Jin-Gou Xu

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.

Preparation of pH-sensitive polymer by thermal initiation polymerization and its application in fluorescence immunoassay

A fluorescence immunoassay for human IgG (Ag) was developed using a pH-sensitive polymer prepared by thermal initiation or redox initiation polymerization as a carrier. In the competitive immunoassay, appropriate quantity of Ag was immobilized on the polymer and the standard Ag (or sample) solution, and a constant amount of fluorescein isothiocyanate labeled goat anti-human IgG antibody (Ab-FITC) was added. Immobilized Ag and the standard (or sample) Ag competed for binding to the Ab-FITC in 37 degrees C in homogeneous format. After changing the pH to separate the polymer-immune complex precipitate, it was re-dissolved and determined by fluorescence method. The results showed that the immobilization efficiency, immunological reaction activities of immobilized Ag and phase transition pH range were improved as Ag was immobilized by thermal initiation instead of redox initiation polymerization. Under optimum conditions, the calibration graphs for the Ag in both methods, thermal initiation and redox initiation, were linear over the concentration range of 0.0-1000ngmL(-1), with detection limits 8 (thermal initiation) and 12ngmL(-1) (redox initiation), respectively. Moreover, some pH-sensitive polymer prepared only in organic solvent or under high temperature could also be used as an immunoreaction carrier by thermal initiation polymerization. Thermal initiation polymerization was a better immobilization mode.

Nanometer fluorescent hybrid silica particle as ultrasensitive and photostable biological labels

Nanometer-sized fluorescent hybrid silica (NFHS) particles were prepared for use as sensitive and photostable fluorescent probes in biological staining and diagnostics. The first step of the synthesis involves the covalent modification of 3-aminopropyltrimethoxysilane with an organic fluorophore, such as fluorescein isothiocyanate, under N2 atmosphere for getting a fluorescent silica precursor. Then the NFHS particles, with a diameter of well below 40 nm, were prepared by controlled hydrolysis of the fluorescent silica precursor with tetramethoxysilane (TMOS) using the reverse micelle technique. The fluorophores are dispersed homogeneously in the silica network of the NFHS particles and well protected from the environmental oxygen. Furthermore, since the fluorophores are covalently bound to the silica network, there is no migration, aggregation and leakage of the fluorophores. In comparison with common single organic fluorophores, these particle probes are brighter, more stable against photobleaching and do not suffer from intermittent on/off light emission (blinking). We have used these newly developed NFHS particles as a fluorescent marker to label antibodies, using silica immobilization method, for the immunoassay of human alpha-fetoprotein (AFP). The detection limit of this method was down to 0.05 ng mL(-1) under our current experimental conditions. We think this material would attract much attention and be applied widely in biotechnology.

A cyclometalated palladium-azo complex as a differential chromogenic probe for amino acids in aqueous solution

Solutions of a cyclometalated palladium-azo complex exhibited differential UV-Vis absorption spectra in the presence of alpha-amino acids with different side chain groups.

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 Thiamine as A Fluorogenic Substrate in the Determination of Hydrogen Peroxide Based on the Catalytic Effect of Hemin

Abstract A rapid and sensitive fluorimetric method has been developed for the determination of trace hydrogen peroxide (0–5.0 × 10−6 mol/l) and, indirectly, glucose (3.0 × 10−7 – 3.5 × 10−5 mol/l), based on the catalytic effect of hemin on the thiamine, a new fluorogenic substrate, and hydrogen peroxide system at pH=8.0. Using the fixed time method, the detection limits of hydrogen peroxide and glucose are 3.0 × 10−8 mol/l and 3.0 × 10−7 mol/l, respectively. This method is simple, inexpensive and has been applied successfully to the determination of glucose in human sera with relative standard deviation of 2.4–3.1%. ∗The State Laboratory for Tumor Cell Engineering. Department of Biology, Xiamen University. Xiamen 361005, P.R. China.

Determination of DNA and RNA by their quenching effect on the fluorescence of the Tb3+-Tiron complex

Abstract The fluorescence quenching of the Tb 3+ -Tiron complex by nucleic acids is reported. Studies involving calf thymus(CT) DNA, salmon(SM) DNA, herring sperm(HS) DNA and yeast RNA revealed that they compete with Tiron for the Tb 3+ ion, which resulted in the fluorescence quenching of the Tb 3+ -Tiron complex because the Tb 3+ -nucleic acid complex is non-fluorescent under the same conditions. The ratio of molar concentrations of Tb 3+ and Tiron appeared to be a key factor, a Tb 3+ : Tiron ratio of 3: 10 being the most suitable. The maximum fluorescence was produced at pH 6.9, with maximum excitation and emission wavelengths at 317 and 546 nm, respectively. Under optimal conditions, the differential value of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range of 0.005–1.0 μg ml −1 for CT DNA, SM DNA, HS DNA, and 0.005–0.7 μg ml −1 yeast RNA. The detection limits were 1 ng ml −1 for CT DNA, 1 ng ml −1 for SM DNA, 0.9 ng ml −1 for HS DNA, and 0.6 ng ml −1 for yeast RNA, respectively. The relative standard deviations (6 replicates) were within 2.5% in the middle of the linear range. The mechanism for the fluorescence quenching was also studied.

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.