Temperature modulated solubility and activity alteration for oligo-(N-isopropylacrylamide)-iron tetrasulfonatophthalocyanine conjugates as a new mimetic peroxidase

Iron tetrasulfonatophthalocyanine (FeTSPc) was covalently bound to the terminus of a temperature sensitive oligomer, oligo-N-isopropylacrylamide (ONIPAAm), to form a new mimetic enzyme (ONIPAAm-FeTSPc) to; mimic the peroxidase activity of horseradish peroxidase. This FeTSPc-based mimetic enzyme exhibits a lower critical solution temperature (LCST) of 32 degrees C in neutral solution. It precipitates from water above the LCST and redissolves when the solution temperature is lowered below the LCST. The peroxidase activity of this mimetic enzyme was studied based on its catalytic effect on the reaction of p-hydroxyphenylpropionic acid and H2O2 The results show that the peroxidase activity of the new mimetic enzyme is higher than that of the free FeTSPc. The possibility of its application in the analytical field was also tested by the determination of H2O2 and ONIPAAm-FeTSPc; the detection limits are 8.2 x 10(-9) and 1.7 x 10(-9) mol L-1, respectively

Mimetic-enzyme fluorescence immunoassay using a thermal phase separating polymer

Poly-N-isopropylacrylamide (PNIP), a water-soluble, thermally precipitated synthetic polymer, has been conjugated together with a monoclonal antibody and utilized as a novel separation method for an immunoassay. PNIP precipitates out of water above a critical temperature of 31 degrees C, enabling a polymer-bound immune complex to be separated from the solution. These characteristics were used to develop a novel polymer-mimetic enzyme immunoassay method for determination of alpha-1-fetoprotein (AFP) with hemin as a labeling reagent to catalyze the reaction of p-hydroxyphenyl acetic acid (HPA) and hydrogen peroxide in alkaline medium. After a one-step competitive immunoreaction, the polymer-antibody-antigen-hemin conjugate moiety was determined by coupling the fluorogenic reaction of HPA and hydrogen peroxide, The calibration graph for AFP was linear over the range of 0-380 ng cm(-3) with a detection limit of 1.0 ng cm(-3). This method combines some advantages of both homogeneous and heterogeneous immunoassays, and has been applied to determine AFP in human blood serum with satisfactory results

Application of a novel fluorescence probe in the determination of nucleic acids

A novel fluorimetric method has been developed for rapid determination of DNA and RNA with hypocrellin A (HA) as a fluorescence probe, based on the fluorescence enhancement of HA in the presence of DNA or RNA. Maximum fluorescence is produced in the pH range 3.4-4.0, with maximum excitation and emission wavelengths at 470 and 600 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range 0-200.0 ng cm(-3) for calf thymus DNA and 13.0-200.0 ng cm(-3) for yeast RNA, respectively. The corresponding detection limits are 5.0 ng cm(-3) for calf thymus DNA and 13.0 ng cm(-3) for yeast RNA. The relative standard deviation of six replicate measurements is 4.5% for 100 ng cm(-3) calf thymus DNA, DNA could be determined in the presence of 20% m/m yeast RNA. The mechanism for the binding of HA to DNA is also studied

Study on fluorometric determination of hydrogen peroxide catalyzed by iron(III)-tetrasulfonato-phthalocyanine with thiamine hydrochloride as a substrate

Iron(III)-tetrasulfonatophthalocyanine(FeTSPc) has been used as a mimetic enzyme in the determination of hydrogen peroxide with thiamine hydrochloride as a fluorogenic substrate. The determinations were carried out in both acidic and basic environments, with different limits of detection and linear ranges. In acidic condition, the linear calibration graph was obtained from 5.0x10(-8) mol/L to 8.0x10(-6) mol/L, with a detection limit of 2.1x10(-8) mol/L H2O2 when Na2HPO4-citric buffer solution (pH 2.8) was used as the reaction medium. It was also found that using one of the three polybasic carboxylic acids such as citric acid, tartaric acid and malonic acid as the catalytic reaction medium can lead to particularly sensitive systems, permitting a detection limit as low as 3.5x10(-9) mol/L H2O2; whereas in basic reaction medium (Na2CO3-NaHCO3 buffer solution, pH = 10.0), the linear range of the calibration graph was from 5.0x10(-8) mol/L to 2.0x10(6) mol/L H2O2 with a detection limit of 1.4x10(-8) mol/L. The applicability of the method to the determination of glucose in human serum was demonstrated by investigating the recovery of the known glucose added to human serum

A new red-region substrate, tetra-substituted amino aluminium phthalocyanine, for the fluorimetric determination of H2O2 catalyzed by mimetic peroxidases

A new red-region fluorogenic substrate, tetra-substituted amino aluminium phthalocyanine, was developed for the selective determination of H2O2 based on the catalytic effect of mimetic peroxidases, viz., hemin or iron tetrasulfonatophthalocyanine (FeTSPc). Under the optimum conditions, the linearity of the calibration graph for the determination of H2O2 with hemin (or FeTSPc) as the catalyst was in the range from 0.0 to 3.0 X 10(-7) mol L-1 (or from 0.0 to 2.0 x 10(-6) mol L-1). The detection limits were 3.7 X 10(-9) and 4.9 x 10(-9) mol L-1 H2O2, respectively. The relative standard deviation (n = 7) was within 1.5% in the middle of the linear range. The peroxidase activity of the mimetic enzymes hemin and FeTSPc, the effects of some experimental conditions and the influence of foreign substances were investigated. With this substrate, 0.0-7.5 x 10(-8) mol L-1 hemin and 0.0-2.0 x 10(-6) mol L-1 FeTSPc can be determined with an accuracy and precision of about 1.3%. The potential application of the reagent was tested by the determination of H2O2 in rainwater

Sensitive fluorimetric determination of formaldehyde by the co-quenching effect of formaldehyde and sulfite on the fluorescence of tetra-substituted amino aluminium phthalocyanine

A novel and sensitive fluorimetric method was developed for the determination of formaldehyde based on the co-quenching effect of formaldehyde and sulfite on the fluorescence of tetra-substituted amino aluminium phthalocyanine. Formaldehyde in the concentration range 0.040-1.19 mug ml(-1) can be determined with a limit of detection of 7.5 ng ml(-1). The relative standard deviation for nine replicate measurements of 80.0 ng ml(-1) formaldehyde is 1.8%. The method was applied to the analysis of real samples with satisfactory results

Novel spectrofluorimetric method for the determination of thiamine with iron(III) tetrasulfonatophthalocyanine as a catalyst

A sensitive, selective and rapid spectrofluorimetric method is proposed for the determination of thiamine by using mimetic enzyme iron(iii) tetrasulfonatophthalocynanine (FeTSPc) as a catalyst for the oxidation reaction between thiamine and hydrogen peroxide. It is based on the oxidation of thiamine in alkaline medium to give an intensively fluorescent compound, which has an excitation wavelength of 375 nm and an emission wavelength of 440 nm. The determination was found to be activated by fluorogenic substrates with a p-hydroxyphenyl structure such as L-tyrosine, tyramine and p-hydroxyphenylpropionic acid. Under optimum conditions, the responses for thiamine were linear from 1.0 x 10(-8) to 1.0 x 10(-4)mol L-1, with a detection limit of 4.3 x 10(-9) mol L-1. The relative standard deviation was 2.2% for 2.0 X 10(-7) mol L-1 thiamine (n = 6). The activation of the p-hydroxyphenyl substrates, the effects of some experimental conditions and the influence of foreign substances were investigated. The potential application of the method was tested by selectively determining thiamine in commercial vitamin B-1, vitamin B complex and rice

Interaction of a novel red-region fluorescent probe, Nile Blue, with DNA and its application to nucleic acids assay

A novel fluorimetric method was developed for the rapid determination of DNA and RNA based on their quenching effect on the cationic red-region fluorescent dye Nile Blue (NB) In the investigation of the interaction of NE with DNA by steady-state polarization measurements, thermal denaturing study, determination of absorption and fluorescence characteristics, salt effect study and electrophoresis experiments, the results supported the suggestion that NE served as an intercalator to the stack base pairs of nucleic acids. Further evidence showed that the quenching could be ascribed to the static quenching mode. A binding constant of about 10(6) M-1 and a binding site size of about three base pairs were obtained by spectral methods. Under optimum conditions, the calibration curves for the determination of calf thymus DNA (CT DNA) and yeast RNA were linear over the ranges 3.0 ng mL(-1)-2.0 mu g mL(-1) and 27 ng mL(-1)-10 mu g mL(-1), respectively, The detection limits were 3.0 ng mL(-1) for CT DNA and 27 ng mL(-1) for RNA. The relative standard deviation (n = 6) was within 2.1% in the middle of the linear range. Interferences from some interesting co-existing substances in the determination of DNA were also examined

Imaging Oxygen Distribution in Marine Sediments. The Importance of Bioturbation and Sediment Heterogeneity

The influence of sediment oxygen heterogeneity, due to bioturbation, on diffusive oxygen flux was investigated. Laboratory experiments were carried out with 3 macrobenthic species presenting different bioturbation behaviour patterns:the polychaetes Nereis diversicolor and Nereis virens, both constructing ventilated galleries in the sediment column, and the gastropod Cyclope neritea, a burrowing species which does not build any structure. Oxygen two-dimensional distribution in sediments was quantified by means of the optical planar optode technique. Diffusive oxygen fluxes (mean and integrated) and a variability index were calculated on the captured oxygen images. All species increased sediment oxygen heterogeneity compared to the controls without animals. This was particularly noticeable with the polychaetes because of the construction of more or less complex burrows. Integrated diffusive oxygen flux increased with oxygen heterogeneity due to the production of interface available for solute exchanges between overlying water and sediments. This work shows that sediment heterogeneity is an important feature of the control of oxygen exchanges at the sediment–water interface

A rapid method for the determination of molar ratio of fluorochrome to protein techniques by fluorescence anisotropy detection

The Determination of molar ratio of fluorochrome to protein is an important part in fluorescent antibody techniques. The conventional method is time consuming and with troublesome manipulations. A rapid homogeneous method based on the anisotropy change of the fluorochrome after reacting with protein was presented here. In our experiments, fluorescein isothiocyanate (FITC) and bovine serum albumin(BSA) were chosen to form the FITC/BSA conjugate. A series of solutions containing the two components were prepared and allow to react according to the standard procedure. Fluorescence anisotropy of the mixtures were detected, a diagram of r-lgc is then obtained, where c is the concentration of protein. Because FITC in the mixture exists in both free form(F) and binding form(B), the fluorescence anisotropy observed is given by r = f(F)r(F) + f(B)r(B), where r(F) and r(B) refer to the anisotropy of free and bound FITC, respectively; and f(F) and f(B) represent the fraction of the free and bound forms, respectively, f(F) + f(B) = 1. A formula as f(B) = (r-r(F))/(r(B)-r(F)) is achieved. Here r(B) correspods to the value of r in the upper platform region of the r-lgc curve, where FITC is bound to protein completely, while r(F) can be obtained by the determination of anisotropy of FITC in the absence of BSA. So, for each of the mixtures, the binding fraction of FITC can then be calculated. Correspondingly, the content of bound form of FITC that was used to calculate the molar ratio of FITC to BSA can be gained. Since the method avoids the tiresome separation procedure, it bears the merit of time saving and can be used to estimate the molar ratio of fluorochrome to protein rapidly. The determination results of samples by this method were compared with that got from a spectrophotometric analysis. The results were in good agreement