João M.M. Leitão

Firefly luciferase inhibition.

Firefly luciferase (Luc) is the most studied of the luciferase enzymes and the mechanism and kinetics of the reactions catalyzed by this enzyme have been relatively well characterized. Luc catalyzes the bioluminescent reaction involving firefly luciferin (D-LH(2)), adenosine triphosphate (ATP), magnesium ion and molecular oxygen with the formation of an electronically excited species (oxyluciferin), inorganic pyrophosphate (PPi), carbon dioxide and adenosine monophosphate (AMP). Luc also catalyzes other non-luminescent reactions, which can interfere with the light production mechanism. Following electronic relaxation, the excited oxyluciferin emits radiation in the visible region of the electromagnetic spectrum (550-570 nm). Among the various possible compounds, several classes of inhibitory substances interfere with the activity of this enzyme: here, we consider substrate-related compounds, intermediates or products of the Luc catalyzed reactions, in addition to anesthetics and, fatty acids. This review summarizes the main inhibitors of Luc and the corresponding inhibition kinetic parameters.

Multiway chemometric decomposition of EEM of fluorescence of CdTe quantum dots obtained as function of pH

The effect of the pH (from 3 to 10) on the excitation emission matrices (EEMs) of fluorescence of CdTe quantum dots (QDs), capped with mercaptopropionic acid (MPA), were analyzed by multiway decomposition methods of parallel factor analysis (PARAFAC), a variant of the parallel factor analysis method (PARAFAC2) and multivariate curve resolution alternating least squares (MCR-ALS). Three different sized CdTe QDs with emission maximum at 555 nm (QDa), 594 nm (QDb) and 628 nm (QDc) were selected for analysis. The three-way data structures composed of sets of EEMs obtained as function of the pH (EEMs, pH) do not have a trilinear structure. A marked deviation to the trilinearity is observed in the emission wavelength order--the emission spectra suffers wavelength shift as the pH is varied. The pH-induced variation of the fluorescence properties of QDs is described with only one-component PARAFAC2 or MCR-ALS models--other components are necessary to model scattering and/or other background signals in (EEMs, pH) data structures. Bigger sized QDs are more suitable tools for analytical methodologies because they show higher Stokes shifts (resulting in simpler models) and higher pH range sensitivity. The pH dependence of the maximum wavelength of the emission spectra is particularly suitable for the development of QDs/EEMs wavelength-encoded pH sensor bioimaging or biological label methodologies when coupled to multiway chemometric decomposition.

Carbon dots from tryptophan doped glucose for peroxynitrite sensing.

Tryptophan doped carbon dots (Trp-CD) were microwave synthesized. The optimum conditions of synthesizing of the Trp-CD were established by response surface multivariate optimization methodologies and were the following: 2.5 g of glucose and 300 mg of tryptophan diluted in 15 mL of water exposed for 5 min to a microwave radiation of 700 W. Trp-CD have an average size of 20 nm, were fluorescent with a quantum yield of 12.4% and the presence of peroxynitrite anion (ONOO(-)) provokes quenching of the fluorescence. The evaluated analytical methodology for ONOO(-) detection shows a linear response range from 5 to 25 μM with a limit of detection of 1.5 μM and quantification of 4.9 μM. The capability of the ONOO(-) quantification was evaluated in standard solutions and in fortified serum samples.

Factorial analysis optimization of a Diltiazem kinetic spectrophotometric quantification method

A Diltiazem kinetic spectrophotometric method was optimized by factorial analysis. The experimental method is based on a two-stage reaction of Diltiazem with hydroxylamine and a ferric salt: in the first stage there is a hydroxamic acid formation; and, in the second stage there is a red colour complex ferric hydroxamate formation. The variables under investigation were: solvent; hydroxylamine, sodium hydroxide and ammonium ferric sulphate concentrations; volume of perchloric acid; and, temperature. The responses of the reactional system were the maximum absorbance, the wavelength and the reaction time at maximum absorbance. Experimental design methodologies were used in the optimization. Fractional and full factorial designs followed by optimization Box-Behnken and central composite experimental designs were used. The observed optimum conditions were: methanol as reaction solvent; hydroxylamine concentration of 9.375%; sodium hydroxide concentration of 18.750%; ferric reagent concentration of 2.000%; minimum volume of perchloric acid to neutralize the sodium hydroxide; and, room temperature as reaction temperature. With this set of experimental conditions a reaction time of 10.5s with maximum colour development at 512nm wavelength was achieved.

Parallel factor analysis of EEM of the fluorescence of carbon dots nanoparticles

The effect of experimental factors [pH and Hg(II)] on the fluorescence excitation emission matrices (EEMs) of nanosensor carbon dots (CDs) was analyzed by multiway decomposition methods based on parallel factor (PARAFAC) analysis. PARAFAC analysis of the EEM structures identifies three components corresponding to two different‐sized CDs with the Hg(II) and pH profiles highly correlated plus a background. Parallel profiles with Linear Dependences (PARALIND) model with three components in the excitation–emission spectral modes and two components in the Hg(II) or pH mode gave similar results as PARAFAC, but is more useful from a theoretical point of view because PARALIND shows that the two different‐sized CDs have similar chemical reactivity toward Hg(II) and pH. PARAFAC2 was used as a trilinear confirmatory test of the data structures under analysis. Copyright

Optimization of Verapamil drug analysis by excitation-emission fluorescence in combination with second-order multivariate calibration.

Excitation emission fluorescence matrices (EEMs) of Verapamil drug were obtained by direct and by derivatization fluorescence spectroscopy. The fluorescence excitation and emission wavelengths were displaced to longer wavelengths and the fluorescence intensity was enhanced upon derivation with respect to the native fluorescence of the drug. The complete EEM of the native fluorescence of the drug and of the derivatization product were rapidly acquired by using a charged-coupled device detector (CCD), which is advantageous in terms of speed in the analysis, with respect to the use of a conventional photomultiplier detector. The EEMs were analyzed by several second-order multivariate calibration methods exploiting the second order advantage. The three-dimensional decomposition methods used, based in different assumptions about the trilinearity of the three way data structure under analysis, were parallel factor analysis (PARAFAC), bilinear least squares (BLLS), parallel factor analysis 2 (PARAFAC2) and multivariate curve resolution—alternating least squares (MCR-ALS). The determination was performed by using the standard addition approach. The figures of merit of the PARAFAC and BLLS methods were calculated, obtaining a lower limit of detection with the derivatization procedure, when compared with the direct measurement of the fluorescence of the drug. In Verapamil drug the best estimations were found with the BLLS and the MCR-ALS models. In the quantification of Verapamil in a pharmaceutical formulation the best estimation, when compared with the result obtained by the US Pharmacopeia high performance liquid chromatography approach, was obtained by direct fluorescence spectroscopy with MCR-ALS and by derivatization fluorescence spectroscopy with the PARAFAC2 model.

Determination of Total Sulfur in Soils and Plants by an Automated Dry Combustion Method

Abstract Determination of total sulfur in environmental samples can be accomplished using automated dry combustion techniques. The analytical performance of an elemental analyzer (CE Instruments) was compared with a method involving dry ashing followed by ion chromatographic detection of sulfate (IC). Samples included certified reference materials, confirmation materials with a known content of sulfur, and several soils and plants. There was a close agreement between both methods. Superior accuracy, precision and detection limits were obtained using the dry combustion method. Current results suggest that the automated dry combustion technique has developed into an adequate method for the determination of the relatively low total sulfur contents commonly encountered in soils and plants.

Chemometric Analysis of Excitation Emission Matrices of Fluorescent Nanocomposites

The performance of multivariate curve resolution (MCR-ALS) to decompose sets of excitation emission matrices of fluorescence (EEM) of nanocomposite materials used as analytical sensors was assessed. The two fluorescent nanocomposite materials were: NH2-polyethylene glycol (PEG200) functionalized carbon dots, sensible to aqueous Hg(II) (CD); and, CdS quantum dots attached to the dendrimer DAB, sensible to the ionic strength of the aqueous medium (CdS-DAB). The structures of these sets of EEM, obtained as function of the Hg(II) concentration and ionic strength, are characterized by collinear properties (CD) and non-linear spectral variations (CdS-DAB). MCR-ALS was able to detect that the source of the collinearities is the presence of different size CD that show similar affinity towards Hg(II). Moreover, MCR-ALS was able to model the non-linear spectral variations of the CdS-DAB that are induced by varying ionic strength. The chemometric pre-processing of the fluorescent data sets using soft-modelling multivariate curve resolution like MCR-ALS is a critical step to transform these nanocomposites with interesting fluorescent proprieties into analytical useful nanosensors.

Flow injection analysis for nitric oxide quantification based on reduced fluoresceinamine

A fluorescence flow injection analysis (FIA) methodology for nitric oxide (NO) quantification was optimized by factorial analysis for the lowest limit of detection of nitric oxide. This methodology is based on the reaction of the NO with the non-fluorescent reduced fluoresceinamine given a high fluorescent oxidized fluoresceinamine. Box–Behnken and central composite optimization experimental design methodologies were used. The factors initially analysed by a screening experimental design methodology were the flow rate of the pump (Q), loop volume (L), reactor length (R), reduced fluoresceinamine concentration (CFl) and cobalt chloride concentration (CCoCl2). The response variables under analysis were the maximum fluorescence intensity, response repeatability and peak width. The optimum conditions were: one flow stream FIA configuration, Q = 0.60 mL min−1, L = 100 μL, R = 2 m, CFl = 1.50 mM and without CoCl2. A linear working range between 5 to 40 μM was evaluated with a limit of detection of 1.20 μM. Hydrogen peroxide, superoxide, nitrite and nitrate did not interfere with the NO detection. Good results were found in the quantification of NO liberated by a NO donor at pH 7.4 and in fortified serum samples.

Luminol-doped nanostructured composite materials for chemiluminescent sensing of hydrogen peroxide

Silica based nanostructured composite materials doped with luminol and cobalt(II) ion were synthesized and characterized, resulting in a highly chemiluminescent material in the presence of hydrogen peroxide. A detection system with the CL light guided from the reaction tube to the photomultiplier tube using a one millimeter glass optical fiber was developed and assessed. A linear response was observed using a semi-logarithm calibration between 50–2000 μM hydrogen peroxide with 1 μM as the limit of detection.