A. Navas Díaz

Sol-gel horseradish peroxidase biosensor for hydrogen peroxide detection by chemiluminescence

Horseradish peroxidase (HRP) was immobilized by microencapsulation in sol‐gel crystals derived from tetramethyl orthosilicate. A sol‐gel biosensor based on the chemiluminescence reaction of the hydrogen peroxide‐luminol‐HRP system was developed. Calibration plots for hydrogen peroxide have been established by chemiluminescence measurements in a cuvette and through an optical fibre modified at its end with the immobilized HRP gel. The detection limit was 6.710 ˇ4 M and the linear range was 0.1‐3.0 mM. The relative standard deviation was 7.1% (na4). This method was applied to hydrogen peroxide determination in disinfectant solutions for contact lenses. # 1998 Elsevier Science B.V.

Free and sol-gel immobilized alkaline phosphatase-based biosensor for the determination of pesticides and inorganic compounds

Alkaline-phosphatase (ALP) catalyses the hydrolysis of 1-naphthyl phosphate to fluorescent 1-naphthol ( λex = 346 nm, λem = 463 nm). This enzymatic reaction was investigated in presence of inhibitors: organochlorine (tetradifon), carbamate (metham-sodium) and organophosphorus pesticides (fenitrothion), heavy metal (Ag + ) and CN − . The fluorescent signal, which is inversely dependent on the inhibitor concentration, is related to the amount of the inhibitor. Detection limits between 4.1 M for tetradifon and 91.2M for metham-sodium were found. The relative standard deviation (R.S.D.) was between 2.6 and 6.2%. Sol–gel matrices derived from tetramethyl orthosilicate were doped with ALP using microencapsulation. The response of the biosensor based ALP sol–gel encapsulated to 1-naphthyl phosphate was reproducible (R.S.D. = 6.6%). Inhibition plots obtained for test pesticides (metham-sodium and tetradifon) display linear calibration in the ranges 194–774 M and 3.5–28M, detection limits of 4.9 and 292.3M and R.S.D. of 3.9 and 7.3% for metham-sodium and tetradifon, respectively. The results show that the system is able to detect class compounds such as pesticides and inorganic compounds.

Sol-gel cholinesterase biosensor for organophosphorus pesticide fluorimetric analysis

Abstract Sol-gel crystals derived from tetramethyl orthosilicate (TMOS) doped with cholinesterase using microencapsulation have been prepared. Organophosphorus pesticides (ORPs) are determined by fluorimetric detection based on enzymatic inhibition of immobilized cholinesterase; the percentage inhibition is correlated to pesticide concentration. The working substrate concentration (indoxyl-acetate) for ORP determination, 1.63 × 10−4 M, is optimized by selecting a linear range in a plot of substrate concentration against signal. Precision as a relative standard deviation (RSD) is 10.2% (n = 7) for the working substrate concentration. Fenitrothion, azinphos-ethyl, methidation, naled and mecarbam are analysed. Calibration plots have been established by measuring in a cuvette and through an optical fibre modified at its end with the immobilized cholinesterase gel. The biosensor performance is evaluated by measuring the percentage inhibition covering concentration ranges from 1.21–11.99 μg ml−1 (naled) up to 4.9–328.9 μg ml−1 (mecarbam). RSDs from 1.5 to 10.3% and detection limits from 0.12 (naled) to 57.6 μg ml−1 (methidation) have been achieved. Mass production and good storage for one month of the immobilized enzyme render the gel disposable after a single use. ORP determination is fast (inhibition time of 5 min) and the enzyme immobilization directly onto the end of the optical fibre enhances the sensitivity.

Hydrogen peroxide assay by using enhanced chemiluminescence of the luminol-H2O2-horseradish peroxidase system: Comparative studies

Abstract p-Iodophenol, p-coumaric acid, phenol and aniline are enhancers of the luminol-H2O2-horseradish peroxidase chemiluminescence. These enhanced and unenhanced chemiluminescences were used to assay low concentrations of hydrogen peroxide. The curves of chemiluminescence intensity maxima against hydrogen peroxide concentration were fitted to third order polynomial regressions, which permit a very good approach to the experimental results. Double the sensitivity (RSD ≤ 4.3%) and a slightly lower detection limit (e.g. 0.08 μM with p-coumaric acid of 0.18 μM without enhancer) were obtained with enhanced chemiluminescence than with unenhanced chemiluminescence.

Enhanced chemiluminescence biosensor for the determination of phenolic compounds and hydrogen peroxide

The enhanced chemiluminescence (CL) reaction of the luminol‐H2O2‐horseradish peroxidase (HRP) system with immobilized HRP using microencapsulation in a sol‐gel matrix has been used to develop a biosensor for p-iodophenol, p-coumaric acid, 2-naphthol and hydrogen peroxide. The detection limits obtained for p-iodophenol, p-coumaric acid and 2-naphthol were 0.83 mM, 15 and 48 nM, respectively. Direct enzyme immobilization onto the end of the optical fibre permits the construction of a remote enhanced CL biosensor. This remote biosensor has been applied to hydrogen peroxide assay (detection limit 52.2 mM and relative standard deviation 4.7% for na 4) and the results have been compared with the biosensor developed in cuvette. # 2001 Elsevier Science B.V. All rights reserved.

Phenol derivatives as enhancers and inhibitors of luminol–H2O2–horseradish peroxidase chemiluminescence

Systematic studies on phenol derivatives facilitates an explanation of the enhancement or inhibition of the luminol‐H2O2‐horseradish peroxidase system chemiluminescence. Factors that govern the enhancement are the one-electron reduction potentials of the phenoxy radicals (PhO ∞ /PhOH) vs. luminol radicals (L ∞ /LH ˇ ) and the reaction rates of the phenol derivatives with the compounds of horseradish peroxidase (HRP-I and HRP-II). Only compounds with radicals with a similar or greater reduction potential than luminol at pH 8.5 (0.8 V) can act as enhancers. Radicals with reduction potentials lower than luminol behave in a different way, because they destroy luminol radicals and inhibit chemiluminescence. The relations between the reduction potential, reaction rates and the Hammett constant of the substituent in a phenol suggest that 4-substituted phenols with Hammett constants (s) for their substituents similar or greater than 0.20 are enhancers of the luminol‐H2O2‐horseradish peroxidase chemiluminescence. In contrast, those phenols substituted in position 4 for substituents with Hammett constants (s) lower than 0.20 are inhibitors of chemiluminescence. On the basis of these studies, the structure of possible new enhancers was predicted.

Thin-layer chromatography and fibre-optic fluorimetric quantitation of thiamine, riboflavin and niacin

Thiamine (vitamin B,), riboflavin (vitamin B,) and niacin (nicotinic acid) were separated by thin-layer chromatography and fluorimetrically determined by using a commercially available fibre-optic-based instrument. Under Ruorimetric monitoring riboflavin shows native fluorescence, but nicotinic acid and thiamine had to be pre-chromatographically converted to fluorescent derivatives. A new fluorescent tracer, fluoresceinamine, isomer II, was used to label the nicotinic acid. Thiamine was converted to fluorescent thiochrome by oxidizing with potassium ferricyanide solution in aqueous sodium hydroxide. The analytes were separated on HPTLC silica gel plates using methanol-water (70:30, v/v) as mobile phase. In these conditions the R, values for the thiamine, riboflavin and niacin derivatives were, respectively, 0.73, 0.86 and 0.91. The developed plate was scanned by a bifurcated fibre-optic that both transmits emission radiation to the plate and collects the emission signal to the fluorimeter. Calibration curves for the determination of thiamine 300-750 ng, riboflavin 48-320 ng and niacin 10-100 ng were established.

Enhancer effect of fluorescein on the luminol–H2O2–horseradish peroxidase chemiluminescence: energy transfer process

The chemiluminescence of the luminol-H2O2-horseradish peroxidase system is increased by fluorescein. Fluorescein produces an enhancement of the luminol chemiluminescence similar to that of phenolphthalein, by an energy transfer process from luminol to fluorescein. The maximum intensity and the total chemiluminescence emission (between 380 and 580 nm) of luminol with fluorescein was more than three times greater than without fluorescein; however, the emission duration was shorter. The emission spectra in the presence of fluorescein had two maxima (425 and 535 nm) and the enhancement was dependent on pH and fluorescein concentration. A mechanism is proposed to explain these effects.

Horseradish peroxidase sol–gel immobilized for chemiluminescence measurements of alkaline-phosphatase activity

Naphthyl phosphate is a pro-enhancer of the luminol-H2O2-horseradish peroxidase reaction. Alkaline-phosphatase hydrolyses the phosphate group and produces 2-naphthol. This compound is an enhancer of the chemiluminescence (CL) reaction. The influence of 2-naphthyl phosphate and the incubation time on the chemiluminescence reaction, have been studied. The horseradish peroxidase (HRP) was immobilized in a sol-gel matrix, obtaining a biosensor for alkaline-phosphatase. The relative standard deviations using free HRP in solution and the immobilized one were 3.2 (n ¼ 4) and 9.3% (n ¼ 3), respectively. The detection limits for alkaline-phosphatase using free and immobilized HRP were 128 and 106 mU/ml, respectively. # 2002 Elsevier Science B.V. All rights reserved.

Micellar liquid chromatography of plant growth regulators detected by derivative fluorometry

Abstract A micellar liquid chromatographic method with fluorimetric detection was developed for the determination of the plant growth regulators indol 3-yl acetic acid (IAA), 2-(1-naphthyl) acetic acid (1-NAA), indol 3-yl propionic acid (IPA), 2-(2-naphthyl) acetic acid (2-NAA), indol 3-yl butyric acid (IBA), 2-(1-naphthyl) acetamide (1-Namide) and indol 3-yl acetic acid ethyl ester (IAA ethyl ester). Extraction of plant hormones was performed with methanol, followed by purification with ethyl acetate, solid-phase extraction and again partitioning against diethyl ether. Sodium dodecylsulphate (10 mM) is used as mobile phase to elute the compounds in a maximum run-time of 23 min. Partially or unresolved peaks are separated by calculation of the 1st derivative chromatogram. Detection limits are between 0.30 μg g−1 and 1.10 μg g−1.