Novel and simple spectrophotometric and distance based procedures for thiols (L-cysteine, N-acetylcysteine, and glutathione) determination in biological fluids and pharmaceuticals have been proposed based on their inhibitory action on the oxidation of catechol in the presence of Agaricus bisporus crude extract (ABE). The influence of L-glycine, L-alanine, L-proline, L-methionine, L-cystine, ascorbic acid, uric acid, and bilirubin on the thiol determination has been investigated. Uric acid, bilirubin, L-cystine (oxidized thiol), and L-amino acids do not interfere with the determination. The interference of ascorbic acid up to 350 mg/L is eliminated by using Cucumis sativus crude extract (CSE) with ascorbate oxidase activity. Distance based microfluidic paper-based analytical device (D mu PAD) has been developed using origami paper device approach and ABE-catechol-3-methyl-2-benzothiazolinone hydrazone (MBTH) system. D mu PAD has the 3D structure of three layers: CSE microzone layer, ABE microzone layer, and chemometer layer (catechol + MBTH). This structure allows sequential sample treatment (ascorbic acid oxidation by CSE) and the following introduction of treated sample to ABE and its substrate to perform ABE inhibition. Separate loading of ABE and its substrate allows preventing their interaction prior to sample loading. D mu PAD thiol determination is performed by measuring the length of uncolored flow channel, which allows very simple thiol determination and enables complete integration of analytical procedure steps: sample treatment and enzymatic determination with visual signal output. The analytical ranges are (0.3-2.5)center dot 10- 4 M and (1.1-10.0)center dot 10- 4 M, the recoveries are 80.5-126.7 % and 92.0-112.0 %, the RSD values are 1.6-19.0 % and 2.7-10.8 %, for spectrophotometric and D mu PAD procedures, respectively. Both easy-to-use procedures have been successfully applied to the determination of total thiol content (all free sulfhydryl groups) in synthetic urine and N-acetylcysteine in a pharmaceutical sample. The values found with D mu PAD and spectrophotometric procedures are in good agreement with values obtained using Ellman's reagent.

Chromogenic enzymatic reactions are very convenient for the determination of various biochemically active compounds. Sol-gel films are a promising platform for biosensor development. The creation of sol-gel films with immobilized enzymes deserves attention as an effective way to create optical biosensors. In the present work, the conditions are selected to obtain sol-gel films doped with horseradish peroxidase (HRP), mushroom tyrosinase (MT) and crude banana extract (BE), inside the polystyrene spectrophotometric cuvettes. Two procedures are proposed: the use of tetraethoxysilane-phenyltriethoxysilane (TEOS-PhTEOS) mixture as precursor, as well as the use of silicon polyethylene glycol (SPG).In both types of films, the enzymatic activity of HRP, MT, and BE is preserved. Based on the kinetics study of enzymatic reactions catalyzed by sol-gel films doped with HRP, MT, and BE, we found that encapsulation in the TEOS-PhTEOS films affects the enzymatic activity to a lesser extent compared to encapsulation in SPG films. Immobilization affects BE significantly less than MT and HRP. The Michaelis constant for BE encapsulated in TEOS-PhTEOS films almost does not differ from the Michaelis constant for a non-immobilized BE. The proposed sol-gel films allow determining hydrogen peroxide in the range of 0.2–3.5 mM (HRP containing film in the presence of TMB), and caffeic acid in the ranges of 0.5–10.0 mM and 2.0–10.0 mM (MT- and BE-containing films, respectively). BE-containing films have been used to determine the total polyphenol content of coffee in caffeic acid equivalents; the results of the analysis are in good agreement with the results obtained using an independent method of determination. These films are highly stable and can be stored without the loss of activity for 2 months at +4 °C and 2 weeks at +25 °C.

The review summarizes the author’s results concerning the complex formation in the phase of modified xerogels synthesized by the sol–gel process for application as sensor materials. The results of studying the complex formation (pH ranges of reactions, light absorption maxima and compositions of complexes, and equilibrium constants) are presented and discussed for 87 systems of two types, that is, immobilized ligand (organic analytical reagent)–metal ion and immobilized metal ion–organic or inorganic compound. The approach proposed by the author to describe the complex formation in the modified xerogel phase is presented. The results of applications of modified xerogels as sensor materials are considered, including their use for determining metal ions; organic compounds; hydrogen peroxide; chloride, fluoride, and oxalate ions in food and hygienic products, pharmaceuticals, biological fluids, and environmental media.

The influence of cetylpyridinium chloride (CPC) on the interaction of triphenylmethane dyes (pyrocatechol violet, eriochrome cyanine R, and chrome azurol S) with silica-titania xerogels has been studied. For all the studied triphenylmethane dyes (TPMD) the formation of colored complexes of CPC and TPMD with titanium(IV) embedded in silica-titania xerogels has been observed using solid phase spectrophotometry. Maximal absorbance increase has been observed for pyrocatechol violet (PV) complexes. The procedure for CPC determination has been proposed using silica-titania xerogel – PV system (analytical range 0.01–0.56 mM, limit of detection 3.6 μM). The recovery test of the proposed solid phase spectrophotometric CPC determination procedure has been used for the procedure validation. The procedure has been applied for the determination of CPC in treated waste water, and cationic surfactants in terms of CPC in working solutions of disinfectants Alaminol and Catamine AB.

In order to design a sensor material for total antioxidant capacity determination we have prepared silica and silica–titania xerogels doped with iron(III) and modified with 1,10-phenanthroline. Titanium(IV) tetraethoxyde content in the precursors (titanium(IV) tetraethoxyde and tetraethyl orthosilicate) mixtures has been varied from 0 to 12.5% vol. Iron(III) concentrations in sol has been varied from 1 to 100 mM. The increase of titanium(IV) content has led to a decrease in BET surface area and average pore diameter and an increase of micropore surface area and volume, which has resulted in better iron(III) retention in the xerogels. Iron(III), immobilized in the xerogel matrix, retains its ability to form complexes with 1,10-phenanthroline and to be reduced to iron(II). Static capacities for 1,10-phenanthroline have been determined for all the iron(III) doped xerogels (0.207 mmol/g–0.239 mmol/g) and they are not dependent on the iron(III) content. Sensor materials—xerogels doped with iron(III) and modified with 1,10-phenanthroline—have been used for antioxidants (catechol, gallic and ascorbic acids, and sulphite) solid phase spectrophotometric determination. Limits of detection for catechol, gallic and ascorbic acids, and sulphite equal 7.8 × 10−6 M, 5.4 × 10−6 M, 1.2 × 10−5 M, and 3.1 × 10−4 M, respectively. The increase of titanium(IV) content in sensor material has led to an increase of the reaction rate and the sensitivity of determination. Proposed sensor materials have been applied for total antioxidant capacity (in gallic acid equivalents) determination in soft beverages, have demonstrated high stability, and can be stored up to 6 months at room temperature.

Salicylhydroxamic acid (SHA) is used as antimicrobic medicine and its concentration has to be monitored in urine. For the first time, silica xerogels doped with iron(III) have been proposed as sensor materials for SHA determination in biological samples. Three xerogels with iron(III) content in the range of 0.04–1.74% wt have been synthesized. BET surface area of these xerogels has varied in the range of 696–529 m2/g and total pore volume has varied in the range of 0.92–0.23 cm3/g. Complex formation between immobilized iron(III) and salicylhydroxamic acid has been investigated with solid phase spectrophotometry and IR spectroscopy. Orange-brown iron(III)-SHA complex with 1:1 stoichiometry is formed at pH 1–4 with half-reaction time of 17 min. Silica xerogel doped with 0.33% wt iron(III)) has been used as sensor material for SHA solid phase spectrophotometric determination (LOD 1.4 mg/L (n = 3), analytical range 4–230 mg/L). Proposed sensor material has been applied for SHA determination in biological samples of synthetic and human urine. The proposed procedure is characterized by a good level of accuracy (recovery values 97–120%) and precision (RSD values 4–9%) and can be recommended for pharmacokinetic–pharmacodynamic studies of hydroxamic acid-based medications.

In the present work crude Agaricus bisporus extract (ABE) has been prepared and characterized by its tyrosinase activity, protein composition and substrate specificity. The presence of mushroom tyrosinase (PPO3) in ABE has been confirmed using two-dimensional electrophoresis, followed by MALDI TOF/TOF MS-based analysis. GH27 alpha-glucosidases, GH47 alpha-mannosidases, GH20 hexosaminidases, and alkaline phosphatases have been also detected in ABE. ABE substrate specificity has been studied using 19 phenolic compounds: polyphenols (catechol, gallic, caffeic, chlorogenic, and ferulic acids, quercetin, rutin, dihydroquercetin, l-dihydroxyphenylalanine, resorcinol, propyl gallate) and monophenols (l-tyrosine, phenol, p-nitrophenol, o-nitrophenol, guaiacol, o-cresol, m-cresol, p-cresol). The comparison of ABE substrate specificity and affinity to the corresponding parameters of purified A. bisporus tyrosinase has revealed no major differences. The conditions for spectrophotometric determination have been chosen and the analytical procedures for determination of 1.4 × 10−4–1.0 × 10−3 M l-tyrosine, 3.1 × 10−6–1.0 × 10−4 M phenol, 5.4 × 10−5–1.0 × 10−3 M catechol, 8.5 × 10−5–1.0 × 10−3 M caffeic acid, 1.5 × 10−4–7.5 × 10−4 M chlorogenic acid, 6.8 × 10−5–1.0 × 10−3 M l-DOPA have been proposed. The procedures have been applied for the determination of l-tyrosine in food supplements, l-DOPA in synthetic serum, and phenol in waste water from the food manufacturing plant. Thus, we have demonstrated the possibility of using ABE as a substitute for tyrosinase in such analytical applications, as food supplements, medical and environmental analysis.

In order to develop a simple, reliable and low cost enzymatic method for the determination of phenolic compounds we studied polyphenol oxidase activity of crude eggplant (S. melongena) extract using 13 phenolic compounds. Catechol, caffeic and chlorogenic acids, and L-DOPA have been rapidly oxidized with the formation of colored products. Monophenolic compounds have been oxidized at a much slower speed. Ferulic acid, quercetin, rutin, and dihydroquercetin have been found to inhibit polyphenol oxidase activity of crude eggplant extract. The influence of pH, temperature, crude eggplant extract amount, and 3-methyl-2-benzothiazolinone hydrazone (MBTH) concentration on the oxidation of catechol, caffeic acid, chlorogenic acid, and L-DOPA has been investigated spectrophotometrically. Michaelis constants values decrease by a factor of 2 to 3 in the presence of MBTH. Spectrophotometric (cuvette and microplate variants) and smartphone-assisted procedures for phenolic compounds determination have been proposed. Average saturation values (HSV color model) of the images of the microplate wells have been chosen as the analytical signal for smartphone-assisted procedure. LOD values for catechol, caffeic acid, chlorogenic acid, and L-DOPA equaled 5.1, 6.3, 5.8 and 30.0 µM (cuvette procedure), 12.2, 13.2, 13.2 and 80.4 µM (microplate procedure), and 23.5, 26.4, 20.8 and 120.6 µM (smartphone procedure). All the variants have been successfully applied for fast (4-5 min) and simple TPC determination in plant derived products and L-DOPA determination in model biological fluids. The values found with smartphone procedure are in good agreement with both spectrophotometric procedures values and reference values. Using crude eggplant extract- mediated reactions combined with smartphone camera detection has allowed creating low-cost, reliable and environmentally friendly analytical method for the determination of phenolic compounds.

The interaction of silica–titania xerogel with triphenylmethane dyes (pyrocatechol violet, chrome azurol S, eriochrome cyanine R) has been investigated to create a new sensor material for solid phase spectrophotometric determination of food oxalates. The complex forming reaction between xerogel incorporated titanium(IV) and triphenylmethane dyes has been studied; half-reaction periods, complex composition, equilibrium constants, and xerogel sorption capacity have been calculated for each dye. Eriochrome cyanine R (ECR) is characterized by the shortest half-reaction period, the smallest equilibrium constant, and the greatest capacity; it has been chosen for the sensor material construction because titanium(IV)-ECR complex is formed faster and can be destroyed easier than other studied complexes. The interaction of this sensor material with oxalates has been described: the presence of oxalates causes sensor material discoloration and the absorbance is used as analytical signal. The analytical range is 35–900 mg/L (LOD 10.5 mg/L, n = 7). High concentrations of interfering inorganic anions, organic acids, and sucrose did not affect oxalate determination. Proposed solid phase spectrophotometric procedure has been successfully applied for the determination of oxalates in food samples (sorrel, spinach, parsley, ginger, and black pepper) and the results are in good agreement with HPLC oxalate determination.

Background: The consumption of antioxidants, including phenolic compounds, is considered important for preventing the oxidative damage diseases and ageing. The total polyphenol content (TPC) is the parameter used to estimate the quality of plant-derived products. Methods: Phenol oxidase activity of green bean (Phaseolus vulgaris) crude extract (in the presence of hydrogen peroxide) and banana (Musa sp.) pulp crude extract has been studied spectrophotometrically using catechol, gallic acid, caffeic acid, ferulic acid, and quercetin as substrates. All studied compounds have been oxidized in the presence of green bean crude extract and hydrogen peroxide; all studied compounds except ferulic acid have been oxidized in the presence of banana pulp crude extract. Michaelis constants (Km) and maximum reaction rates (Vmax) have been determined for oxidation in the presence of green bean crude extract and hydrogen peroxide (Km are 3.8×10-4 M, 1.6×10-3 M, 2.2×10-4 M, 2.3×10-4 M, 1.4×10-4 M and Vmax are 0.046 min-1, 0.102 min-1, 0.185 min-1, 0.053 min-1, 0.041 min-1 for catechol, gallic acid, caffeic acid, ferulic acid, and quercetin, respectively) and for oxidation in the presence of banana pulp crude extract (Km are 1.6×10-3 M, 3.8×10-3 M, 2.2×10-3 M, 4.2×10-4 M and Vmax are 0.058 min-1, 0.025 min-1, 0.027 min-1, 0.015 min-1 for catechol, gallic acid, caffeic acid, and quercetin, respectively). The influence of 3-methyl-2-benzothiazolinone hydrazone (MBTH) on the oxidation reactions kinetics has been studied: Michaelis constants values decrease and maximum reaction rates increase, which contributes to the increase in sensitivity of the determination. Results: Kinetic procedures of Total Polyphenol Content (TPC) determination using crude plants extracts in the presence of MBTH have been proposed (time of analysis is 1 min). For gallic acid (used as a standard for TPC determination) detection limit is 5.3×10-5 M, quantitation limit is 1.8×10-4 M, and linear range is 1.8×10-4 - 1.3×10-3 M for green bean crude extract; detection limit is 2.9×10-5 M, quantitation limit is 9.5×10-5 M, and linear range is 9.5×10-5 - 2.4×10-3 M for banana pulp crude extract. Proposed procedures are characterized by higher interference thresholds for sulfites, ascorbic acid, and citric acid compared to pure enzymes (horseradish peroxidase and mushroom tyrosinase) in the same conditions. Compared with standard Folin-Ciocalteu (FC) method the procedures described in this work are also characterized by less interference and more rapid determination. Conclusion: The procedures have been applied to TPC determination in tea, coffee, and wine samples. The results agree with the FC method for tea and coffee samples and are lower for wine samples, probably, due to sulfites interference.

Data on the synthesis of silica–titania sol–gel materials and their analytical applications as new sensor materials obtained by the author are generalized. The methods of the synthesis of silica–titania xerogels with specified textural characteristics are considered. Data on heterogeneous complexation and redox reactions with the participation of silica–titania xerogels are reported. The new sensor materials based on silica–titania xerogels were used for the development of procedures for the solid-phase spectrophotometric determination of hydrogen peroxide, ascorbic acid, polyphenols, salicylic acid derivatives, and fluoride and oxalate ions and for the test determination of hydrogen peroxide and propyl gallate. The results of the application of the developed procedures to the analysis of food, pharmaceutical preparations, hygienic products, and biological liquids are given.

Ascorbic acid is one of the most important vitamins to monitor in dietary sources (juices and vitamins) and biological liquids.

A silica–titania xerogel obtained by cetylpyridinium chloride (CPC) assisted synthesis has been used as a new sensor material for solid phase spectrophotometric and visual test determination of propyl gallate.

Silica-titania xerogels modified with the pyrocatechol violet have been proposed as new sensor materials for fluoride determination in the artificial saliva. The determination is based on the ability of fluoride ions to disrupt the colored titanium(IV)-pyrocatechol violet complexes. The complex reactions of matrix embedded titanium(IV) with pyrocatechol violet (resulting in blue colored xerogels) and with fluoride ions (resulting in bleaching of colored xerogels) have been investigated. The selected conditions have been used for developing the procedures of fluoride determination in the artificial saliva and water. Varying the volume of the analyzed solution two analytical ranges (0.05-1 ppm (LOD 2 ppm) and 5-100 ppm (LOD 0.02 ppm)) have been obtained. The procedures’ accuracy has been evaluated using the spiked probes of the artificial saliva achieving the RSD of 5-23%. The developed sensor materials can be used for studying the efficiency of various fluor-containing products for medical diagnostics, scientific research, and also for the construction of new dental materials. Keywords: silica-titania xerogels, solid phase spectrophotometry, fluoride ions, artificial saliva. (Russian) DOI: http://dx.doi.org/10.15826/analitika.2015.19.4.001 M.A. Morosanova, Z.V. Samodelov and E.I. Morosanova Lomonosov Moscow State University, Moscow, Russian Federation