Anal. Chem. 2011, 83, 225–230
Fluorescence Detection of Lead(II) Ions Through Their Induced Catalytic Activity of DNAzymes Chi-Lin Li,† Kung-Tien Liu,‡ Yang-Wei Lin,§ and Huan-Tsung Chang*,†
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Department of Chemistry, National Taiwan University, Taipei, Taiwan, Chemical Analysis Division, Institute of Nuclear Energy Research, Taoyuan, Taiwan, and Department of Chemistry, National Changhua University of Education, Changhua, Taiwan We have developed a fluorescence approach for the highly selective and sensitive detection of Pb2+ ions using AGRO100, a G-quadruplex DNAzyme. The sensing strategy is based on Pb2+ ions inducing increased DNAzyme activity of AGRO100 in the presence of hemin, which acts as a cofactor to catalyze H2O2mediated oxidation of Amplex UltraRed (AUR). A test of eight aptamers of various sequences for the detection of Pb2+ ions revealed that AGRO100 performed the best in terms of sensitivity. The AGRO100-AUR probe exhibited high selectivity (>100-fold) toward Pb2+ ions over other tested metal ions. The fluorescence intensity (excitation/emission maxima, ca. 561/ 592 nm) of the AUR product was proportional to the concentration of Pb2+ ions over the range 0-1000 nM, with a linear correlation (R2 ) 0.98). For 5 mM Tris-acetate (pH 7.4) solutions in the presence and absence of 100 mM NaCl, the AGRO100-AUR probe provided limits of detection (signal-to-noise ratio ) 3) for Pb2+ ions of 1.0 and 0.4 nM, respectively. We validated the practicality of the use of the AGRO100AUR probe for the determination of the concentrations of Pb2+ ions in soil samples. This approach allows the determination of the concentrations of Pb2+ ions with simplicity, selectivity, and sensitivity.
tion of Pb2+ ions.3 Nevertheless, these techniques are timeconsuming, expensive, and/or require sophisticated equipment. Therefore, the challenge remains to develop simple and inexpensive methods for the determination of the concentrations of Pb2+ ions in environmental, biological, and industrial samples. DNAzyme-based sensors have been demonstrated for the detection of Pb2+ ions through their induced changes in the catalytic activity of the DNAzymes.4 DNAzymes are non-natural short-stranded nucleic acids that act much like enzymes, catalyzing many biochemical reactions.5 Relative to enzymes, DNAzymes are easier to synthesize, less expensive, and more stable; nevertheless, they are less popular, mainly because very few highly active and specific DNAzymes have been found.6 G-Rich oligonucleotides are DNAzymes that are effective catalysts for oxidations of many molecules (e.g., luminol) in the presence of metal ions, organic molecules, DNA, or proteins.7 The four-stranded structures (G-quadruplexes) of DNAzymes, in which four guanine bases are hydrogen bonded to each other to form a planar arrangement (G-tetrad), are stabilized in the presence of metal ions.8 Alkali metal cations are located in the cavity between two adjacent G-tetrads of a G-quadruplex; they are bound to eight CdO oxygen atoms from the G-tetrads.9 Alternatively, some aromatic planar ligands, including porphyrins and metalloporphyrins, are apt to stack externally on the terminal G-tetrads of G-quadruplexes through
Detection of lead ions (Pb2+) is important because they can have severe effects on human health and the environment.1 Even exposure to very low levels of Pb2+ ions (70%), and (iii) it has long excitation and emission wavelengths (therefore, functioning with less interference from the autofluorescence of biological matrixes).16 Because fluorescence-based sensors are usually at least 100 times more sensitive than colorimetric ones, we expected our AGRO100AUR probe to provide a correspondingly lower LOD. Figure 1A displays fluorescence spectra of mixtures of AUR, hemin, and AGRO100 in the absence and presence of Pb2+ or K+ ions. In the absence of the cations, the catalytic activity of hemin (50 nM) in the absence or presence of AGRO100 (100 nM) was very low (curves a and b). The fluorescence intensity of the mixture of AUR, hemin, and AGRO100 increased significantly after the addition of 1.0 µM Pb2+ (curve c), revealing that Pb2+ ions induced the formation of hemin/Gquadruplex complexes that possessed high catalytic activity for
the H2O2-mediated oxidation of AUR. As a control, we found that K+ ions (100 µM) promoted the DNAzyme activity only slightly under the same conditions (curve d). We subjected the four solutions to UV-vis absorption measurements to study the interactions between hemin and the Pb2+- or K+-stabilized AGRO100. We assigned the Soret absorption band centered at 397 nm to hemin (curve a in Figure 1B); this signal did not change upon the addition of AGRO100 in the absence of Pb2+ or K+ ions. The Soret band of hemin increased and red-shifted, however, after the addition of Pb2+ (1.0 µM; curve c) or K+ (100 µM; curve d) ions, revealing the formation of hemin/Gquadruplex complexes.17 Relative to the K+ ions, the Pb2+ ions induced a larger red shift of the Soret band, suggesting the formation of a more compactly stacked structure between the aromatic planar hemin and the G-quartet of AGRO100 in the presence of Pb2+ ions. The ionic radius of Pb2+ ions (r ) 1.29 Å) is smaller than that of K+ ions (r ) 1.51 Å) but similar to that of the G8 cage formed by the two stacked G-quartets of AGRO100.14b,18 We employed an AGRO100-based molecular beacon (MBAGRO100) that was labeled with the donor carboxyfluorescein (FAM) and the quencher 4-([4-(dimethylamino)phe-
(16) (a) Sˇnyrychova´, I.; Ayaydin, F.; Hideg, E´. Physiol. Plant. 2009, 135, 1–18. (b) Zhou, M.; Diwu, Z.; Panchuk-Voloshina, N.; Haugland, R. P. Anal. Biochem. 1997, 253, 162–168.
(17) Li, T.; Wang, E.; Dong, S. J. Am. Chem. Soc. 2009, 131, 15082–15083. (18) These values are for the octa-coordinate cations: Shannon, R. D. Acta Crystallogr. 1976, A32, 751–767.
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Table 1. G-Rich DNA Sequences of Eight G-Quadruplex Oligonucleotides oligonucleotide
DNA sequence
G-quadruplex structure
ref
AGRO100 T30695 PW17 PS2.M EAD2 TBA15 TBA27 AptATP
GGTGGTGGTGGTTGTGGTGGTGGTGG GGGTGGGTGGGTGGGT GGGTAGGGCGGGTTGGG GTGGGTAGGGCGGGTTGG CTGGGAGGGAGGGAGGGA GGT TGG TGT GGT TGG AGT CCG TGG TAG GGC AGG TTG GGG TGA CT ACC TGG GGG AGT ATT GCG GAG GAA GGT
intermolecular antiparallel intramolecular parallel coexisting or mixed-type hybrid coexisting or mixed-type hybrid intramolecular parallel intramolecular antiparallel intramolecular antiparallel intramolecular antiparallel
25 26 27 28 22a 29 30 31
nyl]azo)benzoic acid (DABCYL) at its 5′ and 3′ termini, respectively, to further study the conformational changes of AGRO100 that were induced by Pb2+ or K+ ions. Relative to K+ ions (100 µM), Pb2+ ions (1.0 µM) induced greater fluorescence quenching efficiency (14% vs 81%) of FAM by DABCYL through fluorescence resonance energy transfer (Figure S1A, Supporting Information), supporting our notion that the Pb2+ ions induced the formation of a more-compact AGRO100 structure. The quenching efficiency was calculated by (IF0 - IF)/IF0; IF0 and IF are the fluorescence intensities of the solutions in the absence and presence of the additional metal ions, respectively. In a control experiment, we observed that Pb2+ ions over the concentration range 100 nM-10 µM did not cause changes in the fluorescence intensity of FAM solutions (100 nM), revealing no fluorescence quenching due to heavy atom effect. To further investigate the interactions between AGRO100 and these two metal ions, we recorded CD spectra (Figure S1B, Supporting Information). In the absence of the metal ions, AGRO100 formed a random-coil structure and, thus, its ellipticity band (positive) at 265 nm was small (curve a). A representative CD spectrum of the solution containing AGRO100 and K+ ions (100 µM) is displayed as curve b. In the presence of 1.0 µM Pb2+ ions, AGRO100 formed monomeric parallel and intermolecular antiparallel G-quadruplex structures, leading to a positive peak at 265 nm with a small shoulder at 295 nm (Figure S1B, curve c, Supporting Information).19 These results further supported our hypothesis that Pb2+ ions induced AGRO100 to form more compact G-quadruplex structures than did K+ ions. Catalytic Activities of Eight Aptamers. Next, we investigated the catalytic activities and selectivities of eight aptamers, including AGRO100, toward Pb2+ ions (Table 1). Figure 2 reveals that AGRO100 was superior to the other aptamers for the detection of Pb2+ ions in terms of its sensitivity (activity) and selectivity. The catalytic activities of the DNAzymes decreased in the following order: intermolecular antiparallel or intramolecular parallel (AGRO100) > intramolecular parallel (EAD2) > intramolecular parallel with a small amount of the antiparallel (T30695) > coexisting or mixed-type hybrid parallel and antiparallel (PW17 and PS2.M) . intramolecular antiparallel (TBA15, TBA27, AptATP).20 The loops of the intramolecular antiparallel G-quadruplex structure bridged either two diago(19) (a) Ðapic´, V.; Bates, P. J.; Trent, J. O.; Rodger, A.; Thomas, S. D.; Miller, D. M. Biochemistry 2002, 41, 3676–3685. (b) Ðapic´, V.; Abdomerovic´, V.; Marrington, R.; Peberdy, J.; Rodger, A.; Trent, J. O.; Bates, P. J. Nucleic Acids Res. 2003, 31, 2097–2107. (20) (a) Kong, D. M.; Yang, W.; Wu, J.; Li, C. X.; Shen, H. X. Analyst 2010, 135, 321–326. (b) Li, T.; Dong, S.; Wang, E. J. Am. Chem. Soc. 2010, 132, 13156–13157.
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nally opposite (diagonal loops) or two adjacent (edgewise loops) antiparallel strands.21 Because the loops were distributed on the top and bottom surfaces, they interacted weakly with hemin through end stacking. The CD spectra of these aptamers (Figures S1B and S2, Supporting Information) in the absence and presence of Pb2+ or K+ ions revealed that the metal-induced aptamers formed various G-quadruplex structures, controlling their catalytic activities and selectivity. For example, Pb2+ ions slightly suppressed the catalytic activities of the hemin complexes with TBA15 and TBA27. EAD2 had good catalytic activity in the presence of Pb2+ ions, but its selectivity for Pb2+ over K+ ions was poor. We point out that the binding stability of the hemin-G-quadruplex DNA complexes does not always correlate with their catalytic activity.22 The selectivity values (100 × ∆IFPb/∆IFK) of AGRO100, EAD2, and T30965 for Pb2+ over K+ ions were 550, 100, and 340, respectively; here ∆IFPb and ∆IFK are the differences in the fluorescence intensities of hemin/AGRO100 solutions in the absence (IF0) and presence (IF) of Pb2+ (1.0 µM) and K+ (100 µM) ions, respectively. Our results revealed that among the eight systems we tested, AGRO100-AUR was undoubtedly the best sensing probe for Pb2+ ions. Effect of Buffer Concentration on Activity and Selectivity of AGRO100-AUR. Further, we investigated the role that the concentration of Tris-acetate buffer played in determining the sensitivity and selectivity of our AGRO100-AUR probe.23 Figure S3 (Supporting Information) reveals that the catalytic activity of AGRO100 in the absence and presence of K+ ions (100 µM) increased slightly upon increasing the concentration of Tris-acetate over the ranges 1-100 and 1-50 mM, respec-
Figure 2. Fluorescence intensities of solutions containing AUR (25 µM), hemin (50 nM), one of the eight aptamers (100 nM), and Pb2+ (1.0 µM) or K+ (100 µM), after the addition of H2O2 (1.0 mM). Other conditions were the same as those described in Figure 1.
Figure 4. Analysis of a representative soil sample using the AGRO100-AUR probe. The samples were spiked with Pb2+ ions at different concentrations (0-500 nM). Error bars represent standard deviations from four repeated experiments. Other conditions were the same as those described in Figure 1.
Figure 3. (A) Fluorescence spectra of solutions containing AUR (25 µM), hemin (50 nM), and AGRO100 (100 nM) in the presence of Pb2+ (0-1.0 µM), after the addition of H2O2 (1.0 mM). Inset: Plot of the fluorescence signal (IF592) at 592 nm of the solutions against the concentration of Pb2+ ions. (B) Selectivity of the AGRO100-AUR probe toward Pb2+ ions. The concentration of Pb2+ ions was 1.0 µM; the concentration of each of the other metal ions was 100 µM. IF0 and IF are the fluorescence intensities of the solutions in the absence and presence of the additional metal ions, respectively. Other conditions were the same as those described in Figure 1.
tively. The hemin/AGRO100 complex became more stable in the presence of Tris+ ions (
