Dong-Hui Li

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 NB 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 NB 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 micrograms mL-1 and 27 ng mL-1-10 micrograms 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.

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) tetrasulfonatophthalocyanine (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 × 10–8 to 1.0 × 10–4 mol L–1, with a detection limit of 4.3 × 10–9 mol L–1. The relative standard deviation was 2.2% for 2.0 × 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 B1, vitamin B complex and rice.

Determination of nucleic acids based on shifting the association equilibrium between tetrasulfonated aluminium phthalocyanine and Acridine Orange

Based on the ability of nucleic acids to shift the association equilibrium of the ion-association complex of Acridine Orange and tetrasulfonated aluminium phthalocyanine, thus leading to an increase in the phthalocyanine fluorescence, a method is suggested for the fluorimetric determination of nucleic acids. Investigations were carried out on the spectral characteristics, order of addition of reagents, selection of the buffer system, effect of pH, influence of reaction time, effect of salt, the usage of reagents, interference of foreign substances and the effect of different acridine derivatives. Under the optimum conditions, the calibration graphs for the determination of calf thymus DNA (CT DNA), salmon DNA (SM DNA) and yeast RNA were linear over the ranges 0.04-1.2, 0.04-1.2 and 0.1-1.2 micrograms cm-1, respectively. The detection limits for CT DNA, SM DNA and RNA were 17, 24 and 98 micrograms cm-3, respectively. The relative standard deviation (n = 6) was within 4.6% for the detection of samples. The method was applied to the determination of Staphylococcus aureus DNA and the result was in agreement with that achieved by a UV method.

Determination of proteins at nanogram levels by a resonance light-scattering technique with tetra-substituted sulphonated aluminum phthalocyanine

This is the first report on the determination of proteins with tetra-substituted sulphonated aluminum phthalocyanine (AlS(4)Pc) by resonance light-scattering (RLS). At pH 3.0, the weak RLS of AlS(4)Pc can be enhanced by the addition of proteins. Based on this, a novel quantitative method has been developed for the determination of proteins in aqueous solutions. Under optimal conditions, the linear ranges of the calibration curves were 0.050-2.0 mug ml(-1) for both human serum albumin (HSA) and human r-IgG. The detection limits were 12.7 ng ml(-1) for HSA and 16.1 ng ml(-1) for human r-IgG. The method has been applied to the analysis of total protein in human serum samples collected from the hospital and the results were in good agreement with those reported by the hospital, which indicates that the method presented here is not only sensitive, simple, but also reliable and suitable for practical applications.

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 pthalocyanine, 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.

A SENSITIVE FLUORIMETRIC METHOD FOR THE DETERMINATION OF NITRITE AND NITRATE IN SEAWATER BY A NOVEL RED-REGION FLUORESCENCE DYE

A novel method for the determination of dissolved inorganic nitrite and nitrate in surface seawater was reported, which is based on the diazotization reaction between nitrite and a novel red-region fluorescent dye, tetra-substituted amino aluminum phthalocyanine (TAAlPc). Nitrate is determined as nitrite after reduction on a cadmium column. Under optimal conditions, the linear range of the calibration curve is 21–840 nM for nitrite. The detection limit is 7 nM for and the relative standard deviation for six replicate measurements of a 350 nM nitrite solution is 3.2%. Since the method employs a red-region fluorescent dye (λex/em = 610 nm/678 nm), the potential fluorescent background resulting from the organic matters in seawater can be eliminated and thus the sensitivity of the method can be enhanced.

Brilliant cresyl blue as a new red region fluorescent probe for determination of nucleic acids

Abstract A fluorescence quenching method was developed for determination of microamounts of nucleic acids by using brilliant cresyl blue (BCB) as a new red region fluorescent probe. In aqueous hexylmethylene tetramine solution, BCB showed maximum excitation and emission wavelengths at 626 and 670 nm, respectively, and the fluorescence of BCB could be greatly quenched by DNA (or RNA). Under optimal conditions, the calibration graphs are linear over the range of 0.02–0.80 μg/ml for SM DNA and 0.25–1.5 μg/ml for yeast RNA. The corresponding detection limits are 7 ng/ml for SM DNA and 25 ng/ml for yeast RNA, respectively. SM DNA can be determinated in the presence of 40% (w/w) RNA, and the relative standard deviation of six measurements is 2.5% for 500 ng/ml SM DNA. The result of the determination of golden staphylococcus DNA by this method was satisfactory.

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

ABSTRACT 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 1.4x10−8mol/L. The appl...

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

The determination of the fluorophore to protein molar ratio is an important part of fluorescent antibody techniques. A rapid and homogeneous method for the determination of fluorophore to protein molar ratio based on the fluorescence anisotropy variation of the fluorophore after reacting with protein is presented. In the work, fluorescein isothiocyanate (FITC) and bovine serum albumin (BSA) were chosen to form a FITC/BSA conjugate. In a series of reacting mixture solutions, the concentration of FITC was kept constant, while the concentration of BSA was varied, and the fluorescence anisotropies of the reacting mixture solutions were measured. By plotting the fluorescence anisotropy of FITC versus the logarithm value of the concentration of BSA, an important parameter which refers to the fluoresecence anisotropy when the fluorophore in the system was bound completely by protein, can be obtained. Combined with another parameter, fluorescence anisotropy in the absence of protein, the fraction of bound fluorophore in the samples can be achieved, and then the molar ratio of flurochrome to protein can be calculated. This method has been employed to the analysis of real samples, and the results well agree with that obtained by traditional method, indicating that this method is not only simple and rapid but also reliable, and can be possibly applied in practice.

Determination of gaseous acidic compounds by measuring fluorescence after their reaction with tetra-substituted amino aluminum phthalocyanine

Based on the fluorescence enhancement of a red-region fluorescent dye, tetrasubstituted amino aluminum phthalocyanine (TAAlPc), in strongly acidic medium, a new method was developed for the detection of four strong acids (HCl, HBr, HNO(3) and H(2)SO(4)). Under optimal conditions the linear ranges of the calibration curves were 0.04-0.67 mol/l (HCl), 0.04-0.67 mol/l (HBr), 0.04-0.80 mol/l (HNO(3)) and 0.02-0.80 mol/l (H(2)SO(4)), respectively. The detection limits were 0.007 mol/l for HCl, 0.006 mol/l for HBr, 0.005 mol/l for HNO(3) and 0.007 mol/l H(2)SO(4.) This method has been applied to the analyses of four artificial samples with satisfactory results.