Simple and sensitive assay for nucleic acids by use of the resonance light-scattering technique with copper phthalocyanine tetrasulfonic acid in the presence of cetyltrimethylammonium bromide

On the basis of enhancement of resonance light scattering (RLS) of copper phthalocyanine tetrasulfonic acid (CuTSPc) by nucleic acids and cetyltrimethylammonium bromide (CTMAB) under suitable conditions, a new RLS method for determination of nucleic acids in aqueous solutions has been developed. At pH 9.80–10.95 and ionic strength 0.01 mol L^–1 (NaCl), the interaction of copper phthalocyanine tetrasulfonic acid with nucleic acids in the presence of cetyltrimethylammonium bromide results in enhanced RLS signals at 282.0 nm, 383.6 nm, and 616.2 nm in the enhanced regions. It was found that the enhanced RLS intensity at 383.6 nm was proportional to the concentration of nucleic acids within suitable ranges. The limits of detection were 10.6 ng mL^–1 for fish sperm DNA and 32.4 ng mL^–1 for calf thymus DNA when the concentration of copper phthalocyanine tetrasulfonic acid was 2.0×10^–6 mol L^–1. This method is rapid, simple and sensitive. In addition, the reagents used are relatively inexpensive, stable, and easily synthesised. The method can be applied to the determination of nucleic acids in the presence of coexisting substances, and we have applied it to the determination of DNA in synthetic samples, with satisfactory results.     

Sensitive Determination of DNA by Resonance Light Scattering with Pentamethoxyl Red

A novel sensitive method for the determination of nucleic acid (DNA) using the resonance light scattering (RLS) spectra of pentamethoxyl red has been developed. It is based on the effects on the resonance light scattering of Pentamethoxyl Red. The effective factors and the optimum conditions were studied, and the enhanced intensity of RLS is in proportion to the concentration of nucleic acids in the range of 0–2.54 µg mL⁻¹ for ct-DNA, 0–4.54 µg mL⁻¹ for hs-DNA. The limits of detection are 1.1 and 2.1 ng mL⁻¹, respectively. Most foreign substances do not interfere in the determination, and the method has good selectivity and high sensitivity. It has been applied to the determination of DNA in synthetic samples and in real samples with satisfactory results.     

Simple and sensitive assay for nucleic acids by use of the resonance light-scattering technique with the anionic dye methyl blue in the presence of cetyltrimethylammonium bromide

This is the first report on the determination of nucleic acids based on the enhancement of resonance light scattering (RLS) of the anionic dye methyl blue (MB) in the presence of cetyltrimethylammonium bromide (CTMAB). In tris(hydroxymethyl) aminomethane buffer of pH 9.0, MB and nucleic acids react with CTMAB to form large particles of complex, which results in strong enhanced RLS signals characterized by three peaks at 334 nm, 393.5 nm and 548 nm. Mechanistic studies show that the enhanced RLS stems from the aggregation of MB on nucleic acids through the bridged and synergistic effect of CTMAB. With the enhanced RLS signals at the best wavelength at 334 nm, the enhanced RLS intensity is proportional to the concentration of nucleic acids in a wide range. The lowest limit of determination was 2.1 ng mL⁻¹, three synthetic samples were analyzed satisfactorily.     

Study on the interaction between nucleic acids and cationic surfactants

The interactions of nucleic acids and cationic surfactants (cetylpyridine bromide (CPB) and cetyltrimethylammonium bromide (CTMAB)) in aqueous solution have been studied using the techniques of resonance light scattering (RLS) spectroscopy, the absorption spectroscopy, zeta potential assay and NMR assignment measurement. It is considered that CPB or CTMAB can assemble on the surface of nucleic acid via electrostatic and hydrophobic forces, which results in the formation of large associate of nucleic acid-cationic surfactant and RLS enhancement of nucleic acid. Besides these forces, the pi-pi stacking force between CPB and nucleic acid also exists in the associate. In comparison with CTMAB, CPB has larger enhancement on RLS of nucleic acid, which is attributed to that the enhancement of the former is only due to the absorption of the bases of nucleic acid, while the enhancement of the latter is own to the synergetic resonance caused by the absorption of both bases of nucleic acid and the pyridyl in CPB. These results have important implication for understanding the influence of surfactants on nucleic acid functionality in life science.     

聚二烯丙基二甲基氯化铵与核酸相互作用的共振光散射光谱及分析应用

实验基于核酸与聚阳离子聚二烯丙基二甲基氯化铵(PDDA)的相互作用导致共振光散射(RLS)增强的现象来测定核酸。考察了pH值、PDDA浓度和离子强度对体系共振光散射强度的影响。在优化条件下,建立了用RLS光谱测定微量核酸的新方法。方法的抗干扰能力较强,可允许大部分的常见金属离子、核苷酸、氨基酸、糖、蛋白质等干扰物质的存在。同时用于合成样品的分析,结果令人满意。     

A simple and sensitive assay of nucleic acids based on the enhanced resonance light scattering of zwitterionics

A new assay of nucleic acids at nanogram level was established based on the enhanced resonance light scattering (RLS) signals of two zwitterionics cocamidopropyl hydroxysultaine (HSB) and lauryl betaine (BS-12). Under optimum conditions, the weak RLS signal of HSB is enhanced by nucleic acids, and the enhanced RLS intensity is proportional to the concentration of nucleic acids in the range of 0.02–7.3 mg l⁻¹ for calf thymus DNA and 0.01–8.6 mg l⁻¹ for fish sperm DNA. The detection limits were 1.5 ng ml⁻¹ for calf thymus DNA and 1.9 ng ml⁻¹ for fish sperm DNA. Plasmid DNA extracted from K-12-HB101 colt was determined with satisfactory results.     

Spectral studies on the interaction of acid chrome blue K with nucleic acids in the presence of cetyltrimethylammonium bromide and the confirmation of combined points

The interaction of Acid chrome blue K (ACBK) with nucleic acids in weak basic medium was studied in the presence of cetyltrimethylammonium bromide (CTMAB) based on the measurements of resonance light scattering (RLS), UV-vis, NMR spectra, etc. In hexamethylene tetramine (HMTA) buffer (pH 7.45), ACBK and nucleic acids react with CTMAB to form a ternary complex, which results in strong enhanced RLS signals characterized by four peaks at 285, 335, 405.5 and 548nm. Mechanistic studies show that the enhanced RLS stems from the aggregation of ACBK on nucleic acids through the bridged and synergistic effect of CTMAB. With the enhanced RLS signals at the best wavelength at 335nm, the enhanced RLS intensity is proportional to the concentration of nucleic acids in a wide range. The lowest limit of determination was 7.52ngml(-1), three synthetic samples were analyzed satisfactorily. And the combined points of the anionic dye ACBK with nucleic acids-CTMAB have been tentatively confirmed through the measurement of 1H NMR spectra.     

Determination of deoxyribonucleic acids by a resonance light scattering technique and its application

For the first time, acetamiprid has been used to determine nucleic acid (DNA) using the resonance light scattering (RLS). The RLS of acetamiprid was greatly enhanced by DNA in the range of pH 1.6-1.8. A RLS peak at 313 nm was found, and the enhanced intensity of RLS at this wavelength was proportional to the concentration of DNA. The linear range of the calibration curve was 0-11.0 microg ml(-1) with the detection limit of 20 ng ml(-1). The nucleic acids in synthetic sample and in rice seedling extraction were determined satisfactorily. The interaction mechanism of acetamiprid and DNA is discussed. Mechanism studies show that the enhanced RLS is due to the aggregation of acetamiprid in the presence of DNA.     

The sensitive determination of nucleic acids using resonance light scattering quenching method

It is found that in hexamethylene tetramine (HMTA)-HCl buffer of pH 7.00, nucleic acids can quench the resonance light scattering (RLS) of europium (III) (Eu3+)-2-thenoyltrifluoroacetne (TTA)-1,10-phenanthroline (Phen) system. Based on this, a sensitive method for the determination of nucleic acids is proposed. The experiments indicate that under the optimum conditions, the quenched RLS intensity is in proportion to the concentration of nucleic acids in the range of 1.0x10(-10) to 2.0x10(-6) g ml-1 for fish sperm (fsDNA), 1.0x10(-11) to 1.0x10(-6) g ml-1 for yeast RNA (yRNA), 5.0x10(-11) to 5.0x10(-7) g ml-1 for calf thymus DNA (ctDNA). Their detection limits (S/N=3) are 0.03, 0.006 and 0.002 ng ml-1, respectively. Therefore, the proposed method is the most sensitive RLS method for the determination of nucleic acids so far. The interaction between nucleic acids and Eu3+-TTA-Phen is also discussed.     

Interactions of Night Blue with Nucleic Acids and Determination of Nucleic Acids Using Resonance Light Scattering Technique

The noncovalent interactions of night blue (NB) with several nucleic acids in buffer medium of Britton‐Robinson at pH 4.1 have been studied by spectroscopic methods. It is shown that the binding of NB with nucleic acids involves the J‐aggregation of NB molecules on the surface of nucleic acids. The aggregation was encouraged by polyanions nucleic acids, in which nucleic acids served for acting templates. In this connection, a new method of nucleic acids with sensitivity at nanogram level is proposed based on the measurement of enhanced resonance light scattering (RLS). The linear range of ctDNA, fsDNA and yRNA is 0.01—2.5, 0.03—2.5 and 0.04—1.0 μg/mL, respectively, and the corresponding detection limits (3s?) are 9.4, 7.3 and 5.7 ng/mL at 2.5 × 10–⁵mol/L of NB. Synthetic and real samples were analyzed with satisfactory results.     

Determination of nucleic acids at nanogram level using resonance light scattering technique with Congo Red

Based on the enhancement of the resonance light scattering (RLS) of Congo Red (CR) by nucleic acid, a new quantitative method for nucleic acid is developed. In the Tris-HCl buffer (pH 10.5), the weak light scattering of CR is greatly enhanced by addition of nucleic acid and CTMAB, the maximum peak is at 560 nm and the enhanced intensity of RLS is in proportion to the concentration of nucleic acid. The linear range is 1.0 x 10(-9) to 1.0 x 10(-6) g ml(-1), 7.5 x 10(-8) to 1.0 x 10(-6) g ml(-1) and 7.5 x 10(-8) to 2.5 x 10(-6) g ml(-1) for herring sperm DNA, calf thymus DNA and yeast RNA, and the detection limits are 0.019, 0.89 and 1.2 ng ml(-1) (S/N = 3), respectively. Actual biological samples were satisfactorily determined.     

Study on the interaction of DNA with resveratrol by resonance light scattering technique and its analytical application

The interaction between resveratrol and DNA has been studied by resonance light scattering (RLS) technique. In strongly acidic solution, resveratrol has a maximum peak at 368 nm and the RLS intensity is remarkably enhanced by trace amounts of DNA due to its interaction with resveratrol. Based on this, a novel assay for nucleic acids has been developed. The characteristics of RLS, fluorescence and UV-VIS absorption spectra, the influential factors and optimum conditions of the reaction have been studied. The enhanced RLS intensity at 368 nm is proportional to the concentration of DNA within the range of 0–1600 μg/L for calf thymus DNA. The determination limit (3σ) is 5.2 ng/mL. The study of foreign substance effect on the determination of DNA indicates that most of metal ions have little effect on the determination of DNA. Three synthetic samples of DNA were analysed with satisfactory results. The results show that the proposed method is very sensitive, convenient, rapid and reproducible.     

Determination of Nucleic Acids Based on their Resonance Light Scattering Enhancement Effect on Metalloporphyrin Derivatives

A new method for the determination of nucleic acids at nanogram per mL level is proposed based on the enhanced resonance light scattering (RLS) signal resulting from the interaction of metalloporphyrins with nucleic acids. Under optimum conditions, the weak RLS signal of metalloporphyrin is enhanced by nucleic acids, and the enhanced RLS intensity is proportional to the concentration of nucleic acids. The detection limits of calf thymus DNA were 3.5ngmL^–1, 2.9ngmL^–1 and 1.0ngmL^–1 for three metalloporphyrins, respectively. Synthetic samples were determined with satisfactory results.     

Investigation of the interaction between curcumin and nucleic acids in the presence of CTAB

It is found that nucleic acid can enhance the resonance light scattering (RLS) enhancement effect of curcumin (CU) in the presence of cationic surfactant cetyltrimethylammonium bromide (CTAB). The investigation indicates that in BR (pH 4.3) buffer, both the positive CTAB and negative yeast RNA (yRNA) combine and form a positive large association, then which is bound on the two carbon atoms of the carbonyls of CU through hydrogen bond and hydrophobic force and form CU-CTAB-yRNA ternary complex, resulting in the RLS enhancement of this system. Based on it, a sensitive method for determination of nucleic acids at ngml(-1) is established.     

Determination of nucleic acids with a near infrared cyanine dye using resonance light scattering technique

A new method for the determination of nucleic acids has been developed based on the enhancement effect of resonance light scattering (RLS) with a cationic near infrared (NIR) cyanine dye. Under the optimal conditions, the enhanced RLS intensity at 823 nm is proportional to the concentration of nucleic acids in the range of 0-400 ng mL-1 for both calf thymus DNA (CT DNA) and fish sperm DNA (FS DNA), 0-600 ng mL-1 for snake ovum RNA (SO RNA). The detection limits are 3.5 ng mL-1, 3.4 ng mL-1 and 2.9 ng mL-1 for CT DNA, FS DNA and SO RNA, respectively. Owing to performing in near infrared region, this method not only has high sensitivity endowed by RLS technique but also avoids possible spectral interference from background. It has been applied to the determination of nucleic acids in synthetic and real samples and satisfactory results were obtained.     

On the factors affecting the enhanced resonance light scattering signals of the interactions between proteins and multiply negatively charged chromophores using water blue as an example

Electrostatic interactions of proteins, including bovine serum albumin (BSA), human serum albumin (HSA), γ-globulin (γ-IgG), α-chymotrypsin (Chy), lysozyme (Lys) and cellulase (Cel), with multiply negatively charged chromophores were investigated based on the measurements of the enhanced resonance light scattering (RLS) signals. Using triply negatively charged water blue (WB) as an example, the factors were discussed that affect the enhanced resonance light scattering signals of the interactions between proteins and the negatively charged chromophores. It was found that the enhanced RLS signals with the maximum light scattering peak at 346.0 nm in these interacting systems are strongly dependent on the isoelectric points of proteins and show adverse linear relationships with increasing ionic strength depending on the positive charges of the inorganic metal ions used to control the ionic strength of the medium, sufficiently disclosing that the electrostatic attraction performs an important role in the combination of proteins with WB. Linear responses were discovered between the enhanced RLS signals and the protein molecular weights (M_w), displaying the dimensions of scattered particles formed by proteins and WB make a key contribution to the RLS enhancements. An empirical equation is proposed which possibly displays the factors affecting the enhanced RLS signals of the interactions between proteins with negatively charged chromophores.     

A Novel Protein Assay With an Azo Dye Using Rayleigh Light Scattering Technioque

ABSTRACT

The interaction of SPADNS [3-(4-sulfophenylazo)-4, 5-dihydroxy-2, 7-naphthalene disulfonic acid] with proteins in acidic solution was studied by spectrophotometric and Rayleigh light scattering (RLS) methods. SPADNS reacts rapidly with albumin at room temperature, and the intensity of the weak RLS of SPADNS was enhanced remarkably and quantitatively by this reaction.

Based on this observation, a novel RLS method for serum albumin determination was developed. The linear range is 0.125-14.9 μg/ml for BSA. The relative standard deviation (n=10) for 5.0 μg/ml BSA was 3.94%. The method was applied to the determination of proteins in human plasma, and the results were compared with the traditional proteins assay (CBB method). There is almost no interference from amino acids, metal ions and other coexistent substances. The reaction mechanism was also discussed.     

聚丙烯酸与核酸相互作用的共振光散射光谱研究及其分析应用

研究了聚丙烯酸(PAA)与脱氧核糖核酸(DNA)相互作用的共振光散射(RLS)光谱.实验结果表明,在pH=2.0的B-R缓冲溶液中,PAA与DNA自身的共振光散射峰均较弱,但当二者发生静电作用形成复合物后,体系的共振光散射峰增强,散射增强程度则各不相同,其相对散射强度的顺序是fsDNA＞ctDNA＞yRNA.在一定范围...     

Molecular spectroscopy study of the reaction of nucleic acids with brilliant cresol blue

The interaction of brilliant cresol blue (BCB) with nucleic acids in aqueous solution has been studied by spectrophotometry and Rayleigh light scattering (RLS) spectroscopy. Under suitable conditions, the RLS spectra of BCB changed significantly due to the presence of nucleic acids. RLS intensity of BCB at 364 nm is greatly enhanced with the addition of nucleic acids, and a new RLS peak is observed at 552 nm. This peak is about half the intensity of that at 364 nm. The results of this study show that BCB interacts with DNA possibly due to the cooperative effect of electrostatic attraction, intercalation, coordination and hydrophobic effect. Under optimum conditions, the increase of RLS at 364 nm of a BCB solution is proportional to the concentration of nucleic acids added. This result is the basis for a new RLS method for determination of nucleic acids. The linear range of ctDNA, fsDNA and yRNA is 0.12-4.70, 0.11-4.64 and 0.43-7.07 microg ml(-1), respectively.     

Interactions of Nile Blue Sulphate with Nucleic Acids as Studied by Resonance Light-Scattering Measurements and Determination of Nucleic Acids at Nanogram Levels

ABSTRACT

The interactions of nile blue sulphate (NBS) with nucleic acids, including calf thymus DNA, fish sperm DNA and yeast RNA, were characterized with resonance light-scattering (RLS) measurements by using a common spectrofluorometer. Accordingly a method for the determination of nucleic acids at nanogram levels was established. At pH's of 7.20～7.60 and ionic strengths lower than 0.012, the interactions of NBS with nucleic acids result in three characteristic RLS peaks at 293.4 nm, 349.4 nm and 560.4 nm. Mechanism study shows that these peaks are ascribed to the long range assembly of NBS on the molecular surface of nucleic acids, which depends on pH, ionic strength and the stranded structure of nucleic acids. A Scatchard plot was constructed by using the RLS data, yielding the assembly number and assembly constant being 6.4 and 7.13x10⁶ mol^?1 1 for NBS assembly on the molecular surface of calf thymus DNA. The same parameters are 6.6 and 4.58x10⁶ mol^?1 1 for the assembly on that of fish sperm DNA, 3.9 and 1.67x10⁶ mol^?1 1 on that of yeast RNA, respectively. Linear relationships were found between the enhanced RLS intensity at 293.4 nm and nucleic acid concentration. If 1.2x10^?5 mol I^?1 NBS was employed, 0～0.80 μg ml^?1 calf thymus DNA and fish sperm DNA, 0.20～0.60 μg ml^?1 yeast RNA can be determined with the determination limits being 3.2 ng ml^?1 for calf thymus DNA, 11.5 ng ml^?1 for fish sperm DNA and 38.3 ng ml^?1 for yeast RNA, respectively. Four synthetic samples were determined with satisfaction.