Articles Anal. Chem. 1995, 67, 1785-1788
Selective Determination of DNA by Its Enhancement Effect on the Fluorescence of the E@-Tetracycline Complex Yun-Xiang Ci,* YuanZong Li, and Xiaa-Jing Liu Department of Chemistry, Peking University, BeJing 100871, China
Fluorescence enhancement of the Eu3+-telracyclinecomplex by DNA or RNA was studied. Studies involving natural and denatured fish sperm DNA, calfthymus DNA, and yeast RNA revealed that double-strandedand singlestranded DNA is capable of enhancing the fluorescence of the Eu3+-tetracyclinecomplex, while RNA showed very little enhancement effect, which founded a basis for selective determination of DNA in the presence of RNA Maximum fluorescence was produced in the pH range 8.0-9.7, with maximum excitation and emission wavelengths at 398 and 615 nm,respectively. Under optimal conditions, the calibration graphs were linear between 0.02 and 1.0 pg mL-' for both double-stranded calf thymus and fish sperm DNA The corresponding detection limits were 0.01 pg mL-l. The relative standard deviation (seven replicates)was within 3.0%in the linear range. DNA could be determined in the presence of yeast RNA. If the time-resolved mode was used, the low end of the linear range could be extended to 0.005 pg mL-', with a detection limit of 0.003pg mL-'. The mechanism for the fluorescence enhancement was also studied. Direct use of the fluorescence emission properties of nucleic acids to investigate their biological properties has been limited,1s2 whereas interest in trivalent lanthanide cations as fluorescence probes of the structure and function of nucleic acids has increased markedly.3-6 In particular, attention has been directed toward two rare earth cations, Tb3+and Eu3+,as their resonance energy levels overlap with the triplet energy states of nucleic acid ligands irradiated with ultraviolet light.7 We previously reported the fluorescence enhancement of Tb3+by nucleic acids and nucleotides in the presence of phenanthr~line,~,~ but the nucleic acids (1) Udenfriend, S.; Zaltzman, P. Anal. Biochem. 1962,3, 49-59. (2) Borresen, H. C. Acta Chem. Scand. 1963,17, 921-929. (3) Kayne, M. S.; Cohn, M. Biochemisty 1974,13, 4159-4165. (4) Pavlick, D.; Formoso, C. Biochemistry 1978,17, 1537-1540. (5) Ringer, D. P.; Howell, B. A.; Kizer, b.E. Anal. Biochem. 1980,103,337332. (6) Topal, M. D.; Fresco, J. R Biochemisty 1980,19, 5531-5537. (7)Sinha, A P. B. Spectroscopy in Inorganic Chemistry; Academic: New York, 1971; Vol. 2, pp 255-288. (8) Ci, Y.-X.; Li, Y.-Z.; Chang, W.-B. Anal. Chim. Acta 1991,248,589-594. (9) Ci. Y.-X.; Li, Y.-Z.; Chang, W.-B. Freseniusj. Anal. Chem. 1992, 342, 9194. 0003-2700/95/0367-1785$9.00/0 0 1995 American Chemical Society
must be singlestranded for energy transfer to be observed. The absence of lanthanide luminescence with double-stranded nucleic acids is attributed to an alteration of the energy of the triplet levels of the bases.1° We now describe the europium fluorescence sensitized by tetracycline, through the formation of an organic chelate, which was used for the sensitive detection of DNA. It is well known that tetracycline is part of a family of antibiotics whose antibacterial action is due to their ability to chelate metal." In the literature, it was reported that organic chelateswith lanthanide ions, especially those of europium, samarium, and terbium, have been used either to enhance the detection sensitivity of the lanthanides themselves or for the sensitive detection of the ligands.12 In this report, we show that the Eu3+-tetracycline complex is a sensitive fluorescenceprobe for determining double stranded and singlestranded DNA without interference from RNA. The detection limits for calf thymus DNA were 0.01 and 0.003 pg mL-' respectively when normal and time-resolved modes were used. EXPERIMENTAL SECTION
Chemicals. Commercially prepared fish sperm (FS) DNA and yeast RNA (Shanghai Biochemical, China) and calf thymus (CT) DNA (97%, from The Factory of Biochemical Reagents, The Institute of Biophysics, Academia Siica) were suspended directly in 50 mM sodium chloride solution at a final concentration of 100 pg mL-l and used without further purification. Native forms of DNA were thermally denatured by incubating them at 100 "C for 10 min, followed by cooling in an ice-water bath. Deionized distilled water was used in preparing the solutions. A stock solution of Eu3+(1.0 mM) was prepared by dissolving europium oxide (Eu203, Beijing Xinxin Chemicals) in concentrated hydrochloric acid, and the solution was evaporated to dryness. The residue was dissolved in 0.1 M hydrochloric acid. A tetracycline stock solution (1.0 mM) was prepared by dissolving 44.4 mg of tetracycline in 30 mL of 0.1 M hydrochloric acid and then diluting to 100 mL with water. More dilute standard solutions were prepared by appropriate dilution with water. A 0.1 M Tris-HC1 buffer solution containing 0.1 M sodium chloride was prepared (10) Cross, D. S.; Simpkins, H. J. Biol. Chem. 1981,256, 9593-9598. (11) Albert, A; Keesy, C. W. Nature 1956,177, 433-434. (12)Hirschy, L. M.; Dose, E. V.; Winefordner, J. D. Anal. Chim. Acta 1983, 147, 311-316.
Analytical Chemisty, Vol. 67, No. 7 1 , June 7, 7995 1785
24
I
I
4
350 390
430
470
610
580
590
830
Wavelength (nm) Figure I. Excitation (left) and emission (right) spectra: (a) 1.O pM Eu3+ 1.OpM tetracycline; (b) 1.OpM Eu3+ 1.OpM tetracycline 1.0 pg mL-I RNA; (c) 1.0 pM Eu3+ 1.0 p M tetracycline 1 ,ug mL-' native FS DNA; and (d) 1.O pM E d t + 1.OpM tetracycline 1 pg mL-' thermally denatured FS DNA.
+
+
+
+
+ +
by dissolving 12.11g of Tris and 5.82 g of sodium chloride in water and adjusting the pH with hydrochloric acid to give a final total volume of 1000 mL. Equipment. Fluorescence intensities were measured with a Hitachi 850 spectrofluorometerwith a quartz cell (1 x 1cm2cross section) equipped with a xenon lamp and a dual monochromator. The fluorescence lifetime and the data in Table 3 were measured on a LS50B luminescence spectrometer. The pH was measured with a Model 821 pH meter (Zhongshan University, China). Procedures. Typically, samples containing appropriate concentrations of tetracycline, nucleic acid, and europium ion were made up to 10 mL in 20 mM Tris-HC1 buffer (PH 8.5). The entrance and exit slits were maintained at 5.0 and 10 nm, respectively,for all fluorescence measurements. The wavelengths were A(ex) = 398.0 nm and l(em) = 615.0 nm. Fluorescence readings are given as net fluorescenceintensities in arbitrary units of the instrument. Background fluorescence (from all the reagents except the one being evaluated) has been subtracted for each value reported except for excitation and emission spectra. RESULTS AND DISCUSSION
Spectral Characteristics. Typical excitation and emission spectra are presented in Figure 1. The spectrum of the Eu3+tetracycline-DNA system differs signX"tly fYom those of both the Eu3+-tetracycline-RNA and Eu3+-tetracycline systems at the same pH, indicating ternary complex formation involving the tetracycline, europium ion, and DNA From the results presented in Figure 1,it can be seen that the native and thermally denatured DNA have nearly the same ability to enhance the fluorescence of the binary complex, whereas RNA hardly enhances the fluorescence of the binary complex at all. The emission spectrum of Eu3+ consists of several bands corresponding to the ~Do-~F, transitions, the most intense being the ~ D o - ~transition F~ at 590 nm and the "DO-~FZ transition at 615 nm. The emission intensity at 615 nm was much stronger than that at 590 nm; therefore, the peak at 615 nm was used for fluorescence intensity measurements. Optimization of the General Procedure. First, the effect of pH on Eu3+emission as its ternary complex with FS DNA and 1786 Analytical Chemisrry, Vol. 67,No. 1 7 , June 7, 7995
tetracycline was studied. The concentrations of DNA, Eu3+,and tetracycline were maintained at 10 pg mL-', 1.0 pM, and 1.0 pM, respectively. The maximum emission of the Eu3+complex occurs in the pH range 8.0-9.7. The fluorescence intensity diminished below pH = 6.7, which may be a result of protonation of the phosphate group of DNA at pH = 7. The fluorescence intensity increases with pH at pH = 6.7, which may be due to the deionization of the phosphate group that facilitates the reaction between DNA and the positively charged binary complex. The fluorescence intensities begin to decrease at pH > 9.7, which is most probably due to the formation of insoluble europium hydroxide. For CT DNA, the variation in fluorescence intensity as a function of pH is very similar to that of the FS DNA system. In the subsequent study, pH = 8.5 was used. Next, the influence of Eu3+concentration on the fluorescence intensity was investigated with constant concentration of FS DNA and tetracycline (5 pg mL-' and 1.0 pM, respectively) at pH = 8.5, the result showing that the maximum fluorescence intensity is reached when the concentration of Eu3+is 1.0 pM. At higher europium concentrations, the fluorescence intensities become smaller, indicating a quenching effect of free Eu3+ ion. This quenching effect is probably through competitive binding of EuBt and Eu3+tetracycline complex to DNA, which decreased the portion of bound Eu3+-tetracycline and therefore decreased the fluorescence intensity of the system. The fluorescence intensity was further studied as a function of the concentration of tetracycline. With a fixed amount of Eu3+ (1.0 pM) and FS DNA (5 ,ug mL-'), a rectilinear relationship was observed between fluorescence intensity and the concentration of tetracycline up to 1.0 p M , which suggests a molar ratio of 1:l for Eu3+and tetracycline. At higher tetracycline concentration, the fluorescence intensity would decrease. If the optimal tetracycline concentration were evaluated in the presence of 2 or 4 pM Eu3+ instead of 1 pM Eu3+,the corresponding optimal tetracycline concentration would be 2 or 4 pM, which again suggests molar ratios of 1:l for Eu3+ and tetracycline. Further studies show that an equimolar increase in the concentrationsof Eu3+and tetracycline could linearly increase the fluorescence intensity and extend the upper limit of the dynamic range. Meanwhile, the low end of the dynamic range was also upshifted, leading to a decrease in the detection limit. This is apparently due to the increase in the background signal. An equimolar decrease in the Eu3+and tetracycline concentrations will decrease the fluorescence intensity and the detection precision. Therefore, in the determination of DNA, LOpM tetracycline is used to obtain a relatively high sensitivity while maintaining a good precision. In summary, the optimal conditions for the determination of DNA are 1.0 pM each Eu3+and tetracycline at pH = 8.5. Determination of Nucleic Acids in synthetic Samples. The fluorescence enhancement of the Eu3+-tetracycline complex by FS DNA was studied over the nucleic acid concentration range of 0.02-50 pg mL-I with concentrations of Eu3+and tetracycline fixed at 1.0 pM. From Figure 2, it can be seen that the Eu3+ emission increased with increasing DNA concentration up to 10 pg mL-'. Above this concentration, the fluorescence is constant. The sensitivity of the fluorometric determination of DNA was determined from the slopes of the calibration graphs for DNAs and the limit of the detection from the stability of the blank measurements (Table 1). It can be observed that the sensitivity of FS DNA is the same as that of CT DNA. For both FS DNA
Table 2. Recoveries of FS DNA in 5 Times Excess of Yeast RNA
180 1 8
F
140
sample no.
120
1 2 3 4 5
.iloo
80
FS DNA (ug mL-l) added found 0.05 0.075 0.23 0.32 0.45
0.048 0.070 0.215 0.34 0.49
recovery (%) 96 95 93.5 106.5 109
E 6 0 40
?-----A 0
0 1 0 2 0 P O 4 0 5 0 5 0 7 0 DNA concentration (ug/ml)
Figure 2. Fluorescence enhancement of the Eu3+-tetracycline complex by FS DNA. Conditions: 1.0 pM Eu3+ and 1.0 pM tetracycline, pH = 8.5. Table 1. Analytical Parameter for the Determination of DNAs
DNA FS
CT
sensitivity linearran e (slope) (LgmL- B) 175 81 174 81
0.02-0.2 0.2-1.0 0.02-0.3 0.3-1.0
LOD" (ugmL-') 0.01 0.01
Ib
RSDC(%)
0.9951 0.9997 0.9940 0.9989
2.08 1.35 2.41 1.81
Limits of detection (signal-to-noiseratio = 2). Correlation coefficient. Relative standard deviation for seven measurements of 0.1 or 0.5 pg mL-' DNAs. (I
and CT DNA, the sensitivities are higher at lower concentration ranges than those at higher concentration ranges. As the method for DNA from different species has the same sensitivity for each, it may be used as a common method for absolute determination of DNA concentration. As the native and thermally denatured DNA have nearly the same ability to enhance the fluorescence of the Eu3+-tetracycline complex, while RNA is not able to enhance the fluorescence, it is expected that DNA could be measured in the presence of RNA. In this experiment, we found that an equivalent amount of RNA did not interfere with the determination of DNA, but excess RNA would result in a decrease in the fluorescence intensity. A possible explanation for this phenomenon is that the phosphate group of RNA may interact with Eu3+,which would reduce the amount of Eu3+ bound to DNA, This hypothesis has been confrmed by experiment. Interference can be eliminated by adding an excess of Eu3+. Experimental results showed that up to 5 times excess of RNA over DNA could be compensated by increasing the concentration of Eu3+. When the RNkDNAweight ratio was 5:1, the interference from RNA could be eliminated by using Eu3+in the concentration range of 1.2-2.2 pM. Large excess RNA cannot be completely compensated by the method described. In the determination of DNA in the presence of RNA, 1.5 pm Eu3+was used. The above procedure was applied to the determination of FS DNA in five synthetic samples. The recoveries are summarized in Table 2. Mechanism for the Fluorescence Enhancement. The most important fact worthy of mention is the selectivity of the Eu3+-
0' " " " " 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 Wavelength (nm) Figure 3. Absorption spectra: (a) 10 pg mL-' FS DNA: (b) 10 pg mL-l FS DNA + 1.OpM Eu3++ 1 .OpM tetracycline: (c) 10 pg mL-' thermally denatured FS DNA; and (d) 1Opg mL-' thermally denatured FS DNA 1.O,uM Eu3+ 1.OpM tetracycline.
+
+
tetracycline complex toward DNA (in both natural and denatured forms) in the fluorescence-enhancing reaction. This specificity is different from that of lanthanide ions and Eu3+) or their complexes, which show a specific fluorescence reaction to singlestranded DNA and RNA.lo To understand the basis of the difference, further experiments were carried out. The fact that both natural and denatured DNA could equally enhance the fluorescence of the Eu3+-tetracycline complex suggests that the interaction between them is not an intercalation type, like that between natural DNA and ethidium bromide (EB) .I3 One possible explanation is that natural DNA is denatured under the experimental conditions. The absorption spectra (Figure 3) of native and denatured DNA before and after addition of the Eu3+tetracycline complex showed that no enhancement in absorption is observed, indicating that no denaturation occurred upon addition of the complex to natural DNA solution. The existence of the Eu3+-tetracycline (1.0 pM each) complex does not quench the fluorescence of the DNA-EB complex, which also suggests a non-intercalation mechanism between natural DNA and the Eu3+-tetracycline complex and no denaturation of DNA by the Eu3+-tetracycline complex. Therefore, the fluorescence enhancement mechanism is different from that described earlier for Tb3+ or E d + , in which energy transfer from nucleic acid bases occurred. In this study, the energy transfer appears to be from tetracycline to Eu3+both in the presence and in the absence of DNA. The fluorescence lifetime of tripositive lanthanide ions ranges from 1 ps to > 2 ms, depending on the emitting ion and the
m3+
(13) Latomer, L. J. P.; Leet, J. S. J. Bid. Chem. 1991, 266, 13849-13551.
Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
1787
Table 3. Analytical Parameters for the Determination of CT DNA in Normal and Time-Resolved Modes on a Perkin-Elmer LS-50B Luminometer
80
measuring mode
normal
linear range GgmL-')
LODn GgmL-')
0.05-0.2
0.02
0.2-1.0
time-resolved 0.005-0.2 0.2-1.0
20
0.003
P
instrument
RSDC parameters
0.9984 0.9950 0.9991
2.1 1.8 3.0
0.9962
2.5
slits, 10 nm
slits, 10 nm; delay time, 0.1 ms gate time, 1.0 ms
Limits of determination (signal-to-noise ratio = 2). * Correlation coefficient. C Relative standard deviation for seven measurements of 0.1 or 0.5 pg mL-' DNAs.
0
0
0.04
0.08
0.12
0.16
0.2
Time (ms) Figure 4. Fluorescence decay curves for the fluorescence of the with 1 0 p mL-l Eu3+-tetracycline complex (a, 0) without and (b, I) RNA, (c, A) with 1Opg mL-' denatured FS DNA, and (d, 0 ) with 10 ,ug mL-l native FS DNA.
medium. George et have shown that the fluorescence enhancement observed for the Eu3+-tetracycline chelate on addition of Triton X-100 in water is directed to an increase in the lifetime of Eu3+ emission. The decay curves (Figure 4), corresponding to the Eu3+-tetracycline chelate and that in the presence of RNA, natural FS DNA, and thermally denatured FS DNA were recorded, giving values of 48, 56, 89, and 87 ps, respectively. As shown in Figure 4, in the presence of RNA, the fluorescence lifetime of Eu3+is increased slightly, whereas in the presence of native DNA and thermally denatured DNA, the fluorescence lifetime is greatly increased. A possible explanation of this phenomenon is the formation of a large hydrophobic energy protection shell'5 against nonradioactive deactivation of Eu3+ions in the presence of DNA; therefore, the fluorescence is increased. At present, we are still not able to give a reasonable explanation for the specificity of the fluorescence enhancement reaction to DNA. Perhaps the sequence specificityand conformation of DNA in solution play important roles in the reaction, as nucleotides in DNA and RNA are unable to enhance the fluorescence of the Eu3+- tetracycline complex. Increasing of Sensitivity and Dynamic Range by Using Time-Resohred Mode. Figure 4 clearly shows the advantages (14) Georges, J.; Ghazarian, S. Anal. Chim. Acta 1993,276,401-409. (15) Zhu, G. Y.; Yang, J. H.; Si, Z. K J Chin. Rare Earth SOC.1989,2, 73-78.
1788 Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
of using the time-resolved mode. Therefore, CT DNA was studied on a Perkin-Elmer LS50B luminometer in both normal and timeresolved modes. The results are summarized in Table 3, which shows that both the assay sensitivity and the dynamic range could be increased about 10 times. In conclusion sensitization of the europium fluorescence by tetracycline via the formation of an organic chelate was used for the sensitive detection of double-stranded and single-stranded DNA in the presence of RNA The sensitivity of the method is similar to that in the detection for DNA by ethidium bromide, but EB is a strong carcinogenic reagent, and EB binds well to both RNA and DNA duplexes, showing little selectivity to DNA.I5 So the study of the fluorescence enhancement of the Eu3+tetracycline complex in the presence of DNA is very significant providing a sensitive detection method for either double-stranded or single-stranded DNA or for total concentration of DNA without iduence from partial denaturation of DNA. By using a timeresolved technique, the sensitivity of the method could be increased and the dynamic range extended. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China. Received for review December 5, 1994. Accepted March 10, 1995.@ AC941177C @
Abstract published in Advance ACS Abstracts, April 15, 1995
