Nucleic Acid Analysis with UV-vis and NMR - Spectroscopy

38 Spectroscopy 23(11) November 2009 www.spectroscopyonline.com
Intensity
450
400
350
300
250
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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