Solid-substrate room-temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing dibromofluorescein luminescent molecules and analytical application.

Luminescent 20-nm silicon dioxide nanoparticles containing dibromofluorescein (D; particles denoted D-SiO(2)) were synthesized by the sol-gel method. In the presence of Pb(Ac)(2) as a heavy atom perturber, the particle can emit the intense and stable room temperature phosphorescence(RTP) signal on a polyamide membrane at the wavelength of lambdaex(max)/lambdaem(max) = 460/623 nm for D. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with D-SiO(2) and human IgG can be carried out quantitatively on a polyamide membrane, and the phosphorescence intensity was evidently enhanced after the immunoreaction. Thus a new solid substrate-room temperature phosphorescence immunoassay (SS-RTP-IA) for determination of human IgG was established. The linear range of this method is 0.0624-20.0 pg spot(-1) of human IgG (corresponding to a concentration range of 0.156-50.0 ng ml(-1), sample volume: 0.40 microl spot(-1)). The regression equation of the working curve is DeltaIp = 94.39 + 17.00 m IgG (pg spot(-1)) (460/623 nm, r = 0.9998). Detection limit calculated as 3 Sb/k is 0.015 pg spot(-1). After elevenfold replicate measurement, RSD are 3.2% and 2.4% for samples containing 0.156 and 50.0 ng l(-1) IgG, respectively. This method is sensitive, accurate, and of high precision. And it has been applied to the determination of IgG in human serum with satisfactory results. Meanwhile, the mechanism of SS-RTP-IA based on an antibody labeled with nanoparticles containing dibromofluorescein luminescent molecules was discussed.

Comparison of properties on non-protected fluid room temperature phosphorescence of some tetra-ring aromatic hydrocarbons.

A comparative study of the photoluminescence properties of three kinds of tetra-ring aromatic hydrocarbon (1-sodium pyrenesulphonate, benz[alpha]anthracene and chrysene) solution in the absence of any protecting medium is described. It was found that a room temperature phosphorescence signal with different intensities can be induced for these solutions, using only TlNO3 or KI as a heavy atom perturber (HAP) and Na2SO3 as a deoxygenator. An appropriate amount of organic solvent added to the systems of pyrene, benz[alpha]anthracene and chrysene is necessary for increasing the solubility and phosphorescence intensity, and the preferable solvent is acetonitrile. For the pyrene, pyrenesulphonate and chrysene systems, a delayed excimer fluorescence accompanied with the room temperature phosphorescence (RTP) emission can be observed, but that for benz[alpha]anthracene cannot. The ratio of delayed excimer fluorescence and phosphorescence signals for pyrene, pyrenesulphonate and chrysene systems can be controlled by adjusting the concentration of luminophor, kinds and amount of both organic solvents and HAP. Under the optimal conditions, the RTP signals are proportional to the concentration of the four aromatic hydrocarbons, which means that the RTP properties of the four tetra-ring aromatic hydrocarbons can be used for quantitative analysis.

Determination of traces of bismuth by quenching of solid-substrate room-temperature phosphorescence from morin-labeled silicon dioxide nano-particles.

Silicon dioxide nano-particles, diameter 50 nm, containing morin (morin-SiO2) have been synthesized by the sol-gel method. They emit strong and stable room-temperature phosphorescence (SS-RTP) on filter paper as substrate, and bismuth can quench the intensity of the SS-RTP. On this basis a new morin-SiO2 solid-substrate room-temperature phosphorescence-quenching method has been established for determination of traces of bismuth. Reduction of phosphorescence intensity (DeltaI(p)) is directly proportional to the concentration of bismuth in the working range 0.16-14.4 ag spot(-1) (sample volume 0.40 muL spot(-1), corresponding to the concentration range 0.40-36.0 fg mL(-1)). The regression equation of the working curve is DeltaI(p)=14.86+5.279x[Bi3+] (ag spot(-1)) (n=6, r=0.9982). The detection limit of this method is 0.026 ag spot(-1) (corresponding to a concentration of 6.5 x 10(-17) g mL(-1)).This sensitive, reproducible and accurate method has been used for successful analysis of real samples.

Determination of human IgG by solid substrate room temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing Rhodamine 6G luminescent molecules.

Luminescent silicon dioxide nanoparticles (R-SiO2) with size of 50 nm containing Rhodamine 6G (R) were synthesized by sol-gel method. In the presence of Pb(Ac)2 as a heavy atom perturber, the particle can emit intense and stable room temperature phosphorescence signal of R, respectively, on polyamide membrane, with the lambda(ex)(max)/lambda(em)(max) = 470/635 nm for R. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with R-SiO2 and human IgG can be carried on polyamide membrane quantitatively, and the phosphorescence intensity was enhanced after the immunoreactions. Thus, a new method of solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA) for the determination of human IgG was established basing on antibody labeled with the nanoparticles containing binary luminescent molecules. The linear range of this method is 0.0624-20.0 pg spot(-1) of human IgG (corresponding concentration, 0.156-50.0 ng mL(-1); sample volume, 0.40 microL spot(-1)). The regression equations of working curves are delta I(p) = 88.16. + 16.79 m(IgG) (pg spot(-1)) (485/646 nm, r = 0.9997). Detection limits calculated by 3Sb/k are 0.017 pg spot(-1). For samples containing 0.156 and 50.0 ng mL(-1) of IgG, we measured repeatedly for 11 times, RSDs are 3.9 and 2.8%, respectively. This method is sensitive, accurate and of high precision.

Determination of trace mercury by solid substrate room temperature phosphorescence quenching method based on lead carboxymethyl cellulose (Pb(CMC)(2)) particles containing luminescent salicyl fluorones molecules.

Luminescent particles of lead carboxymethyl cellulose (Pb(CMC)(2)), which contains salicyl fluorones (THBF), Pb(CMC)(2)-THBF, were synthesized by sol-gel method. Pb(CMC)(2)-THBF can emit intense and stable solid substrate room temperature phosphorescence (SS-RTP) on filter paper. EDTA can chelate the Pb(2+) in Pb(CMC)(2)-THBF, causing it decompose into aqueous soluble components PbY(2-), CMC(-) and THBF, and these components can react with Hg(2+) to form (CMC)(2)Hg-THBF, causing decrease of phosphorescence intensity. Based on the facts above, a new method for the determination of trace mercury by SS-RTP quenching method was established. The linear range of this method is 2.0-40.0fgspot(-1) (5.0-100.0pgml(-1)) of Hg(2+), with a detection limit (LD) of 0.26fgspot(-1), and the regression equation of working curve is [Formula: see text] (fgspot(-1), 0.4mul spot(-1)), r = 0.9994. This method has been applied to the determination of trace mercury in water sample with satisfactory results. The mechanism of SS-RTP emission is also discussed.

Determination of human IgG by solid-substrate room-temperature phosphorescence immunoassay based on an antibody labeled with nanoparticles containing rhodamine 6G luminescent molecules.

Luminescent 50-nm silicon dioxide nanoparticles containing both types of rhodamine 6G (R; particles denoted R-SiO2) were synthesized by the sol-gel method. In the presence of Pb(Ac)2 as a heavy atom perturber the particle can emit the intense and stable room-temperature phosphorescence (RTP) signal of R on a polyamide membrane, with lambda(ex)max/ lambda(em)max=470/635 nm for R. Our research indicates that the specific immune reaction between goat-anti-human IgG antibody labeled with R-SiO2 and human IgG can be carried out quantitatively on a polyamide membrane, and the phosphorescence intensity was enhanced after the immunoreaction. Thus a new method for solid-substrate room-temperature phosphorescence immunoassay (SS-RTP-IA) for determination of human IgG was established on the basis of antibody labeled with the nanoparticles containing binary luminescent molecules. The linear range of this method is 0.0624-20.0 pg spot(-1) of human IgG (corresponding to a concentration range of 0.156-50.0 ng mL(-1), sample volume 0.40 microL spot(-1)). The regression equations of the working curves are DeltaIp = 71.27+7.208 m(IgG) (pg spot(-1)) (r = 0.9996). Detection limits calculated as 3 Sb/k are 0.022 pg spot(-1). Compared with the same IA using fluorescein isothiocyanate (FITC) as the marker the new method was more sensitive and had a wider linear range. After elevenfold replicate measurement RSD are 4.5 and 3.6% for samples containing 0.156 and 50.0 ng mL(-1) IgG, respectively. This method is sensitive, accurate, and of high precision.

Determination of human complement3 by solid substrate room temperature phosphorescence immunoassay with a labelled avidin-biotin bridge.

A solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA) for the determination of human complement3 (C3) based on a sandwich type assay and a labelled avidin-biotin (LAB) type assay was described. The anti-human complement3 and avidin were labeled with eosin5-isothiocyanate. On a polyamide membrane (PM), SS-RTP signals (lambda(ex)/lambda(em) = 535/678 nm) of immune complexes obtained by both assays were linear with the concentration of complement3 in the range of 6.25-100 ng/ml. The detection limits are 1.37 ng/ml for sandwich assay and 2.74 ng/ml for labeled avidin-biotin assay. (For a sample volume of 0.4 microl per spot, the mass detection limits are 0.546 and 1.09 pg/spot, respectively. If the molecular weight of human complement3 is 185,000, the detection limits are 2.95 and 5.91 amol per spot.) The results of determination of complement3 in 20 human sera obtained by labeled avidin-biotin SS-RTP-IA are correlated well with those obtained by ELISA. This study shows that SS-RTP-IA by whichever direct, sandwich or labeled avidin-biotin type assay can combine very well the characteristics of both the high sensitivity of SS-RTP and specificity of the immunoreaction.

Determination of trace mercury by solid substrate-room temperature phosphorimetry based on catalytic effect of Hg(2+) on formation of the ion association complex [Fe(bipy)(3)](2+)[(FinBr(4))(2)](2-).

A new solid substrate-room temperature phosphorimetry method for the determination of trace mercury has been established. It bases on the fact that in acetic acid medium, Hg(2+) ion can catalyze the substitute reaction of CN(-) ligand in [Fe(CN)(6)](4-) by 2,2'-bipyridyl (bipy), and the resultant [Fe(bipy)(3)](2+) cation can react with FinBr(4)(-) anion of tetrabromofluorescein (HFinBr(4)) to form ion association complex [Fe(bipy)(3)](2+)[(FinBr(4))(2)](2-) which can emit phosphorescent signal on filter paper substrate. Under the optimum condition, the linear dynamic range of this method is 1.6-16fg per spot with a detection limit (LD) of 0.18fg per spot (0.4mul sample solution per spot), and the regression equation of working curve is DeltaI(p)=1.058+7.671 C(Hg(2+)) (fg per spot ), n=7, correlation coefficient is 0.9990. This method has been applied to the determination of trace mercury in hair and cigarette samples with satisfactory result. The reaction mechanism for formation of the ion association complex is also discussed.

[The medium effects of fluorescence of eosin-5-isothiocyanate as a luminescent marker].

In this paper, the medium (pH, organic solvent and surfactant) effects on fluorescence of eosin-5-isothiocyanate (eosin-ITC) have been studied in detail. The results show that the pH and polarity of system studied are two great important influence factors on fluorescence of eosin-ITC. The fluorescence intensity of eosin-ITC in weak acidic medium is higher obviously than that of in weak basic medium, but it is sensitive to change of pH, be careful to use. The reaction between surfactant and eosin-ITC shows a good electric selectivity, and the effect of CTMAB on fluorescence of eosin-ITC can be classified as two stages: monomer quenching (below cmc) and micelle sensibilization (above cmc). In the presence of micelle of CTMAB, the fluorescence intensity of eosin-ITC in weak basic medium is even higher than that of in weak acidic medium. In the presence of high organic solvent content, the fluorescence intensity of eosin-ITC is also increased obviously. These properties are available for use.

Medium effects on fluorescence of ciprofloxacin hydrochloride.

The medium (pH, organic solvents, cyclodextrin (CD) or surfactants) effects on the fluorescence of ciprofloxacin hydrochloride (CPFX.HCl) were studied in detail. It is found that the three acid constants of ciprofloxacin (CPFX) are near to each other. Therefore the relation curve between pH and fluorescence intensity has no strident change and keeps relative stable in the pH range of 2-7. When pH was in the range of 5.5-6.0, the fluorescence intensity of CPFX reached the max. The kind and amount of organic solvent added to the luminescent system have various effects. Ethanol quenched fluorescence and the fluorescence excitation wavelength is red shift at first and then blue shift. Acetone has complicated effects on the fluorescence properties of CPFX.HCl solution. The experiment result shows that acetone is really a quencher when its volume content in the system is from 0 to 20%, but when its content is 90%, the signal intensity is unexpectedly one and a half times as much as that of no acetone. This means that there is a strong interaction between the acetone and CPFX; CPFX.H(+) could be included into the gamma-CD but the capping effect is not notable. The effect of cationic surfactant cetyltrimethylammonium bromide and non-ionic surfactant TX-100 and TX-80 on CPFX fluorescence was unimpressive, but the anionic surfactant's effect is aberrant. The fluorescence intensity of CPFX.HCl solution experiences three stages of increasing, decreasing and increasing in turn, as sodium dodecyl sulfate is adding gradually. But for sodium lauryl sulfonate, there are only two stages of decreasing and increasing with the concentration increasing. It is problematic to illustrate clearly the effect mechanism of acetone and anionic surfactant at present. Undoubtedly, the experimental results in this paper should be useful in practice works and the research is worth studying still further.

Syntheses of steroid-based molecularly imprinted polymers and their molecular recognition study with spectrometric detection.

Recognition of five steroid compounds, beta-estradiol, ethynylestradiol, estradiolbenzoate, testosterone and methyltestosterone were studied using a synthesized molecularly imprinted polymer (MIP). When beta-estradiol was used as the template molecule, the polymer was synthesized with methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross linking agent through non-covalent interactions. It is found that the kind of porogen solvent and the polymerization conditions greatly affected the binding ability of a MIP to a certain molecule. Releasing of the template was performed by continuous extraction with methanol containing 10% acetic acid in a Soxhlet extractor. Our results indicated that such carefully synthesized MIP showed specific affinity toward beta-estradiol in the adsorption process.

Studies on properties and application of non-protected room temperature phosphorescence of propranolol.

A direct and simple non-protected room temperature phosphorimetry (NP-RTP) for determine propranolol, which using I- as a heavy atom perturber and sodium sulfite as a deoxygenator, has been developed. The phosphorescence peak wavelength maxima lambda(ex)/lambda(em) = 288/494, 522 nm. The analytical curve of propranolol gives a linear dynamic range of 8.0 x 10(-8)-2.0 x 10(-5) mol l(-1) and a detection limit of 3 x 10(-8) mol l(-1). The influence of I- concentration on RTP lifetime of propranolol was studied and the luminescence kinetic parameters were calculated. It is found that the relation between I- concentration (x) and RTP lifetime (tau) can be expressed as tau = 1.25e(-0.477x) and the rate constants of phosphorescence emission k(p) was 0.800 per ms. The method was applied directly to determination of propranolol in urine and drug tablets with a satisfactory result. The recoveries were 96.6-97.4% and the relative standard deviation was 2% for the 1.00 x 10(-6)-4.00 x 10(-6) mol l(-1) propranolol in spiked urine sample.

[Study on the properties of solid substrate room temperature phosphorescence of the antibody labeled with fluorescein isothiocynate].

The properties of room temperature phosphorescence (RTP) of the labeled goat anti-human antibody(with fluorescein isothiocynate GAHAb-FITC) and labeled rabit anti-goat antibody(RAGAb-FITC) with FITC in different dilution ratios on many kinds of solid substrates were studied in detail. It is found that on the polyamide membrane, only using Pb(Ac)2 as a heavy atom perturber, both the labeled antibodies and their antigen-antibody conjugated compound with the human immunoglobulin G remained excellent RTP properties of isothiocyanate fluorescein, lambda ex(max)/lambda em(max) = 525/650 nm, the relation of RTP intensity to the concentration was linear in a certain range. The conditions for immunological reaction and RTP emission were optimized. The result means that it is possible to develop a new immunoassay named as solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA), which combines both the specificity of immunological reaction and high sensitivity of luminescence analysis.