Nucleic Acid Analysis with UV-vis and NMR - Spectroscopy

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38 Spectroscopy 23(11) November 2009 www.spectroscopyonline.com Intensity 450 400 350 300 250 200 150 100 50 0 250 300 350 400 450 500 550 600 650 700 Wavelength (nm) Figure 4: Effect of surfactants on the I RLS value of IC-DNA-CTMAB system. Conditions: CIC, 3.0 X 10 -6 mol/L; pH, 5.47; CDNA, 500 µg/L. 1 = CTMAB, 2 = CPB, 3 = TX-100, 4 = SDBS, 5 = SDS. Surface tension (mN/m) 100 80 60 40 20 0 1 2 3 4 5 6 7 8 9 C CTMAB (10-5 ) Figure 5: Effect of CTMAB concentration on surface tension. Conditions: CIC, 3.0 X 10 -6 mol/L; pH, 5.74; CDNA, 500 µg/L. I RLS 450 400 350 300 250 200 150 100 50 0 0 0.5 1 1.5 2 Indigo carmine concentration Figure 6: Effect of IC concentration on the ΔI RLS intensity. Conditions: CCTMAB, 3.0 X 10 -5 mol/L; pH, 5.47; CDNA, 500 µg/L. Top: I RLS of the system with DNA; bottom: I RLS of the system without DNA). 1 2 3 4 5 tering spectrum depends on the nature of the system, also reflects the characteristics of the instrument (28), the RLS peak around 470 nm is considered to be possibly the emission of the xenon lamp in this region (29). The RLS intensity at 336 nm is the maximum, so 336 nm was selected in the further study. Effect of buffer and surfactant The effects of buffer and surfactant are examined. Tris is the best buffer tested, HMTA-HCl (88.7); NH 4 Ac-HAc (19.2); NH 3 -NH 4 Cl (11.5). It can be seen from Figure 4 that CTMAB is the best surfactant. The effects of buffer and surfactant are examined (presented in Figure 4) and we know the enhanced RLS signals strongly depend upon the concentration of CTMAB. If the concentration of CTMAB is too small, the enhanced RLS intensity is not significant. However, when the concentration of CTMAB increases, the enhanced RLS signals are very strong. When the concentration of CTMAB is above 4.0 X 10 -5 mol/L, perhaps CTMAB reacts with IC directly and forms larger particles of IC-CTMAB which is not favorable for further reaction with DNA, so the ΔIRLS decreases. From the measurement of surface tension (shown in Figure 5), we can obtain the critical associate concentration (CAC) in this system, which is 3.0 X 10 -5 mol/L. So 3.0 X 10 -5 mol/L CTMAB is selected for further research. The effects of buffer are examined, and the dependence of light-scattering upon pH might be relevant to the form of IC and DNA. When the pH is below 4.50, both the free base form of IC and DNA are protonized, which is not favorable for positively charged CTMAB to be close to the DNA molecule. At the same time, the concentration of IC with two negative charges is low, which is not favorable for the reaction of IC and CTMAB. When the pH is above 6.50, the ΔI RLS of IC- CTMAB-DNA system begins to decline markedly, perhaps as a result of the direct reaction CTMAB with IC. So pH 5.47 is chosen for further research. Effect of indigo carmine concentration The effect of IC concentration on ΔI RLS of the IC-CTMAB-DNA system is investigated and shown in Figure 6. As is
www.spectroscopyonline.com November 2009 Spectroscopy 23(11) 39 indicated by the figure, the IC concentration has an obvious effect on ΔI RLS of the IC-CTMAB-DNA system. It is found that when the concentration of IC is in the range 2.0 X 10 -6 to 4..0 X 10 -6 mol/L, the ΔI RLS of the IC-CTMAB-DNA system reaches a maximum. So we select 3.0 X 10 -6 mol/L IC for further research. Effect of the ionic strength The ionic strength of the medium has an effect on the interaction of IC, CTMAB, and DNA. As Figure 7 shows, the ΔI RLS of the IC-CTMAB-DNA system decreased slightly when NaCl was at a low concentration, while it decreased markedly with the increasing ionic strength. The reason could be that Na+ shielded the phosphorus negative charge of DNA, which weakened the combination of DNA and CTMAB, so the ΔI RLS decreased, despite the strong dependence of the enhanced light scattering of IC by DNA upon ionic strength ranges from 0 to 0.05. Reaction time and stability The binding reaction of DNA and IC with CTMAB occurs rapidly at room temperature after less than 2 min, and the ΔI RLS remains a constant for about 4 h. It shows that the reaction does not require any crucial timing and has good stability. Table I: Interference of foreign substances Foreign substance Conc. Coexisting (µg/L) Relative error (%) Foreign substance Conc. Coexisting (µg/L) Relative error (%) Ca(II) 5.0 -0.66 Fe(III) 48 -0.98 Al(III) 1.5 3.88 KH 2 PO 4 30.0 -2.76 Mg(II) 2.0 7.63 Co(III) 0.10 9.01 Ni(II) 0.90 -1.92 Pb(II) 0.10 -6.12 EDTA 12.0 3.40 L-Leucine 0.08 0.97 Adenine 16.0 -2.74 DL-Alanine 15.0 2.33 20.0 -3.34 Sucrate 20.0 -4.65 D-Galactose 40.0 -5.02 DL-Cystine 7.5 3.27 L-Lysine 30.0 7.11 L-Methionine DL-Tryptophan 40.0 2.18 DL-Histidine 20.0 2.16 Thymine 15.0 -5.23 Tolerance of Foreign Coexisting Substances The effect of substances such as metal ions, amnion acids, galactose, and adenine were examined for interference. The results are summarized in Table I. It can be seen that most of the metal ions in biological systems, such as K + , Na + , and Ca 2+ , can be tolerated at high concentrations because they are hard ions (30) and tend to bind almost exclusively with the phosphate groups, stabilizing the Watson–Crick double helix of DNA, so their effect on the interaction of IC is related to the shielding effects of counter ions on the negative backbone of nucleic acids, which shows that this method is very useful and valuable. Calibration Under the optimum conditions, the dependence of ΔI RLS upon the concentration of DNA is determined. The analytical parameters of this method are listed in Table II, which shows that there was a good
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