Chen Zhanguang, Liao Xuhong, Li Dan, Ding Weifeng
(Department of Chemistry, Shantou University, Shantou 515063, China)

Abstract This is the first report of the sensitizing effect of Cetyltrimethylammonium bromide (CTMAB) on the enhanced resonance light scattering (RLS) resulted from the interaction of organic dye Victoria blue B (VBB) with nucleic acids. In the buffer solution (pH 6.80), the interactions of VBB with nucleic acids gave two characteristic peaks of RLS at 390.0 and 444.0 nm. The interaction was sensitized by the presence of CTMAB. Under the optimum conditions, the enhanced RLS intensity at 390.0 nm was proportional to the concentrations of nucleic acids in the range 0.05 - 2.0mg mL^-1 for fsDNA , ctDNA, and 0.02 - 1.5mg mL^-1 for yRNA. The detection limits (3s) were 5.61, 7.22, 3.03 ng mL^-1 for fsDNA, ctDNA and yRNA, respectively. Synthetic samples were determined with good reproducibility.
Keywords Cetyltrimethylammonium bromide; Nucleic acids; Victoria blue B; resonance light scattering

1 INTRODUCTION
As the material base of genetic inheritance, nucleic acid analysis is critical to chemical and biological studies since it can offer reference information for the measurements of other components. Many methods have been studied; the fluorometric methods are the most widely used methods for determining nucleic acids. Resonance light scattering (RLS) technique is a newly developed method for nucleic acid determination by using a common spectrofluorometer ^[1-4]. The basis for the method is the strong enhancement of nucleic acids on RLS signal of the complex of organic dyes such as safranine T ^[5], azur A ^[6], nile blue sulfate^[7], and rhodaming B ^[8]. Organic dyes with positive charge can feasibly act as condensing agents of nucleic acids polyanions, particularly at a low molar ratio of nucleic acids to organic dyes and low ionic strength, and that suprahelical helixes of nucleic acids could be formed^[9].
    Victoria blue B (VBB) is an easily available triphenylmethane dye. Although VBB can be bound to the polyanion of DNA through chromophore coupling between VBB and DNA bases owing to the positive charge on its molecular structure, the RLS intensity cannot be enhanced significantly. In our experiment, we found that the interactions between VBB and nucleic acids can be sensitized by the presence of CTMAB. Based on this phenomenon, a novel assay of nucleic acids was established. In this paper, the nature of resonance light scattering of the ion-association complex of VBB-CTMAB - nucleic acids system and the mechanism of the reaction have been studied for the first time. The relationship between DI_RLS and nucleic acid concentration was established. Synthetic samples have been determined by this assay, and the results are reproducible and reliable.

2 EXPERIMENTAL
2.1 Reagents
The stock solutions of nucleic acids were prepared by dissolving commercial products in doubly distilled water. The concentration of the working solution was 25 mg mL^-1. The nucleic acids used in this study are fsDNA (Sigma Chemicals Co., USA), ctDNA (Sino-American Biotechnology Co., China), and yRNA (Sigma Chemicals Co., USA). These stocks needed to be stored at 0–4ºC. The absorbances ratios A₂₆₀/A₂₈₀ were 1.83 (DNA) and 2.0 (RNA).
    A 8.0×10^-6 mol L^-1 working solution of VBB was prepared by dissolving commercial product (Shanghai Chemical Reagent Co., Shanghai, China) in 10% ethanol.
    A 8.0×10^-4 mol L^-1 working solution of surfactant CTMAB was prepared by dissolving its crystal product (Shanghai Chemical Reagent Co.) in doubly distilled water.
    NaAc buffer was prepared by dissolving sodium acetate in doubly distilled water, and the pH value was adjusted to 6.80 with NaOH. Its working solution was 2.0 mol L^-1. All chemicals used were of analytical grade or the best grade commercially available and doubly distilled water was used throughout.
2.2 Apparatus
The RLS spectra and intensities were measured with a Perkin-Elmer LS55 luminescence spectrometer with a quartz cuvette (1×1 cm). All absorbance measurements were measured on a Shimadzu UV-2501 ultraviolet spectrophotometer (Kyoto, Japan). A SA 720 laboratory instrument (Orion Research) was used to measure the pH value of the solution.
2.3 General Procedure
Into a 10ml standard flask were added 1.5 mL of NaAc buffer, 0.5 ml of VBB, 0.4 ml of CTMAB, and appropriate standard nucleic acids or sample solution. The mixture was mixed thoroughly after each addition of the interacting reagents, and then diluted to the mark with doubly distilled water. 10 min's incubation time was needed.
    RLS spectra were obtained by scanning simultaneously the excitation and emission monochromators of the LS55 spectrofluorometer from 250.0 to 600.0 nm with Dl= 0 nm. Both the excitation and emission slit widths were kept as 10.0 nm. The RLS intensities were measured at 390.0 nm.

3 RESULTS AND DISSUSIONS
3.1 Features of RLS spectra
Fig.1 displays the RLS spectra of fsDNA and VBB respectively. It can be seen that VBB has two wide weak RLS peak located at 390 and 430 nm. Although VBB exists mainly as R⁺ cationic monomer in the medium of pH 6.80, and nucleic acid exists as a big polyvalent anionic, the RLS intensity of VBB was just enhanced a little by the addition of fsDNA (Fig.1).
    As Fig.2 shows, VBB has two absorption bands characterized at 370 and 615 nm. The absorption intensity increases with the increasing of VBB concentration. This indicates that VBB has aggregate tendency in the reaction medium. According to the RLS theory ^[1,2,10], the RLS peak at 390 nm is ascribed to the absorption band at 370 nm. The weaker RLS in the wavelength range > 500 nm, however, is ascribed to the molecular absorption band of VBB over 500-700 nm on one hand, and to the steep decrease of light scattering intensity with wavelength increase on the other.

Fig.1 RLS spectra of the interaction of VBB and fsDNA Concentrations: curve 1: VBB 4.0×10-7 mol L-1; curve 2: fsDNA 1.0mg mL-1, VBB 4.0 ×10-7 mol L-1; pH, 6.80	Fig. 2 Absorption spectra of VBB. Concentrations: VBB, (from top to bottom, ×10-7 mol L-1) 4.0, 3.0 2.0; pH, 6.80

3.2 Optimal conditions of the interaction
3.2.1 Effect of CTMAB
Strong RLS signals can be observed when CTMAB is added to the reaction system. Fig. 3 displays the role that CTMAB played in the interactive system. As can be seen, CTMAB has little effect on the RLS intensity of VBB because of the electrostatic repulsion. This indicates that the presence of CTMAB sensitized the reaction. The RLS intensity of VBB - CTMAB - fsDNA increases with the CTMAB concentration increasing. The optimum CTMAB concentration is in the range 3.0 × 10^-5 - 3.7 × 10^-5 mol L^-1, and when the concentration of CTMAB is greater than 3.7 × 10^-5 mol L^-1, the formation of micelle resulted in the decrease of RLS intensity.

Fig. 3 Dependence of RLS intensity on CTMAB concentration. Conditions: VBB, 4.0 ×10^-7 mol L^-1; fsDNA, 1.0mg mL^-1; pH, 6.80

3.2.2 Effect of pH and buffers
The dependence of RLS intensity on pH values of the interaction medium has been studied. The optimum pH range is from 6.68 to 6.90. Any pH value out of the range leads to a lower RLS intensity. Thus, pH 6.80 is always used for the interactive medium. Experiments indicate that the different buffers have different effects on the RLS intensity. The following buffers were tested: Tris - HCl, KH₂PO₄ - HCl, NaH₂PO₄ - NaOH and NaAc - NaOH. The results show that NaAc is the best buffer and the optimum volume of buffer is 1.5 mL. In this work, the optimal addition order of the reagents is as follows: buffer solution -VBB-CTMAB-nucleic acids.
3.2.2 Effect of VBB concentration
VBB concentration also has significant effects on the interactive system. RLS intensity increases when VBB concentration is less than 4.0×10^-7 mol L^-1, and decreases when VBB concentration is greater than 4.0×10^-7 mol L^-1. So we selected 4.0×10^-7 mol L^-1 as the optimum VBB concentration for determination of nucleic acids in this method.
3.2.3 Effect of ion strength
We used NaCl to control the ion strength of the reaction system. The result showed that the enhanced RLS intensity is very sensitive to the change of ionic strength. When the ion strength was lower than 0.015 mol L^-1, the RLS intensity had no significant change. But when the ion strength was higher than 0.015 mol L^-1, the RLS intensity increase and the reaction system became unstable. So, in our experiment, we need to control the ion strength at a low level.
3.3 Tolerance of foreign substances
The effects of foreign substances such as metal ions, proteins, sugars, nucleotides and surfactants on the RLS intensity of the interactive system were tested. The results are listed in Table 1. It can be seen that proteins, sugar and nucleotides do not interfere the DNA. The common metal ions in biological fluids, such as Na⁺, Ca²⁺, Mg²⁺ and K⁺ can be tolerated at high concentration. However, easily hydrated metal ions, such as Fe(III), Al (II), Cu(II) in near-neutral solution, could form Fe(OH)₃, Al(OH)₃ and Cu(OH)₂ particles respectively, and result in light scattering, thus they have a positive effect on the light scattering intensity of the studied system. Therefore, these metal ions have lower tolerances.

Table 1 Tolerance of foreign substances

Superscripts a and b in the Table indicate % and mg mL^-1, respectively. Concentrations: VBB, 4.0×10^-7 mol L^-1; CTMAB, 3.4×10^-5 mol L^-1; fsDNA, 1.0mg ml^-1; pH, 6.80.

4. Analytical applications
4.1. Calibration and detection limit
Under the optimum conditions according to above the standard procedure, good linear relationships were obtained between the enhanced RLS intensity at 390.0 nm and the concentration of nucleic acids. All the regression parameters are presented in Table 2. The sensitivities of the RLS method for different nucleic acids have the sequence: fsDNA＞yRNA＞ctDNA.

Table 2 Analytical parameters of the present method

All the data were obtained by using 25mg mL^-1 nucleic acid solution; VBB, 4.0×10^-7 M; CTMAB, 3.4×10^-5 mol L^-1; pH, 6.80. All the data were obtained at 390.0 nm.

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