highly versatile tool for pre-targeting tumors with various molecular profiles, thereby enabling the development of personalized medicine.

Targeted PLGA-Chitosan Nanoparticles for NIR-Triggered Phototherapy and Imaging of HER2-Positive Tumors.

Targeted medicine uses the distinctive features of cancer cells to find and destroy tumors. We present human epidermal growth factor receptor 2 (HER2)-targeted PLGA-chitosan nanoparticles for cancer therapy and visualization. Loading with two near-infrared (NIR) dyes provides imaging in the NIR transparency window and phototherapy triggered by 808 nm light. Nile Blue (NB) is a biocompatible solvatochromic NIR dye that serves as an imaging agent. Laser irradiation of IR-780 dye leads to a temperature rise and the generation of reactive oxygen species (ROS). Resonance energy transfer between two dyes allows visualization of tumors in a wide range of visible and IR wavelengths. The combination of two NIR dyes enables the use of nanoparticles for diagnostics only or theranostics. Modification of poly(lactic-co-glycolic acid) (PLGA)-chitosan nanoparticles with trastuzumab provides an efficient nanoparticle uptake by tumor cells and promotes more than sixfold specificity towards HER2-positive cells, leading to a synergistic anticancer effect. We demonstrate optical imaging of the HER2-positive mouse mammary tumor and tumor-specific accumulation of PLGA-IR-780-NB nanoparticles in vivo after intravenous administration. We managed to achieve almost complete suppression of the proliferative activity of cells in vitro by irradiation with an 808 nm laser with a power of 0.27 W for 1 min at a concentration at which nanoparticles are nontoxic to cells in the dark.

Hydroxyapatite/L-Lysine Composite Coating as Glassy Carbon Electrode Modifier for the Analysis and Detection of Nile Blue A.

An amperometric sensor was developed by depositing a film coating of hydroxyapatite (HA)/L-lysine (Lys) composite material on a glassy carbon electrode (GCE). It was applied for the detection of Nile blue A (NBA). Hydroxyapatite was obtained from snail shells and its structural properties before and after its combination with Lys were characterized using X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and Brunauer-Emmett-Teller (BET) surface area analyses. The coupling of Lys to HA was attributed to favorable interaction between negatively charged -COO- groups of Lys and divalent ions Ca2+ of HA. Electrochemical investigations pointed out the improvement in sensitivity of the GCE/Lys/HA sensor towards the detection of NBA in solution. The dependence of the peak current and potential on the pH, scan rate, and NBA concentration was also investigated. Under optimal conditions, the GCE/Lys/HA sensor showed a good reproducibility, selectivity, and a NBA low detection limit of 5.07 × 10-8 mol L-1. The developed HA/Lys-modified electrode was successfully applied for the detection of NBA in various water samples.