[Determination of protein by synchronous fluorometric method with a new indole homodimeric cyanine as fluorescence probe].

Using a new homodimeric hydrophilic indole dye (Dye-I) as fluorescence probe, a sensitive synchronous spectrofluorometric determination for protein was developed. Characteristics of the fluorescence reaction between DYE-1 and BSA protein were investigated. Effects of the concentration of the hydrophilic dye, pH value of the buffer solution, and ion-intensity of NaCl were also studied and the optimum condition was gained. At pH of 2.50, electrostatic interactions of positive charges of the BSA chain and negative charges on the sulfonic groups of DYE-1 were carried out. The interactions of the indole group of DYE-1 and some active groups of BSA (viz. amido, carboxyl or sulfhydryl) were also achieved, and resulted in the combination of indole group of dye at the chain of BSA, which caused a notable increase in synchronous fluorescence with an observable shift to the longer emission wavelength. Effects of the concentration of indole dye on the determination of BSA were also investigated. With the augmentation of BSA, the alpha-helix structure of BSA molecular would change from the unwrapped state to the enfolded state, which was in favor of restraining free-oscillation of fluorescence probe in the solution and maintaining a high energy transfer efficiency. Such a fact would fuel a high fluorescence enhancement and the change in fluorescence intensity (deltaF) gained the peak at 3.00 micromol x L(-1). The influences of ion-intensity of NaCl on the fluorescence of BSA-DYE-1 system was visible. Effects of coexistent substances such as amino acid and metal ions such as Cu2+, K+, Ca2+, Mg2+, Al3+, and Zn2+ were also investigated. Most substances showed no notable influences on the determination of BSA except Zn2+ and Cu2+ ions. Under the optimum conditions, good calibration curves of the protein were also obtained in the range of 5.00 x 10(-7) -2. 50 x 10(-5) g x mL(-1) (BSA) with a detection limit of 3 x 10(-8) g x mL(-1). Applied to simulant protein samples at the level of 1.00, 2.00, and 5.00 microg x mL(-1) of BSA, the recoveries were in the range of 98.6%-103.0% with the RSD of 1.1%-1.9%. Compared with the UV standard method, the relative deviation was obtained in the range of 0.4%-3.9%.

[Spectrofluorometric detection of protein with a novel hydrophilic cyanine dye].

A sensitive fluorescence quantitative determination for bovine serum albumin (BSA) or human serum albumin (HSA) has been developed by using a new hydrophilic cyanine dye 1, 1'-sulfonopropyl-3,3,3', 3'-tetramethylindolium-5,5'-disulfonic potassium (STDP) as a fluorescence probe. Using BSA as a representative protein, characteristics of the fluorescence reaction of STDP with protein were investigated. Effects of the concentration of the hydrophilic cyanine dye, pH value of the buffer solution, and ion-intensity of NaCl were also studied as well as the ratio of ethanol. In the citrate-HCl buffer solution, the fluorescence emission wavelength of BSA-STDP system was 562 nm with the maximum excitation wavelength of 548 nm, and the Stokes displacement was 14 nm. With the pH ranging from 1.0 to 2.0, the fluorescence was increasing and up to the maximum at pH 2.0. However, in the pH range of 3.0-5.0, the interaction of BSA and STDP was weakened due to the decrease in positive charge on the BSA chain, which resulted in an observable decrease of the enhancement of the fluorescence intensity. At the optimum pH of 2.0, electrostatic interactions of positive charges of the BSA chain and negative charges on the sulfonic groups of STDP were carried out. The interactions of the indole group of STDP and some active groups of BSA (viz. amido, carboxyl or sulfhydryl) were also achieved, and resulted in the combination of indole group of cyanine dye into the chain of BSA. So the hydrophobic effect and the protection provided by the skeleton chain of BSA were both improved to prevent the fluorescent energy of STDP from losing in the solution, which caused a notable fluorescence increase with an observable shift to the longer emission wavelength. Furthermore, with the augmentation of BSA, the alpha-helix structure of BSA molecular turned from the unwrapped state to the enfolded state, in favor of restraining free-oscillation of fluorescence probe in the solution and maintaining a high energy transfer efficiency. Such a fact fueled a highly enhancement of the fluorescence too. Besides, effects of the concentration of cyanine dye on the determination of BSA were also investigated. The fluorescence intensity (DeltaF) was enhanced with the increase in the quantity of STDP and gained the peak at 1.00 micromol x L(-1). However, when STDP ranged from 1.50 to 5.00 micromol x L(-1), some negative congregate effects on the nature of cyanine dye might happen and resulted in a too high fluorescence background. A rapid decrease of the fluorescence intensity was observed. The effects of ion-intensity of NaCl and ethanol on the fluorescence of BSA-STDP system were obvious. Though the fluorescence still remained high at the level of NaCl of 0.025 mol x L(-1), a rapid decrease happen at the level of NaCl from 0.05 to 0.15 mol x L(-1). With the addition of ethanol, the dissolvation capacity of both STDP and BSA was improved and their interactions were accelerated. An increasing fluorescence with the augment of ethanol was obtained and the maximum was achieved with the ratio of ethanol at 10%. Influences of coexistent substances such as amino acid, metal ions such as Cu2+, Na+, Ca2+, Mg2+, Al3+ and Fe3+ were also investigated. Most substances had no notable influences on the determination of BSA except Fe3+ and Cu2+ ions. Under the optimum conditions, the fluorescence of STDP was enhanced markedly with the addition of the BSA or HSA protein. Good calibration curves of the proteins were obtained in the range of 0.20-15.00 microg x mL(-1) for BSA and 0.20-12.00 microg x mL(-1) for HSA with detection limits (3sigma/K) of 0.01 microg x mL(-1). Applied to simulant BSA samples, this method was adaptable. And the results were satisfied with good recoveries ranging from 94.5% to 103.3% at the revels of 4.00, 6.00 and 8.00 microg x mL(-1) respectively.

[Research of Hg2+ sol-gel membrane made of organically modified silicates].

A photochemical sensor for the determination of Hg2+ consisting of organically modified silicates (ormosils) was developed. Hg(2+)-sensitive fluorescent sol-gel films were prepared by co-condensation of tetramethoxysilane (TMOS) and dimethyldimethoxysilane (DMOS), and the fluorescent indicator 5,10,15, 20-tetra (p-sulfonatophenyl) porphyrin sodium was embedded in sol-gel in the form of ion pairs with cetyltrimethyl ammonium bromide. The optimization condition of preparation and response performance of sol-gel membrane were investigated. The experimental results showed a linear range of Hg2+ concentrations from 1.0 x 10(-5) to 1.0 x 10(-4) mol x L(-1) in a Tris-HCl buffer at pH 8.0 with fast response and good repetition. The indicators in the sol-gel membrane did not leak out and the sensor showed good selectivity over other metal ions.

[Study of the pH sensitive polymer].

A pH sensitive polymer was prepared by copolymerization of methacrylic acid as monomer, diethylene glycol dimethacrylate as cross-linking reagent, heptane as porogen, and fluorescent dye eosin as indicator. The factors of influence on the preparation, and the character of the pH sensitive polymer for pH were studied. The maximal emission wavelength of eosin was red shifted in the polymer than in solution, the apparent Ka largened, and the dissociation equilibrium of indicator was shifted to acidity direction, because the polarity of polymer diminished. Under the optimal condition, the calibration curve of the pH sensitive polymer covered the range of pH 0-3.0 with good reproduction and reversibility.

[Determination of nucleic acids using toluidine blue as a fluorescence probe].

A new fluorimetric method has been developed for the determination of deoxyribonucleic acid (DNA) with toluidine blue (TB) as a fluorescence probe. It is based on the fluorescence quenching of toluidine blue in the presence of DNA. In Tris-HCl buffer solution (pH 8.5), the calibration graph was linear over the range 0.10-6.00 microg x mL(-1) for ctDNA, and the detection limit was 27 ng x mL(-1). The result of the determination of DNA of camphor tree's leaf by this method was satisfactory.

Thioacetamide chemically immobilized on silica gel as a solid phase extractant for the extraction and preconcentration of copper(II), lead(II), and cadmium(II).

Thioacetamide immobilized on silica gel was prepared via the Mannich reaction. The extraction and enrichment of copper(II), lead(II), and cadmium(II) ions from aqueous solutions has been investigated. Conditions for effective extraction are optimized with respect to different experimental parameters in both batch and column processes prior to their determination by flame atomic absorption spectrometry (FAAS). The optimum pH ranges for quantitative adsorption are 4.0-8.0, 2.0-7.0, and 5.0-10.0 for Pb(II), Cu(II), and Cd(II), respectively. Pb(II) and Cd(II) can be desorbed with 3 mol/L and 0.1 mol/L HCl/HNO3, and Cu(II) can be desorbed with 2.5% thiourea. The adsorption capacity of the matrix has been found to be 19.76, 16.35, and 12.50 mg/g for Pb(II), Cu(II), and Cd(II), respectively, with the preconcentration factor of approximately equal to 300 for Pb(II) and approximately equal to 200 for Cu(II) and Cd(II). Analytical utility is illustrated in real aqueous samples generated from distilled water, tap water, and river water samples.

[Study on beta-cyclodexdrin sensitizing catalytic kinetic spectrophotometric determination of Mn(II)].

A new catalytic spectrophotometric method for the determination of trace amount of Mn(II) has been developed. The method is based on the oxidation of alizarin green(AG) by potassium periodate in the presence of beta-cylodexdrin(beta-CD) as a sensitizer and nitrilotriacetic acid as an activator. The linear range of the method was 0-2.4 ng x mL(-1) for Mn(II), the determination limit was 0.097 ng x mL(-1), the relative standard deviation was 0.45% (n = 12) for 1.6 ng x mL(-1) of Mn(II), and the sensitivity was increased by a factor of 2.5 compared to that in the absence of the beta-CD. The method was used for the determination of Mn(II) in cereal samples and wine samples with satisfactory results. The mechanism of this reaction system was also presented.

[Study of direct fluorophotometric and flow-injection fluorophotometric methods based on the inhibitory effect for the determination of trace formaldehyde].

Sulfite reacts with o-phthalaldehyde in the presence of ammonium forming the highly fluorescing isoindole-1-sulfonate in neutral or weakly acid solution, and formaldehyde has inhibitory effect on it. Based on this principle, the authors developed the direct fluorophotometric and flow-injection fluorophotometric methods for the determination of trace formaldehyde. The maximum excitation wavelength and the maximum emission wavelength are 320 and 390 nm respectively. With the direct fluorophotometric method, formaldehyde in the concentration range of 0.10-1.60 microg x mL(-1) can be determined with a detection limit of 0.046 microg x mL(-1). With the flow-injection fluorophotometric method, formaldehyde in the concentration range of 0.10-2.00 microg x mL(-1) can be determined with a detection limit of 0.085 microg x mL(-1). The methods were applied respectively to the analysis of river water with satisfactory results.

[Direct fluorophotometric and flow-injection fluorophotometric methods for the determination of trace ammonia].

Direct fluorophotometric and flow-injection fluorophotometric methods for trace ammonia measurement involving the fluorescent reaction of ammonia with o-phthaldlaldehyde (OPA) and 2-mercaptoethanol (ME) producing derivant of iso-indol in alkaline solution are described. Triton X-100 was added to improve the stability of the system. The maximum excitation wavelength is 415 nm, while the maximum emission wavelength is 486 nm. With direct fliuorophotometric method, the calibration curve's regression equation is deltaI = 22.286 + 785.71c(NH3) (microg x mL(-1)) (r = 0. 9995) in the range of ammonia concentration from 0.2 to 1.0 microg x mL(-1), and deltaI = -27.429 + 2 371.4c(NH3) microg x mL(-1)) (r = 0.9965) in the range of ammonia concentration from 0.02 to 0.10 microg x mL(-1). With flow-injection fluorophotometric method, the calibration curve's regression equation is deltaI = 1188c(NH3) (microg x mL(-1)) (r = 0. 9998) in the range of ammonia concentration of ammonia 0-0.7 microg x mL(-1). The mechanism of reaction was also briefly discussed.

[Studies and application of the interaction of pentamethyl-p-rosaniline hydrochloride 6B and nucleic acids system].

A cationic dye pentamethyl-p-rosaniline hydrochloride 6B was used as a chromic probe and its binding reaction with nucleic acids was studied. In the weak alkalescent medium, the cationic dye showed a decrease in absorption at its maximal absorption peak (584.5 nm) on binding to nucleic acids. Based on this fact, a new UV-Vis spectrophotometric method for the determination of nucleic acids has been developed. Effects of ion-intensity, surfactant and organic solvent were investigated to establish optimum reaction conditions, and then nucleic acids were detected with quantitative analysis. The calibration curves of nucleic acids such as hs-DNA, smDNA, ct-DNA and yeast-RNA were linear over the ranges 0.50-4.00 microg x mL(-1), 0.20-5.00 microg x mL(-1), 0.20-5.00 microg x mL(-1) and 0.20-4.50 microg(-1) x mL(-1), respectively. The detection limits (3sigma/K) were 0.082, 0.037, 0.038 and 0.041 microg x mL(-1). In applications to quantitative analysis of nucleic acids synthesis samples, the recoveries of DNA or RNA ranged from 93.5% to 105.0%. Furthermore, the mechanism of interaction of pentamethyl-p-rosaniline hydrochloride 6B and nucleic acids system was also discussed.

[Studies on a new fluorescence-enhanced system of nucleic acids-morin-Al(III)].

A fluorescence-enhanced system was developed for the determination of nucleic acids by using morin-aluminum(III) complex as a new fluorescent probe. In aqueous solution, morin-aluminum(III) complex showed maximum excitation and emission wavelengths at 420.0 nm and 532.8 nm, respectively, and its fluorescence could be greatly enhanced in the presence of nucleic acids. Under optimal conditions, the calibration graph was linear over the range 0.25-1.50 microg x mL(-1) for fish sperm DNA, 0.10-1.60 microg x mL(-1) for salmon sperm DNA, 0.25-2.00 microg x mL(-1) for calf thymus DNA and 0.25-2.00 microg x mL(-1) for yeast RNA. The corresponding detection limits are 3 ng x mL(-1), 2 ng x mL(-1), 2 ng x mL(-1) and 3 ng x mL(-1), respectively. Applied for the determination of nucleic acids in synthetic samples, the relative standard deviation for five replicates is less than 3.6%, and the recovery ranges from 93.3% to 107.9%. Additionally, the interaction mechanism of morin-aluminum(III) with nucleic acids is also discussed.