Qing-Zhi Zhu

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.

Flow injection fluorescence immunoassay for gentamicin using sol-gel-derived mesoporous biomaterial

Sol-gel-derived mesoporous biomaterials were used for the first time in the flow-injection fluorescence immunoassay system. Anti-gentamicin antibody was immobilized in a mesoporous sol-gel material using tetramethoxysilane as a precursor and poly(ethylene glycol) as a template. The sol-gel glass was used to develop an immunoaffinity column for the flow-injection immunoassay of gentamicin. Little unspecific adsorption of gentamicin on the sol-gel and no antibody leaching under harsh elution conditions were found. The immunoassay is based on the competition between gentamicin and fluorescein isothiocyanate-labeled gentamicin for a limited number of encapsulated antibody binding sites. NaOH solution of 5 x 10(-3)mol/L is used for the regeneration of encapsulated antibody binding sites after each measurement, which allows the immunoreactor to be used for up to 20 times without any loss of reactivity. Sample preconcentration is not needed and a single assay can be performed within 10 min. The calibration for gentamicin has a working range of 250-5000 ng/mL with a detection limit of 200 ng/mL, which is close to that of the fluorescence immunoassay and fluorescence polarization immunoassay using the same reactants. Comparison of the results from this method with that obtained from HPLC showed an excellent correlation.

Study on the interaction between rivanol and DNA and its application to DNA assay

Abstract Rivanol (RVN) binds to the double helical DNA with a high affinity, as deduced from the absorption and fluorescence spectral data. Extensive hypochromism and red shifts in the absorption spectra were observed when RVN binds to calf thymus DNA (CT DNA), which suggested the intercalation mechanism of RVN into DNA bases. Upon binding to DNA, the fluorescence from RVN was efficiently quenched by the DNA bases, with no shifts in the emission maximum. The large increases in the polarization upon binding to CT DNA supported the intercalation of RVN into the helix. Iodide quenching studies showed that the magnitude of Ksv of the free RVN was higher than that of the bound RVN. The results of competitive binding studies showed that RVN can be displaced by ethidium bromide. Thermal denaturation experiments exhibited that the quenching of the fluorescence from RVN by single strand (ssDNA) was smaller than that by double strand (dsDNA). The results of all above further studies also proved the intercalation of RVN into DNA base stack. Quenching of fluorescence from RVN by DNA can be employed for sensitive detection of DNA. The limit of detection for CT DNA was 16 ng ml−1.

Application of vitamin K3 as a photochemical fluorescence probe in the determination of nucleic acids

ABSTRACT An in situ photochemical fluorescence probe method for the determination of nucleic acids with vitamin k3(VK3) as the photochemical fluorescence probe was developed for the first time. It was based on the conversion of VK3 into an intensively fluorescent product on irradiating with UV radiation. The photochemical reaction is decelerated by nucleic acids. The determination can be carried out by measuring the fluorescence intensity at a fixed time. The calibration graph was linear in the range of 0– 1.5 μg/ml for CT DNA and 0–2.0μg/ml for yeast RNA, the limit of detection was 10 ng/ml for CT DNA and 26 ng/ml for yeast RNA. The kinetic behaviour of the photochemical reaction and the effects of some experimental conditions were investigated and discussed in detail. CT DNA could be determined in the presence of 40%(w/w) yeast RNA and yeast RNA was determined when the content of CT DNA in synthetic samples was below 6%(w/w).

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.

Vanadium uptake and translocation in dominant plant species on an urban coastal brownfield site

This study, conducted at a brownfield site in New Jersey, USA, investigated factors controlling V uptake and translocation in naturally assembled plant species. Six dominant species were collected from 22 stations in the study area. We found that V concentration in the plants decreased in a sequence of root>leaf>stem. No significant differences were found among the six dominant plant species in terms of root V uptake efficiency (V BCF) and V root to shoot translocation (V TF). Although soil pH and TOC did not show significant impact on V accumulation in the roots, soil labile V content showed significant positive linear correlation (p<0.05) with plant root V. Non-linear regression analysis indicates that V translocation efficiency decreases with increasing concentration in the soil, implying that excessive V in the soil might inhibit its absorption by the plant roots. Leaf V concentration was constant in all the plant species regardless of the variation in soil V concentration. The study shows that the six dominant plant species on site had limited amount of V translocated to the aerial part of the plant.

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.

Lead accumulation and association with Fe on Typha latifolia root from an urban brownfield site

Synchrotron X-ray microfluorescence and X-ray absorption near-edge microstructure spectroscopy techniques were applied to Typha latifolia (cattail) root sections and rhizosphere soils collected from a brownfield site in New Jersey to investigate lead (Pb) accumulation in T. latifolia roots and the role of iron (Fe) plaque in controlling Pb uptake. We found that Pb and Fe spatial distribution patterns in the root tissues are similar with both metals present at high concentrations mainly in the epidermis and at low concentrations in the vascular tissue (xylem and phloem), and the major Pb and Fe species in T. latifolia root are Pb(II) and Fe(III) regardless of concentration levels. The sequestration of Pb by T. latifolia roots suggests a potential low-cost remediation method (phytostabilization) to manage Pb-contaminated sediments for brownfield remediation while performing wetland rehabilitation.

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.