Study on the interaction between nucleic acid and Eu3+-oxolinic acid and the determination of nucleic acid using the resonance light scattering technique

Article abstract of DOI:10.1016/j.saa.2005.03.022

At pH 9.75, the resonance light scattering (RLS) intensity of OA-Eu ³⁺ system is greatly enhanced by nucleic acid. Based on this phenomenon, a new quantitative method for nucleic acid in aqueous solution has been developed. Under the optimum condition, the enhanced RLS is proportional to the concentration of nucleic acid in the range of 1.0 × 10^-9 to 1.0 × 10^-6 g/ml for herring sperm DNA, 8.0 × 10 ^-10 to 1.0 × 10^-6 g/ml for calf thymus DNA and 1.0 × 10^-9 to 1.0 × 10^-6 g/ml for yeast RNA, and their detection limits are 0.020, 0.011 and 0.010 ng/ml, respectively. Synthetic samples and actual samples were satisfactorily determined. In addition, the interaction mechanism between nucleic acid and OA-Eu³⁺ is also investigated.

Full text of DOI:10.1016/j.saa.2005.03.022

Spectrochimica Acta Part A 62 (2005) 896–901
Study on the interaction between nucleic acid and Eu3+–oxolinic acid and
the determination of nucleic acid using the resonance
light scattering technique
Xia Wua, Shuna Suna, Jinghe Yanga,∗, Minqin Wangb, Liyan Liuc, Changying Guoa
a
Key Laboratory of Colloid and Interface Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering,
Shandong University, Jinan 250100, Shandong, PR China
School of Life Science, Shandong University, Jinan 250100, Shandong, PR China
b
c
Jinan Petrochemical Design Institute, Jinan 250100, Shandong, PR China
Received 16 March 2005; received in revised form 31 March 2005; accepted 31 March 2005
Abstract
At pH 9.75, the resonance light scattering (RLS)intensity ofOA–Eu3+ system is greatly enhanced bynucleic acid. Based onthis phenomenon,
a new quantitative method for nucleic acid in aqueous solution has been developed. Under the optimum condition, the enhanced RLS
is proportional to the concentration of nucleic acid in the range of 1.0 × 10−9 to 1.0 × 10−6 g/ml for herring sperm DNA, 8.0 × 10−10
to 1.0 × 10−6 g/ml for calf thymus DNA and 1.0 × 10−9 to 1.0 × 10−6 g/ml for yeast RNA, and their detection limits are 0.020, 0.011 and
0.010 ng/ml, respectively. Synthetic samples and actual samples were satisfactorily determined. In addition, the interaction mechanism between
nucleic acid and OA–Eu3+ is also investigated.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Nucleic acids; Oxolinic acid; Resonance light scattering
1. Introduction
of nucleic acids [4,5]. Subsequently, more studies of quan-
titative determination of macromolecules including nucleic
acids [6–8] and proteins [9,10] and high sensitivity detection
of DNA hybridization [11] by RLS have been published. Up
to now, most of the probe applied in the determination of
nucleic acids are organic dyes [12,13] by their aggregation
on DNA, these methods used above probe are sensitive, inex-
pensive and safe, but they also have the disadvantage of easy
to be interfered by foreign ions and narrow linear range.
In this paper, our focus is to develop the complex of Eu3+
and oxolinic acid (OA) as a sensitive RLS probe for the
determination of trace amount of nucleic acids. OA is one
of quinolone antibiotics used in treating bacterial diseases
in aquatic species. Eu3+–OA complex and nucleic acid have
drastic interaction. The detection limit reaches 10−11 g/ml for
nucleic acids. And its high ability to tolerance foreign ions
and broad linear range also overcome the limitation on selec-
tivity and narrow linear range of other method used dye. So
study this question is important since it not only improved the
The quantitative analysis for micro amounts of nucleic
acids is required in many fields such as biochemistry, molec-
ular biology, biotechnology, and medical diagnostics. Since
using the intrinsic fluorescence and ultraviolet absorption of
nucleic acids for their determination has been severely lim-
ited by low sensitivity and serious interference [1]. Therefore,
some probes based on the interaction between nucleic acids
and extrinsic reagents have been employed in the determina-
tion of nucleic acids using the spectral methods. Among these
spectral methods, resonance light scattering (RLS) technique
has given rise to strong interest among chemists and bio-
chemists since Pasternack et al. pioneering work [2,3] using
a common spectrofluorometer. Huang et al. first used this
technique for analytical purposes to determine trace amounts
∗
Corresponding author. Tel.: +86 5318365459; fax: +86 5318564464.
E-mail address: yjh@sdu.edu.cn (J. Yang).
1386-1425/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2005.03.022

X. Wu et al. / Spectrochimica Acta Part A 62 (2005) 896–901
897
detection limit of nucleic acid but also developed the applied
3. Results and discussion
range of RLS.
3.1. RLS spectra and absorption spectra of
Eu3+–OA–DNA system
2. Experimental
Fig. 1showsthelightscatteringspectraofEu3+–OA–DNA
system. Both OA and Eu3+–OA show very weak RLS signals
over the wavelength range of 200–700 nm. However, when
Eu3+–OA mixed with DNA in pH 9.75, the RLS intensity
is strongly enhanced indicating an interaction between DNA
and Eu3+–OA. Fig. 2 is the absorption spectra of the system.
It can be seen from line 1 that the absorption peak of OA
located in 258, 266, 326 and 340 nm. Compare Fig. 1 with
Fig. 2 according to the theory of RLS [2,14], the RLS peak at
2.1. Apparatus
The RLS spectra and the intensity of RLS were mea-
sured with a F-4500 spectrofluorimeter (Hitachi, Japan). All
absorption spectra were measured on a UV-4100 spectropho-
tometer (Hitachi, Japan). All pH measurements were made
with a Delta 320-S pH meter (Mettler Toledo).
2.2. Chemicals
Stock solutions of nucleic acids (100 ␮g/ml) were pre-
pared by dissolving commercially purchased herring sperm
DNA (fsDNA, Sigma, Germany), calf thymus DNA (ctDNA,
Beijing Baitai Reagent Company, Beijing, China), and yeast
RNA (yRNA, Sigma, Germany) in doubly deionized water.
Working standard solutions were obtained by appropriate
dilution of the stock solutions.
Stock standard solution of Eu3+ (1.0 × 10−2 mol/l) was
prepared by dissolving the corresponding oxide (99.9%) in
hydrochloric acid and heating until nearly dry then diluting
with doubly deionized water.
Stock solution of OA (5.0 × 10−3 mol/l) was prepared
by dissolving the appropriate amount of OA with 0.1 mol/l
NaOH, and diluted with 3.0 × 10−3 mol/l NaOH. Working
standard solution was obtained by appropriate dilution of the
stock solution.
A 0.05 mol/l Tris–HCl buffer solution was prepared by
dissolving of 3.03 g Tris in 500 ml volumetric flask with water
and adjusted the pH to 9.75 with HCl.
Fig. 1. Resonance light scattering spectra of the Eu3+–OA–fsDNA
system. (1) OA; (2) OA–Tris; (3) OA–Tris–fsDNA; (4) OA–Eu3+
;
(5) Eu3+–OA–fsDNA; (6) OA–Eu3+–Tris; (7) OA–Eu3+–Tris–fsDNA;
(8) OA–Eu3+–Tris–yRNA. Conditions: OA, 1.0 × 10−4 mol/l; Eu3+
,
1.0 × 10−5
,
0.05 mol/l; Tris–HCl, 1.0 ml (pH 9.75); fsDNA, 5.0 ×
10−7 g/ml; yRNA, 5.0 × 10−7 g/ml.
All reagents were of analytical reagent grade without
further purification, and doubly deionized water was used
throughout.
2.3. General procedure
To a 25 ml test tube, working solutions were added in
the following order: 1.0 ml of 1.0 × 10−4 mol/l Eu3+; 1.0 ml
of 1.0 × 10−3 mol/l OA; 1.0 ml of 0.05 mol/l Tris–HCl (pH
9.75); and appropriate amount of nucleic acid solutions.
The mixture was diluted to 10 ml with doubly deionized
water and shook to react efficiently and allowed to stand
for 5 min. All RLS spectra were obtained by scanning
simultaneously the excitation and emission monochroma-
tors (namely, ꢀλ = 0 nm) from 200 to 700 nm. The intensity
of RLS was measured at λ = 365 nm in a 1 cm quartz cell
with slit width at 10 nm for the excitation and emission. The
enhanced RLS intensity of Eu3+–OA system by nucleic acids
was represented as ꢀIRLS = IRLS − IR0LS, here IRLS and IR0LS
were the intensities of the system with and without nucleic
acids.
Fig. 2. Absorption spectra of Eu3+–OA–fsDNA system. (1) OA; (2)
OA–fsDNA; (3) OA–yRNA; (4) Eu3+–OA; (5) Eu3+–OA–fsDNA; (6)
Eu3+–OA–yRNA. Conditions: OA, 1.0 × 10−4 mol/l; Eu3+, 1.0 × 10−5
,
0.05 mol/l; Tris–HCl, 1.0 ml; fsDNA, 5.0 × 10−7 g/ml.

898
X. Wu et al. / Spectrochimica Acta Part A 62 (2005) 896–901
Fig. 4. Effect of the concentration of OA on the RLS intensity. Condi-
tions: Eu3+, 1.0 × 10−5, 0.05 mol/l; Tris–HCl, 1.0 ml (pH 9.75); fsDNA,
5.0 × 10−7 g/ml.
Fig. 3. Effect of pH on the RLS intensity. Conditions: OA, 1.0 × 10−4 mol/l;
Eu3+, 1.0 × 10−5, 0.05 mol/l; Tris–HCl, 1.0 ml; fsDNA, 5.0 × 10−7 g/ml.
3.4. Effect of Eu3+ concentration
300 and 368 nm comes from the cooperation of absorption of
DNA and absorption of OA at 266 and 340 nm, respectively.
In Fig. 1, the enhancement of RLS intensity of Eu3+–OA
by yRNA is also observed, and its pattern of the RLS spec-
tra profiles is similar to that of Eu3+–OA–fsDNA. However,
the RLS intensity is little weaker than that of caused by
fsDNA. Since RLS intensity at 368 nm is the maximum in
both Eu3+–OA–fsDNA and Eu3+–OA–yRDNA systems, so
368 nm was selected in the further study.
The effect of Eu3+ concentration on the ꢀIRLS(%) of the
system was tested with OA concentration of 1.0 × 10−4 mol/l
was shown in Fig. 5. It can be seen that the maximum ꢀIRLS
intensity of the system occurs at 1.0 × 10−5 mol/l Eu3+.
3.5. Effect of addition order
The effect of addition order on the ꢀIRLS(%) of the
system was tested. Result shows that the order of Eu3+–
OA–Tris–DNA is the best.
3.2. Effect of pH and buffer solutions
Fig. 3 shows the relationship between the ꢀIRLS intensity
and solution pH of the system. When pH is in the range from
9.5 to 10.2, the ꢀIRLS reaches the maximum and remain
constant. Therefore, the change of pH had a great influence
on ꢀIRLS intensity of the system. As shown in Fig. 3, pH
9.75 is the optimum condition for obtaining the maximum
light scattering from Eu3+–OA–DNA complex.
3.6. Incubation time and the stability of the system
The influence of incubation time on the RLS enhanced
by fsDNA was investigated. The experiment shows that the
Under the condition of pH 9.7 0.1, the effect of different
kinds of buffers on ꢀIRLS(%) of the system was tested, and
theꢀIRLS(%)forTris–HCl, NH3–NH4Cl, Na2HPO4–NaOH,
HMTA, and Na2B4O7·7H2O–NaOH buffer systems were
100, 36.1, 36.2, 73.8, and 33, respectively. The results show
that the enhanced ꢀIRLS is the highest in Tris–HCl buffer
solution and obtain maximum when the concentration of the
Tris–HCl buffer is 0.05 mol/l. Therefore, 1.0 ml of 0.05 mol/l
Tris–HCl buffer of pH 9.75 is selected for further study.
3.3. Effect of OA concentration
Effect of the OA concentration on the ꢀIRLS(%) was
shown in Fig. 4. It can be seen that the maximum ꢀIRLS(%)
obtained when concentration of OA is 1.0 × 10−4 mol/l. So
in further experiments, the concentration of OA was fixed at
1.0 × 10−4 mol/l.
Fig. 5. Effect of the concentration of Eu3+ on the RLS intensity. Con-
ditions: OA, 1.0 × 10−4, 0.05 mol/l; Tris–HCl, 1.0 ml (pH 9.75); fsDNA,
5.0 × 10−7 g/ml.

X. Wu et al. / Spectrochimica Acta Part A 62 (2005) 896–901
899
Table 1
the linear relationship between ꢀIRLS and the concentration
of nucleic acids over a wide range. The limits of detection all
reached 10−11 g/ml. A comparison of this method with other
well-known methods, in terms of sensitivity is summarized
in Table 3. It can be seen that the sensitivity of this method is
much higher than most other methods. Therefore, this method
is rapid, simple and sensitive, and will be a valuable tool for
studying the biological properties of nucleic acids.
Thepresentmethodwasappliedtodeterminenucleicacids
in synthetic samples constructed based on the tolerance of
co-existing species: 1.0 × 10−7 g/ml, BSA; 1.0 × 10−5 mol/l,
thymine; 2.0 × 10−5 mol/l, Val; 3.0 × 10−6 mol/l, ZnCl2;
5.0 × 10−6 mol/l, KCl. The recovery of the synthetic sample
is 96.5–104.1%, and the results obtained were satisfactory.
The proposed method was also used to determine the DNA
in an actual sample, which was isolated from common wheat
(Triticum aestivum L.) derived from granular embryogenic
calli according to the CTAB method [26]. The results, which
obtained by this method and the UV method, were shown in
Table 4. It can be seen that this method is reliable, precise
and simple.
Effects of foreign substances on the RLS intensities
Foreign substances (mol/l)
ꢀIRLS (%)
BSA, 4.6 × 10−6 (g/ml)
+4.9
+5.0
+7.6
−4.3
−5.2
−4.4
−4.8
+4.7
−5.1
+5.0
+4.6
−4.8
+5.1
−2.0
−4.8
−3.4
−5.0
−2.6
+4.6
+4.3
+4.8
+4.8
Val, 4.0 × 10-4
HSA, 2.0 × 10−6 (g/ml)
l-Phe, 3.0 × 10−4
Na+, Cl−, 2.5 × 10−4
l-Asp, 4.5 × 10−4
K+, Cl−, 1.2 × 10−4
Cys, 1.0 × 10−5
Ba2+, Cl−, 3.0 × 10−5
Uracil, 5.0 × 10−6
Al3+, Cl−, 2.0 × 10−6
Thymine, 1.03 × 10−4
Fe3+, Cl−, 8.0 × 10−7
l-His, 4.0 × 10−4
Mg2+, SO42−, 6.4 × 10−6
dl-Thr, 3.0 × 10−4
Zn2+, SO42−, 4.4 × 10−5
l-Ala, 3.0 × 10−4
Gd3+, 1.78 × 10−6
AMP, 1.8 × 10−6
Y
3+, 2.5 × 10−6
Tb3+, 2.0 × 10−6
OA, 1.0 × 10−4 mol/l; Eu3+, 1.0 × 10−5, 0.05 mol/l; Tris–HCl, 1.0 ml (pH
3.9. The mechanism of interaction of Eu3+–OA–DNA
system
9.75); fsDNA, 5.0 × 10−7 g/ml.
ꢀIRLS intensity reaches the maximum within 5 min after the
mixing of the solutions and remains stable for at least 24 h,
which means that the system is very stable for the determi-
nation of nucleic acids.
The fluorescence spectra of the system were shown in
Fig. 6. It can be seen that OA can remarkably enhance the
fluorescence intensity of Eu3+ in aqueous solution, which
indicated that there was strong interaction between Eu3+ and
OA. It also showed that fsDNA can remarkably quench the
fluorescence intensity of Eu3+–OA complex. From above
phenomenon, we predict that interaction between Eu3+–OA
and fsDNA caused the enhancement of RLS intensity of
Eu3+–OA. From absorption spectra, we can see that the
addition of fsDNA only lead to a slightly increasing of the
absorption of Eu3+–OA, without the phenomenon of absorp-
3.7. Interference study
The influence of various ions, proteins, nucleotides and
other microbiomoleculars was tested at pH 9.75 when the
concentration of fsDNA was 5.0 × 10−7 g/ml. The results are
listed in Table 1. As we can see, most of them have little
interference with the determination of nucleic acids, so good
selectivity is found for this method.
3.8. Analytical applications
Underoptimalconditions, linearrelationshipsbetweenthe
enhanced RLS (ꢀIRLS) and the nucleic acid concentrations
(c) are constructed. All the analytical parameters have been
regressed, and are presented in Table 2. It demonstrated that
Table 2
Analytical parameters for the determination of nucleic acids
Nucleic acids
Linear range (g/ml)
ra
LODb (ng/ml)
fsDNA
ctDNA
yRNA
1.0 × 10−9 to 1.0 × 10−6
8.0 × 10−8 to 1.0 × 10−6
1.0 × 10−9 to 1.0 × 10−6
0.9959
0.9932
0.9929
0.020
0.011
0.010
Conditions: OA, 1.0 × 10−4 mol/l; Eu3+, 1.0 × 10−5, 0.05 mol/l; Tris–HCl,
Fig. 6. Fluorescence spectra of the system. (1) Eu3+; (2) Eu3+–fsDNA; (3)
Eu3+–OA; (4 and 5) Eu3+–OA–fsDNA. Conditions: OA, 1.0 × 10−4 mol/l;
1.0 ml (pH = 9.75).
a
Eu3+
,
1.0 × 10−5
, 0.05 mol/l; Tris–HCl, 1.0 ml (pH 9.75); fsDNA,
Correlation coefficient.
Limit of detection (S/N = 3).
b
1.0 × 10−6 g/ml, 3.0 × 10−6 g/ml.

900
X. Wu et al. / Spectrochimica Acta Part A 62 (2005) 896–901
Table 3
Common probes for nucleic acids
Probes
Method
Nucleic acids
LODa (ng/ml)
References
Hoechst 33258
Tb3+-phenanthroline
Pico green
FLb
FL
FL
FL
FL
DNA
10
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[4]
ctDNA/fsDNA/yRNA
dsDNAc
ctDNA/smDNA/yRNA
ctDNA/ssDNA/fsDNA
DNA
100/200/200
0.025
5.0/6.0/13.0
0.75/0.66/1.49
10
19.9/12.6
1.4/1.4/2.7
0.095/1.3/8.5
3.4/6.2/4.1
6.5/2.1/3.5
4.1/4.6/6.7f
0.020/0.011/0.010
Phosphin 3R
Ru(bipy)2(dppx)2+
Ethdium bromide
Azur A
FL
RLS
RLS
RLS
RLS
RLS
RLS
RLS
ctDNA/fsDNA
TAAlPcd
ctDNA/fsDNA/yRNA
ctDNA/fsDNA/yRNA
ctDNA/fsDNA/yRNA
fsDNA/ctDNA/yRNA
ctDNA/fsDNA/yRNA
fsDNA/ctDNA/yRNA
Acridine Red
Morin-CTMAB
Berberine
TAPPe
This probe
a
Limits of detection.
Fluorimetric method.
Double strand DNA.
b
c
d
Tetra-substituted amino aluminum phthalocyanine.
␣,␤,␥,␦-tetrakis[4-(trimethylammoniumyl)phenylporphine.
×10−8 mol/l.
e
f
Table 4
The determination of DNA actual sample
Sample
Wheat DNA found (×10−7 g/ml)
Average (×10−7 g/ml)
R.S.D. (%)
0.33
UV method
This method
1
3.75
3.73, 3.78, 3.70, 3.72, 3.76
3.74
the IRLS of both the Eu3+–OA and Eu3+–OA–DNA systems
tion reducing and red shift of peak. So we consider that
Eu3+–OA has not insert into the base sequence of DNA. As
we known, DNA possessed negative charges due to the phos-
phate group, so we consider that there was strong electrostatic
interaction between DNA and Eu3+–OA, and Eu3+ as a bridge
combined fsDNA with OA to form a complex, which lead to
the significant increasing of IRLS. The influence of PO34− on
the system also demonstrated the electrostatic action between
DNA and Eu3+–OA as shown in Fig. 7. It can be seen that
are increased when the concentration of PO34− is low and
reached maximum at 7.0 × 10−6 mol/l PO43−. Then the IRLS
of Eu3+–OA–DNA system reduced with the increasing of
concentration of PO34−, while the IRLS of Eu3+–OA system
tend to changeless. We consider that a little bit of PO34− can
neutralize the Eu3+ whose coordinate number is not saturated,
which contribute to the broadening of the complex across
area that lead to the increase of IRLS. But excessive PO43−
will compete with DNA for combining with Eu3+, so the
IRLS of Eu3+–OA–DNA system was decreased. Figs. 8 and 9
are the transmission electronic microscope (TEM) pictures of
fsDNA and Eu3+–OA–DNA, respectively. From the pictures
Fig. 7. Effect of the concentration of PO34− on the RLS intensity.
(1) Eu–OA–Tris–HCl; (2) Eu–OA–fsDNA–Tris–HCl. Conditions: OA,
1.0 × 10−4 mol/l; Eu, 1.0 × 10−5 mol/l; Tris–HCl, 1.0 ml (pH 9.75),
0.05 mol/l; fsDNA, 5.0 × 10−7 g/ml.
Fig. 8. TEM of fsDNA at pH 9.75.

X. Wu et al. / Spectrochimica Acta Part A 62 (2005) 896–901
901
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Fig. 9. TEM of Eu3+–OA–DNA at pH 9.75. Conditions: OA,
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Acknowledgement
This work was supported by the Natural Science Founda-
tion of Shandong Province (Y2003B02).