Synthesis and Characterization of Oxazine-doped Silica Nanoparticles for Their Potential Use as Stable Fluorescent Reagents

The synthesis process to obtain silica nanoparticles (NPs) doped with two oxazine dyes, nile blue and cresyl violet, has been investigated using a modification of the reverse micelle microemulsion method and a procedure based on the Stöber method. A micellar medium provided by the non-ionic surfactant Triton X-100 in a hexanol:water mixture and an ethanol:water mixture, have been used to provide the synthesis medium in each case. Tetraethoxysilane has been used as the initiator of the polymerization and condensation reactions after its hydrolysis in basic medium using ammonium hydroxide. Dye-silane precursor NPs have been also synthesized in order to compare their potential advantages against the NPs obtained by the direct encapsulation of the oxazine dyes. Size distribution and fluorescence of the synthesized NPs, which were monitored using Transmision Electron Microscopy (TEM) and a microplate reader, respectively, depend on the molar ratio and total concentration of the reagents involved in the synthesis. NPs obtained using the developed synthesis procedures had sizes below 400 nm in most instances and the best luminescent properties were observed for NPs with sizes ranging from 100 to 300 nm. Lower sizes result in a decrease in the fluorescence intensities of these nanomaterials. Parameters related with the luminescence features of these NPs were calculated in order to compare the feasibility of both synthesis approaches. The repeatability of the reverse-micelle microemulsion procedure performed in different days gave a relative standard deviation of 10% for the fluorescence intensity values.     

Determination of fluoroquinolone antibiotics by microchip capillary electrophoresis along with time-resolved sensitized luminescence of their terbium(III) complexes

We report on the time-resolved detection of the three fluoroquinolone (FQs) antibiotics ciprofloxacin (CIP), enrofloxacin (ENR) and flumequine (FLU). On addition of terbium(III) ions, the terbium(III)-FQs chelates are formed in-situ in an on-capillary derivatization reaction of a microfluidic system. The laser-induced terbium(III)-sensitized luminescence of the chelates is measured at excitation/emission wavelengths of 337/545 nm. The analytes can be separated and quantified within less than 4 min. A solid phase extraction step for analyte preconcentration can be included prior to chelation and microchip capillary electrophoresis. The analytical ranges of the calibration graphs for CIP, ENR and FLU are from 10.6 to 60.0, 10.3 to 51.0, and 11.5 to 58.8 ng mL^?1, respectively, and the detection limits are 3.2, 3.1 and 3.6 ng mL^?1, respectively. The precision was established at two concentration levels of each analyte and revealed relative standard deviations in the range from 3.0 to 10.2 %. The method was applied to the analysis of FQ-spiked water samples. Figure

We report on the time-resolved detection of the three fluoroquinolone antibiotics. The analytes can be separated and quantified within less than 4 min. A solid phase extraction step for analyte preconcentration can be included prior to chelation and microchip capillary electrophoresis.     

Determination of soy proteins in food samples by dispersive solid-phase immunoextraction and dynamic long-wavelength fluorometry

We report on a method for the determination of soy proteins in food samples via dispersive solid-phase immunoextraction using gold-coated magnetic nanoparticles (NPs) as a support. Soy proteins were first extracted using anti-soy protein antibodies immobilized on the NPs, and then quantified by measuring the increase in fluorescence of the long-wavelength fluorophore cresyl violet in the presence of the anionic surfactant sodium dodecyl sulfate at neutral pH in a flow system. The method involves the use of two standard or sample aliquots. The fluorescence intensity of one aliquot is directly measured whereas that of the other aliquot is measured after immunoextraction. The difference between the peak heights of both aliquots serves as the analytical information that is directly proportional to the protein concentration. The limit of detection is 0.35 mg L^?1, the linear range is from 1 to 15 mg L^?1, and the relative standard deviation is <?5 %. Proteins such as bovine serum albumin and globulins do not interfere at the same concentration level. The method was applied to the analysis of soy-based beverages and gave recoveries in the range between 80.0 and 107.3 %.

Application of time-resolved luminescence to dry reagent chemical technology

A time-resolved luminescence method describing the use of terbium(III) in the dry reagent format is presented for the first time. Paper strips previously treated with sucrose are used as solid substrates where terbium(III) is immobilised by adsorption. The strips are stable for at least 6 months and they can be easily stored under a desiccant medium. Only the addition of the buffered sample is required for the analysis. This methodology has been applied to the determination of two local anaesthetics, namely benzocaine and procaine. These compounds release p-aminobenzoic acid after a hydrolysis step in alkaline medium, which reacts to terbium(III) giving a luminescent chelate. The luminescence intensity measurements are obtained at λ_ex 286 and λ_em 545 nm by using the time-resolved mode of the instrument. The method presents a linear range from 1.1 to 21.9 μM and the calculated LOD is 0.4 μM. It has been satisfactorily applied to the analysis of pharmaceutical samples and the recoveries obtained are in the range 88-108%.     

Photometric determination of thioglycolic acid in cosmetics by using a stopped-flow reverse flow-injection system and the formation of gold nanoparticles

The positive effect of thiol compounds as reducing agents in the synthesis of gold nanoparticles in the presence of a micellar medium of Triton X-100 has been photometrically studied using a reverse flow-injection system which operates in the stopped-flow mode (SF-rFIA). The analytical usefulness of this new system has been assessed by its application to the determination of thioglycolic acid (TGA), which was chosen as the analyte model. The dynamic range of the calibration graph was 5.97–80 μmol L^?1, and the detection limit was 1.73 μmol L^?1. The behaviour of other thiol compounds on the system has been also studied. The precision of the method, expressed as relative standard deviation, ranged between 1.5 and 2.3%. The method was applied to the determination of TGA in several cosmetic samples with acceptable recoveries in all instances, which ranged between 90.32 and 101.46%.     

An Optimized Protocol for the Synthesis of Peptides Containing trans-Cyclooctene and Bicyclononyne Dienophiles as Useful Multifunctional Bioorthogonal Probes

Despite the great advances in solid-phase peptide synthesis (SPPS), the incorporation of certain functional groups into peptide sequences is restricted by the compatibility of the building blocks with conditions used during SPPS. In particular, the introduction of highly reactive groups used in modern bioorthogonal reactions into peptides remains elusive. Here, we present an optimized synthetic protocol enabling installation of two strained dienophiles, trans-cyclooctene (TCO) and bicyclononyne (BCN), into different peptide sequences. The two groups enable fast and modular post-synthetic functionalization of peptides, as we demonstrate in preparation of peptide-peptide and peptide-drug conjugates. Due to the excellent biocompatibility, the click-functionalization of the peptides can be performed directly in live cells. We further show that the introduction of both clickable groups into peptides enables construction of smart, multifunctional probes that can streamline complex chemical biology experiments such as visualization and pull-down of metabolically labeled glycoconjugates. The presented strategy will find utility in construction of peptides for diverse applications, where high reactivity, efficiency and biocompatibility of the modification step is critical.     

Long-Wavelength Fluorescence Detection of Flavonoids in Orange Juices by LC

A new post-column liquid chromatographic reaction system for the determination of flavonoids in orange juices is based on the use of the long wavelength fluorophor cresyl violet and cerium(IV) in a cetyl trimethylammonium bromide micellar medium. Two flavone aglycones (quercetin and kaempferol), a flavonone aglycone (naringenin), one flavone-O-glycoside (rutin) and two flavanone-O-glycosides (hesperidin and naringin) have been used as analyte models. The reaction process involves the interaction between the analyte, cerium(IV) and cresyl violet giving rise to a decrease in the fluorescence, measured at λ _ex 585, λ _em 625 nm, which is proportional to the analyte concentration. Dynamic ranges of the calibration graphs and detection limits, obtained with standard solutions of the analytes are (ng mL^?1): quercetin (12.2–4,000, 3.7), kaempferol (3.5–1,000, 1.0), naringenin (6.7–1,000, 2.0), rutin (5.0–800, 1.5), hesperidin (10.1–1,000, 3.0), and naringin (17.8–800, 1.8). The determination coefficients were higher than 0.993 in all instances. The precision of the method, expressed as RSD%, was established at two concentration levels, with values ranging between 2.8 and 6.2%. The practical usefulness of the developed method is demonstrated by the analysis of natural and commercial orange juices, which were filtered, diluted and directly injected into the chromatographic system, with apparent recoveries between 86.9 and 107.0%.     

Determination of some hydroxybenzoic acids and catechins in white wine samples by liquid chromatography with luminescence detection

A liquid chromatographic method with luminescence detection for the determination of eight phenolic compounds is reported. The method involves postcolumn derivatization with terbium(III). This derivatization is based on the reaction between phenolics and terbium(III) to form luminescent chelates, which were determined at lamda ex 295 and lamda em 545 nm using the fluorescence mode. The long wavelength emission of lanthanide chelates can minimize interferences from background sample matrix, which usually emit at shorter wavelengths. Also, the chromatographic separation allows the individual determination of phenolics, which cannot be done using the direct measurement of the fluorescence of their corresponding terbium chelates. Dynamic ranges of the calibration graphs and detection limits, obtained with standard solutions of analytes were (microg/mL): gallic acid (0.9-40, 0.3), protocatechuic acid (0.05-7, 0.016), catechin (0.2-40, 0.07), vanillic acid (0.25-40, 0.08), p-hydroxybenzoic acid (0.8-40, 0.25), syringic acid (0.17-40, 0.05), epicatechin (0.3-40, 0.09) and salicylic acid (0.07-12, 0.02). The precision was established at two concentration levels of each analyte and expressed as the percentage of RSD with values ranging between 1.0 and 6.5%. The practical usefulness of the method was demonstrated by the analysis of white wine samples, which were diluted two-fold and directly injected into the chromatographic system. The recovery values obtained ranged between 93.3 and 108.0%.     

Sensitive capillary GC-MS-SIM determination of selective serotonin reuptake inhibitors: reliability evaluation by validation and robustness study

A simple, fast, selective and very sensitive capillary GC-MS method for the simultaneous determination of five antidepressant drugs is described. Fluoxetine, fluvoxamine, citalopram, sertraline and paroxetine belong to the newest and most important drug group termed selective serotonin reuptake inhibitors. Imipramine was used in this method as an internal standard for quantification. Optimum parameters for GC separation were investigated, i.e., flow rate, column head pressure, injector temperature, injection splitless conditions and oven temperature program. MS detection was performed in SIM mode to increase the sensitivity. Stability of the solutions, linear concentration range, accuracy, precision, LOD, LOQ (3.6-41.5 mg/L) and specificity were examined in the presence of excipients for checking the reliability of this method. The robustness was evaluated with a matrix of 15 experiments (seven factors and three levels) using Plackett-Burman fractional factorial experimental design, and Youden and Steiner statistical treatment. The method was applied to the analysis of these antidepressants in nearly all their pharmaceutical formulations, obtaining recoveries between 98.1% and 102.7% with regard to the claimed values.