Flourescent Switch Constructed Based on Hemin ... - ACS Publications

To testify the universality and practicality of the fluorescent switch, a series of label-free DNA-related sensing platforms are developed, containing...
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Anal. Chem. 2009, 81, 3544–3550

Flourescent Switch Constructed Based on Hemin-Sensitive Anionic Conjugated Polymer and Its Applications in DNA-Related Sensors Bingling Li, Chuanjiang Qin, Tao Li, Lixiang Wang,* and Shaojun Dong* State Key Laboratory of Electroanalytical Chemistry and State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Graduate School of Chinese Academy of Sciences, Changchun, 130022, People’s Republic of China Here, a fluorescent switch is constructed combining hemin, hemin aptamer, and a newly synthesized anionic conjugated polymer (ACP), poly(9,9-bis(6′-phosphatehexyl) fluorenealt-1,4-phenylene) sodium salt (PFHPNa/ PFP). In the “off-state”, the fluorescence of PFP is sensitively quenched by hemin, with a high Ksv value of ∼107. While in the “on-state”, the formation of the aptamer/hemin complex recovers the fluorescence intensity. The fluorescent switch is sensitive and selective to hemin. To testify the universality and practicality of the fluorescent switch, a series of label-free DNArelated sensing platforms are developed, containing three DNA sensing strategies and one ATP recognition strategy. The fluorescent switch developed is simple, sensitive, and universal, which extends applications of the anionic conjugated polymers. Conjugated polymers/polyelectrolytes (CPs/CPEs) are characterized as a collection of short, conjugated units kept in close proximity by virtue of the polymer backbone.1,2 Compared to their small molecule counterparts, this kind of polymer can coordinate the action of a large number of absorbing units3 and provide much more amplified optical efficiency in either excited-state electron transfer or Fo¨rster resonance energy transfer (FRET).4-6 Watersoluble CPs have a sufficient density of polar substituents to make them soluble in water and display very high photoluminescence (PL) quantum efficiencies. Thus, they are considered as one kind of ideal signal producers in fluorescence based assays, especially bioassays.4-6 Over the past decade, many water-soluble CPs have * Corresponding author. Prof. Shaojun Dong, e-mail: [email protected]. Fax: (+86)-431-85689711. Prof. Lixiang Wang, e-mail: [email protected]. Fax: (+86) 431-85685653. (1) Webber, S. E. Chem. Rev. 1990, 90, 1469–1482. (2) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. Rev. 2000, 100, 2537– 2574. (3) Swager, T. M. Acc. Chem. Res. 1998, 31, 201–207. (4) (a) Achyuthan, K. E.; Bergstedt, T. S.; Chen, L.; Jones, R. M.; Kumaraswamy, S.; Kushon, S. A.; Ley, K. D.; Lu, L.; McBranch, D.; Mukundan, H.; Rininsland, F.; Shi, X.; Xia, W.; Whitten, D. G. J. Mater. Chem. 2005, 15, 2648–2656. (b) Zeng, Q.; Lam, L. W. Y.; Jim, C. K. W.; Qin, A. J.; Qin, J. G.; Li, Z.; Tang, B. Z. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 8070–8080. (5) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954–10957. (6) Thomas, S. W.; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339– 1386.

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been employed to make the sensing process more versatile and applicable,6 such as water-soluble polythiophenes (PTs),7 polyfluorenes (PFs),8 poly(p-phenylene ethynylenes) (PPEs),9 and poly(p-phenylenevinylenes) (PPVs).10 The targets are rather diversiform, including metal ions,7,10,11a small molecules,9 drugs,4 and biomolecules (e.g., enzymes,12,13 proteins,14 and DNAs).5 Among these various analytical applications with water-soluble CPs, the homogeneous detections in relation with DNA have always attracted great attention. They contain several areas in dire need of intense investigation, such as classical DNA analysis,5,15,16 single nucleotide polymorphisms (SNPs) detection,17,18 DNA cleavage monitoring,19 and DNA delivery investigation.20 Besides these, peptide nucleic acids (PNAs)5,21 and aptamers7,22 (functional ssDNA/RNA molecules which can specifically bind different targets23-33) are also imported to CPs based assays, recently. Both detecting strategies and targets are enlarged. However, in most (7) Ho, H. A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384–1387. (8) He, F.; Tang, Y. L.; Wang, S.; Li, Y. L.; Zhu, D. B. J. Am. Chem. Soc. 2005, 127, 12343–12346. (9) Fan, Q. L.; Zhou, Y.; Lu, X. M.; Hou, X. Y.; Huang, W. Macromolecules 2005, 38, 2927–2936. (10) Kim, I. B.; Bunz, U. H. F. J. Am. Chem. Soc. 2006, 128, 2818–2819. (11) (a) Qin, C. J.; Cheng, Y. X.; Wang, L. X.; Jing, X. B.; Wang, F. S. Macromolecules 2008, 41, 7798–7804. (b) Qin, C. J.; Tong, H.; Wang, L. X. Sci. China, Ser. B, accepted. (12) Achyuthan, K. E.; Lu, L. D.; Lopez, G. P.; Whitten, D. G. Photochem. Photobiol. Sci. 2006, 5, 931–937. (13) Feng, F. D.; Tang, Y. L.; He, F.; Yu, M. H.; Duan, X. R.; Wang, S.; Li, Y. H.; Zhu, D. B. Adv. Mater. 2007, 19, 3490. (14) Fan, C. H.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2002, 124, 5642–5643. (15) Hong, J. W.; Henme, W. L.; Keller, G. E.; Rinke, M. T.; Bazan, G. C. Adv. Mater. 2006, 18, 878–882. (16) Chi, C. Y.; Mikhailovsky, A.; Bazan, G. C. J. Am. Chem. Soc. 2007, 129, 11134–11145. (17) Duan, X. R.; Li, Z. P.; He, F.; Wang, S. J. Am. Chem. Soc. 2007, 129, 4154. (18) Gaylord, B. S.; Massie, M. R.; Feinstein, S. C.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 34–39. (19) Tang, Y. L.; Feng, F. D.; He, F.; Wang, S.; Li, Y. L.; Zhu, D. B. J. Am. Chem. Soc. 2006, 128, 14972–14976. (20) Tang, G. P.; Yang, Z.; Zhou, J. J. Biomater. Sci., Polym. Ed. 2006, 17, 461– 480. (21) Al Attar, H. A.; Norden, J.; O’Brien, S.; Monkman, A. P. Biosens. Bioelectron. 2008, 23, 1466–1472. (22) Wang, Y. Y.; Liu, B. Analyst 2008, 133, 1593–1598. (23) Ellington, A. D.; Szostak, J. W. Nature (London, U.K.)) 1990, 346, 818– 822. (24) Robertson, D. L.; Joyce, G. F. Nature (London, U.K.) 1990, 344, 467–468. (25) Tuerk, C.; Gold, L. Science 1990, 249, 505–510. (26) Hamula, C. L. A.; Guthrie, J. W.; Zhang, H.; Li, X.-F.; Le, X. C. Trends Anal. Chem. 2006, 25, 681–691. 10.1021/ac900110a CCC: $40.75  2009 American Chemical Society Published on Web 04/03/2009

Scheme 1. Structures of Hemin (A) and PFP (B)

such existing DNA-related sensors, especially in the label-free ones,7,15,16,18,22,34,35 the sensing signals are mainly based on fluorescence of CPs that is quenched by the polyanionic DNA strands via charge or energy transfer. Therefore, only opposite cationic CPs (CCPs) are applicable. Until now, few anionic conjugated polymers (ACPs) have been used in DNA-related assays. It seriously restricts the comprehensive applications of the CPs. Whitten et al. developed two DNA sensors36,37 with anionic PPE, employing ssDNA or ssPNA as probes. The sensors worked well and have eliminated the nonspecific quenching of CCPs by the DNA molecules. However, labeling fluorescent dyes on DNA molecules was required, which was relatively complex and expensive to operate. Therefore, further developments are needed to make the ACPs more practical in label-free DNA-related assays. Recently, we synthesized a series of water-soluble ACPs11 with high PL quantum efficiencies. They have already been applied in sensitive detections of Fe3+,11a methylviologen, meso-5,10,15,20tetrakis-(N-methyl-4-pyridyl) porphyrine (TMPyP4), and cytochrome C.11b Here, we employ one of these ACPs, poly(9,9bis(6′-phosphatehexyl) fluorenealt-1,4-phenylene) sodium salt (PFHPNa/PFP)11 (Scheme 1), as the fluorescent producer to realize a series label-free DNA-related detections. First, a fluorescent switch is constructed combining PFP, hemin, and hemin aptamer (Scheme 2). In the “off-state”, the fluorescence of PFP can be sensitively quenched by hemin. While in the “on-state”, hemin aptamer reacts with hemin and the formation of the aptamer/hemin complex will recover the fluorescence intensity. The fluorescent switch is very sensitive and selective to hemin. Then, to testify the universality and practicality of the fluorescent switch, a series of DNA-related sensing processes are developed, containing three DNA sensing strategies and one ATP recognition strategy. Through this sensing system derived from the fluores(27) Mairal, T.; Ozalp, V. C.; Sanchez, P. L.; Mir, M.; Katakis, I.; O’Sullivan, C. K. Anal. Biochem. 2008, 390, 989–1007. (28) Huang, C. C.; Huang, Y. F.; Cao, Z.; Tan, W.; Chang, H. T. Anal. Chem. 2005, 77, 5735–5741. (29) Liu, J.; Mazumdar, D.; Lu, Y. Angew. Chem., Int. Ed. 2006, 45, 7955–7959. (30) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Anal. Chem. 2006, 78, 2268– 2271. (31) Song, S. P.; Wang, L. H.; Li, J.; Zhao, J. L.; Fan, C. H. TrAC, Trends Anal. Chem. 2008, 27, 108–117. (32) Xiao, Y.; Lai, R. Y.; Plaxco, K. W. Nat. Protoc. 2007, 2, 2875–2880. (33) Centi, S.; Messina, G.; Tombelli, S.; Palchetti, I.; Mascini, M. Biosens. Bioelectron. 2008, 23, 1602–1609. (34) Wang, S.; Gaylord, B. S.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 5446– 5451. (35) Leclerc, M. Adv. Mater. 1999, 11, 1491–1498. (36) Kushon, S. A.; Ley, K. D.; Bradford, K.; Jones, R. M.; McBranch, D.; Whitten, D. Langmuir 2002, 18, 7245–7249. (37) Kushon, S. A.; Bradford, K.; Marin, V.; Suhrada, C.; Armitage, B. A.; McBranch, D.; Whitten, D. Langmuir 2003, 19, 6456–6464.

cent switch, a simple and universal sensing platform is presented, in which all of the small molecules (hemin, ATP) and DNA can be detected, and the commonly used labeling, modification, and separation steps are avoided. In addition, ACPs are successfully applied into both label-free aptamer based recognitions and DNA sensing processes. It helps to prove that besides CCPs, ACPs also possess promising potential in these fields. EXPERIMENTAL SECTION Materials. All the DNA sequences were listed in Table 1. These DNAs and hemin were purchased from Sangon Biotechnology Co., Ltd. (Shanghai,China). Triton X-100 was purchased from Sigma-Aldrich (St. Louis, MO). The stock solution of hemin (5 mM) was prepared in DMSO, stored in the dark at -20 °C, and diluted to the required concentrations with the 1× binding buffer A (25 mM HEPES, pH 7.4, 20 mM KCl, 200 mM NaCl, 0.05% (w/v) Triton X-100, 1% (v/v) DMSO), or 1× binding buffer B (25 mM Tris-HCl, pH 8.3, 20 mM KCl, 300 mM NaCl, 0.05% (w/v) Triton X-100). The poly(9,9-bis (3′-phosphatepropl) fluorenealt-1,4-phenylene) sodium salt (PFP) was synthesized according to our reported method11 and then dissolved with the TE buffer A (10 mM Tris-HCl, 0.1 mM EDTA, 20 mM KCl) or TE buffer B (25 mM Tris-HCl, pH 8.3, 20 mM KCl, 300 mM NaCl) to 5 mg/ mL as stock solution. Instrumentation. Fluorescent emission spectra were recorded on a Perkin-Elmer LS55 Luminescence Spectrometer (PerkinElmer Instruments U.K.). Construction of Fluorescent Switch. The aptamer Hemin18 was prepared in the 1× binding buffer A and heated at 88 °C for 10 min to dissociate any intermolecular interaction, then gradually cooled to room temperature. Then the hemin stock solution was diluted to required concentrations in 1× buffer or the Hemin-18 solution, in which the aptamer was 55 µM. After 1 h of reaction at 25 °C, the mixtures were diluted in 2.5 × 10-4 mg/mL PFP solution at 1:100 (v/v) for fluorescence detection. In control experiments, 3.2 µM hemin, cytochrome C, and Fe4,4′,4′′,4′′′-tetracarboxy phthalocyanine (FePc (COOH)4) were diluted in 92 µM Hemin-18 solution, respectively. After 1 h of reaction at 25 °C, the mixtures were diluted in 2.5 × 10-4 mg/ mL PFP solution in TE buffer at 1:100 (v/v) for fluorescence detection. Optimization of the Hemin Aptamer Strands. All the seven DNA strands used were prepared in the Tris-HCl buffers with different kinds of salts and heated at 88 °C for 10 min to dissociate any intermolecular interaction, then gradually cooled to room temperature. Then the hemin stock solution was diluted to TE buffers or DNA solutions at 1:1 concentration ratio of hemin and DNA (both 4.5 µM). After 1 h of reaction at 25 °C, the mixtures were diluted in 2.5 × 10-4 mg/mL PFP solution at 1:76 (v/v) for fluorescence detection. DNA Sensing Strategy I. AG4 strands was prepared in TE buffer and heated at 88 °C for 10 min to dissociate any intermolecular interaction, then gradually cooled to room temperature. Then C-AG4 was added into the AG4 solutions to different concentrations, in which concentration of the AG4 strand was 4.5 µM. After 1 h of reaction at 25 °C, the mixtures were diluted in 2.5 × 10-4 mg/mL PFP solution at 1:76 (v/v) for fluorescence detection. Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

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Scheme 2. Schematics for Principle of the Fluorescent Switch Combining PFP, Hemin, and Hemin Aptamer

Table 1. Sequences of Aptamers and Other ssDNA Used used in constructing the fluorescent switch Hemin-18: 5′-GTGGGTAGGGCGGGTTGG-3′ used in optimization of the hemin aptamer strands Hemin-18: 5′-GTGGGTAGGGCGGGTTGG-3′ AG4: 5′- TGGGTAGGGCGGGTTGGGAAA-3′ AG5: 5′- TTGGGTTTTGGGTTTTGGGTTTTGGGTT-3′ Random-18: 5′- TCACGTCCAACCCGCCCT-3′ HUM24: 5′-TTAGGGTTAGGGTTAGGGTTAGGG-3′ HUMIN-24: 5′- GTGGGTCATTGTGGGTGGGTGTGG-3′ TBA: 5′- GGTTGGTGTGGTTGG-3′ used in DNA detection AG4: C-AG4: S1: S2: T1: S3: T2: M2:

5′- TGGGTAGGGCGGGTTGGGAAA-3′ 5′- TTTCCCAACCCGCCCTACCCA-3′ 5′-CGA TTC GGT ACT GGC TCA AAA TGT GGA GGG T-3′ 5′-AGG GAC GGG AAG AAA GAT AATGCG CAT GCT CAA-3′ 5′-TTG AGC ATG CGC ATT ATC TGA GCC AGTACC GAA TCG-3′ 5′-CCCTACCCAGCCTTAACTGTAGTACTGGTGAAATTGCTGCCATTTGGGTAGGGCGGGTTGGG-3′ 5′-AATGGCAGCAATTTCACCAGTACTACAGTTAAGGC-3′ 5′-AATCGCAGCAATTTCACCAGTACTACAGTTAAGGC-3′

used in ATP recognition MBA: blocker:

3′-GGGTTGGGCGGGATGGGTTTCATTTTTTGGGGAGGCGTTCGTTATGAGGGGGTCCA-5′ 5′-TACCCAAAGATTTTTTTCCTTCCTC-3′

DNA Sensing Strategy II. DNA sequences S138 and S2 were prepared in the TE buffer A. The two DNA solutions were mixed at a 1:1 volume ratio and heated at 88 °C for 10 min to dissociate any intermolecular interaction, then gradually cooled to room temperature. An equal volume of the hybridization buffer (50 mM HEPES, pH 7.4, 40 mM KCl, 400 mM NaCl, 0.1% Triton X-100, 2% DMSO) was added to the DNA mixture. At room temperature, this mixture was allowed to fold overnight and then incubated with hemin of the same molar concentration for over 12 h to form the hemin/G-quartet complex. Finally, the mixture was treated with required concentrations of T1 overnight at 25 °C, in which S1, S2, and hemin were all 4.5 µM. After 12 h of reaction at 25 °C, the mixtures were diluted in 2.5 × 10-4 mg/mL PFP solution in TE buffer at 1:76 (v/v) for fluorescence detection. DNA Sensing Strategy III. DNA sequence S339 was prepared in the 1× binding buffer and heated at 88 °C for 10 min to dissociate any intermolecular interaction, then gradually cooled to room temperature. Then sequences T2 and M2 were diluted in S3 solution to required concentrations, respectively, in which (38) Li, T.; Dong, S. J.; Wang, E. K. Chem. Commun. 2007, 4209–4211. (39) Xiao, Y.; Pavlov, V.; Niazov, T.; Dishon, A.; Kotler, M.; Willner, I. J. Am. Chem. Soc. 2004, 126, 7430–7431.

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S3 was 4.5 µM. After hybridization for 2 h at 25 °C, hemin was added into each mixture to a final concentration of 4.5 µM. After 1 h of reaction, the mixtures were diluted in 2.5 × 10-4 mg/mL PFP solution in TE buffer at 1:76 (v/v) for fluorescence detection. ATP Recognition. A mixture containing 9.0 µM MBA40 and 11 µM blocker in binding buffer was heated at 88 °C for 10 min to dissociate any intermolecular interaction, then gradually cooled to room temperature. ATP was diluted to different concentrations in the above mixture at 1:25 (v/v) and kept at room temperature for 2 h. Then 9 µM hemin was diluted in all the ATP/DNA solutions at 1:1 volume ratios. After 1 h of interaction, the hemin/ ATP/DNA solutions were, respectively, diluted into 3.2 × 10-4 mg/ mL PFP solution at 1:100 (v/v) for fluorescence detection. RESULTS AND DISCUSSION Construction of the Fluorescent Switch. Hemin (Scheme 1) is an enzyme with iron-containing porphyrin structure, which is made from red blood cells and plays an important role as an active cofactor for a variety of enzymes such as catalases and (40) Li, D.; Shlyahovsky, B.; Elbaz, J.; Willner, I. J. Am. Chem. Soc. 2007, 129, 5804–5805.

Figure 1. (A) Fluorescence response of the fluorescent switch (λex ) 353 nm): (a) 2.5 × 10-4 mg/mL PFP solution; (b) 2.5 × 10-4 mg/mL PFP solution with 32 nM hemin and 920 nM Hemin-18; (c) 2.5 × 10-4 mg/mL PFP solution and 32 nM hemin. (B) Control experiment between 32 nM hemin, 32 nM cytochrome C, and 32 nM FePc(COOH)4 in the presence of 920 nM Hemin-18 and 2.5 × 10-4 mg/mL PFP.

Figure 2. (A) Ksv plot of PFP in the presence of hemin, F′. (B) Ksv plot of PFP in the presence of hemin and 550 nM Hemin-18. CPFP ) 2.5 × 10-4 mg/mL. The y-axis was normalized with the fluorescence of AG4/hemin/PFP as 1. I 0 represented the fluorescence of the original solution with only PFP.

peroxidases.38 In this work, we find that fluorescence emission of PFP can be sensitively quenched by hemin. The fluorescent switch is constructed based on this phenomenon. As shown in Figure 1A, when PFP was added into hemin solution (32 nM here), its fluorescence emission at 420 nm decreased dramatically, about 1/3 of the original intensity, and 5 min is confirmed enough for the quenching (data not shown). This process is defined as the “off-state” of the fluorescent switch. Through measurements (Figure 2A), the lowest limit for quenching response of hemin is 0.2 nM. In a concentration range of 2-50 nM (of hemin), a linear quenching range is gotten. The Stern-Volmer constant (Ksv) quantified from eq 114 is ∼5.5 × 107 at the present condition, indicating the high efficiency of the “off-state”. The quenching mechanism may be speculated as the following reasons (Scheme 2). At first, the positive center containing Fe3+ in hemin approaches the negatively phosphatic backbone of the PFP. Then two processes occur. One is the fast photoinduced electron transfer (ET) from the PFP to Fe3+ in the hemin, according to the previous work.14 The possible mechanism is shown in eq 2, where PFP* stands for the excited state of the polymer and hemin Fe(III) and hemin Fe(II) are the ferric and ferrous states of hemin, respectively. In this photoinduced ET process, the light energy of PFP* is transferred to chemical energy. The energy that light emission requires is diminished, and the fluorescence is thus quenched. The other quenching process is the FRET. In general, hemin and a lot of hemin

containing complexes possess one of the similar absorbance bands from 415 to 430 nm. So the optical properties of the PFP (γem ) 420 nm) and hemin just favor FRET from PFP (donor) to hemin (acceptor). This was proven in our previous work,11b in which a porphyrin, TMPyP4, obviously quenched the fluorescence of PFP. As described, it is a integrated factor of both ET and FRET that leads to a finally quenched fluorescence. I 0 /I ) 1 + Ksv[quencher]

(1)

hν

PFP 98 PFP*PFP* + hemin (Fe(III)) f PFP + hemin (Fe(II)) (2) The “on-state” of the fluorescent switch is a higher emission state with respect to the “off-state”. It requires existence of hemin aptamers. According to previous literatures,39,41 hemin can combine with several guanine (G)-rich DNA aptamers to form a new kind of complex, which is usually used as a synthetic DNAzyme with high peroxidase activity. Here, one of the hemin aptamers (Hemin-18) is chosen as a model to test the fluorescent switch but without making use of the DNAzyme function of the hemin/ aptamer complex.42 In Figure 1A, too, when a mixed sample of 32 nM hemin and 920 nM aptamer was added into the PFP solution, the fluorescence emission would be obviously recovered (41) Travascio, P.; Li, Y. F.; Sen, D. Chem. Biol. 1998, 5, 505–517. (42) Li, T.; Li, B. L.; Dong, S. J. Anal. Biochem. 2007, 389, 887–893.

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Figure 3. (A) Fluorescence spectrum of hemin/DNAs/PFP solution in TE buffer with 20 mM KCl, 300 mM NaCl. The DNAs contained Hemin18, Hemin-24, HUM24, AG5, T2, TBA, and AG4. The blank represented hemin/PFP solution without any DNA strands. (B) Kinetics measurements of the hemin/PFP solution after AG4, Hemin-18, or Hemin-24 was added. CPFP ) 2.5 × 10-4 mg/mL, CDNA ) C hemin ) 59 nM.

(almost to the original intensity) compared to the condition only with 32 nM hemin. This fluorescence recovery results from the protecting function of Hemin-18 to hemin. In neutral buffer, Hemin-18 folds into a three-layer G-quartet to interact with hemin. The G-quartet is negative charge-rich and can shelter hemin from approaching to the PFP. Thus, quenching ability of the hemin itself is dramatically decreased. As shown in Figure 2B, the Ksv of hemin in 550 nM Hemin-18 is about 2.3 × 106, which is about 1 /20 of the “off-state”. This result reflects that the aptamerinduced fluorescence recovery is effective. Meanwhile, it illuminates such fluorescence recovery is directly related to the ratio of CHemin-18/Chemin. Only when the amount of Hemin18 is over 15 times higher than that of hemin can the PFP fluorescence be fully recovered. That may be attributed to that even in the presence of aptamer, some dissociative hemin can still remain in the PFP in the solution. In the control experiments, the “on-state” is also proven selective to hemin. Shown in Figure 1B, all of the hemin, cytochrome C, and FePc(COOH)4 (with the same concentration) could quench the PFP fluorescence at different degrees, but only solutions with hemin gets emission recovery in the presence of Hemin-18. After the kinetic measurements, 35 min is proven enough for Hemin18-hemin interaction (Figure 3B). However, considering the occasional change of the room temperature, 1 h is used in further experiments. Through above descriptions, a fluorescent switch is successfully constructed combining PFP, hemin, and hemin aptamer. With only hemin, the switch takes on an “off-state”. While with both hemin and the aptamer, the switch takes on an “on-state”. Both the two states are sensitive, and the “on-state” is selective to hemin. We hope this fluorescent switch is practical in DNA-related sensor fabrications. Therefore, a series of applications are further developed based on it. Optimization of the Hemin Aptamer Strands. Besides Hemin-18, some other G-quartets may also bind hemin as aptamers. Before further sensor fabrications, we primarily investigate several aptamer strands and try to find a relatively better one in constructing the fluorescent switch. Seven DNA strands are stochastically chosen as examples (Table. 1). As reported, except for the Random-18 strand, all of the Hemin-18,41 Hemin-24,41 HUM24,43 AG4,43 AG5,44 and TBA7 (43) Kong, D. M.; Wu, J.; Ma, Y. E.; Shen, H. X. Analyst 2008, 133, 1158–1160.

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can fold into G-quartet structures at certain conditions. The fluorescence recovery induced by these seven DNA strands are compared at the same concentration. As shown in Figure 3A, except Random18 and TBA strands, all of the Hemin-18, Hemin-24, HUM24, AG4, and AG5 can lead to fluorescence recovery (in the TE buffer with 20 mM KCl and 300 mM NaCl). The order of the recovering response from high to low is AG4, Hemin-18, Hemin-24, AG5, and HUM24. It illustrates that these five strands can bind hemin at different degrees, and thus all of them can be used in the fluorescent switch. Random-18 is a control strand that cannot fold into G-quartet to combine the hemin, so it cannot lead to the fluorescence recovery. An exception is the TBA. We have proved that TBA could interact with the hemin at a low affinity,45 but in the present condition, it was not observed from the fluorescent recovery. Therefore, it cannot be used in the fluorescent switch in this condition. Because the fluorescent recovering response of all the Hemin-18, Hemin-24, and AG4 are higher than the other strands, we further compare these three strands in kinetic measurements. It is observed that the AG4 binds hemin with the shortest time among these three DNA strands, less than 30 min (Figure 3B). Therefore, because of the highest (Figure 3A) and fast response (Figure 3B) of the AG4, it is preferentially used in the fluorescent switch for sensor fabrications. What should be noted is that through the ion-dependence investigation, fluorescence recovery produced by AG4 is highly dependent to K+ in the buffer and 20 mM is proven enough for it to get a sensitive response. Therefore, we still use 20 mM KCl in the sensing process. DNA Sensing Strategy I. To test the practicability of the fluorescent switch in DNA-related assays, we endow it to DNA sensing systems. First, complementary strands to the hemin binding aptamers (G-quartets) are recognized. AG4 complementary strand (C-AG4) is taken as a model. The sensing principle is simple (Scheme 3A). AG4 can increase the fluorescence of the PFP/hemin solution and lead to a “on state” of the fluorescent switch. When C-AG4 exists, it hybridizes with AG4 to form a more stable duplex than the G-quartet structure folded only by the AG4. Hemin is thus released from the AG4/hemin complex, ultimately leading to a quenched fluorescence (“off-state”). By measurement (44) Zhou, Q.; Li, L.; Xiang, J. F.; Tang, Y. L.; Zhang, H.; Yang, S.; Li, Q.; Yang, Q. F.; Xu, G. Z. Angew. Chem., Int. Ed. 2008, 47, 5590–5592. (45) Li, T.; Wang, E. K.; Dong, S. J. Chem. Commun. 2008, 3654–3656.

Scheme 3. Schematics for DNA Sensing Strategies I, II, and III

of the fluorescence decrease of the PFP/hemin/AG4 solution in the presence of C-AG4, C-AG4 can be detected. As shown in Figure 4A, a linear range from 1.4 to 50 nM is received, with a lowest detection limit (LOD) of 1.4 nM. DNA Sensing Strategy II. As described above, DNA strategy I is only applicable to detect complementary strands of the heminbinding G-quartets. Accordingly, the sensing system is improved to another strategy, in which the targets are extended to any free ssDNA strand. The schematic is depicted in Scheme 3B. In given buffer, two G-rich single-strand DNAs (S1, S2)38 could fold into a G-quartet structure (Kd ) 130 mM2)38 with two hemin aptamer parts (dark green) and two free nucleic acid parts (dark blue). The aptamer parts are used to form the fluorescent switch with hemin and PFP. The two free nucleic acid parts are combined as the recognition part complementary to the target (T1). In the absence of T1, hemin can react with the aptamer parts of the G-quartet structure and the fluorescent switch displays the “onstate”, leading to a relatively high fluorescence emission of the PFP. With the addition of T1, hybridization between T1 and the recognition parts will destroy the hemin/aptamer complex and expose hemin to the PFP, turning the fluorescent switch to the “off-state”. A decreased fluorescence emission is received. As shown in Figure 5A,B, with the same concentration of hemin, S1, and S2, the fluorescence intensity was decreased obviously when T1 increased from 9 to 150 nM. The LOD is 9 nM. This detection limit is not very ideal, and after optimization, the single-mismatch discrimination is still difficult. To get a more satisfied detection, another sensing strategy is designed as shown in Scheme 3C. DNA Sensing Strategy III. Strategy III is a signal-on process. S339 is a ssDNA strand containing an aptamer part (dark green, derivated from AG4) and a recognition part (dark blue) partly complementary to the target strand (T2). At given conditions, this strand keeps an optimized conformation of part self-hybridization. In this case, the aptamer part is relatively restricted and could not react with hemin. The fluorescent switch is in an “off-state”, with the PFP fluorescence highly quenched by hemin. However, if with T2 the target is added, it could hybridize with the recognition part of S3 and release the aptamer part. In this case, the hemin/aptamer complex forms and the fluorescent switch is on. Thus, the PFP emission is partly recovered. As shown in Figure 5C, with the same concentration of hemin and S3, the fluorescence intensity increases when T2 was increased from 0.6

to 50 nM. The LOD is 0.6 nM, which is as low as most label-free DNA sensors. We also verify the single-mismatch discrimination of this strategy. Figure 5D shows that when the T2 is displaced by the single-mismatch strand, with the same targets concentrations, the fluorescence intensity with the single-mismatch strand is less than T2, displaying the potential for single-mismatch discrimination at low target concentrations. The relative lower sensitivity of strategy II compared to strategy III may be due to this supramolecular complex formed by S1, S2, and hemin is not so stable as the AG4/hemin complex. ATP Sensing Strategy. To enlarge the application of the fluorescent switch, an aptamer-based small molecule sensing strategy is also developed, taking ATP as a model molecule. As shown in Scheme S1 in the Supporting Information, DNA sequence 1 (MBA)40 was designed as an integrated aptamer of both ATP (dark blue) and hemin (dark green), in which the hemin aptamer part is derived from AG4. Another DNA sequence 2 (PCS) is used as a blocking strand (blocker, pink), which shares several partly complementary sequences to the ATP aptamer part and hemin aptamer part, respectively. In the absence of ATP, the MBA and PCS form stable part duplexes and the hemin aptamer part could not form an effective G-quartet structure to bind hemin. Therefpre, the fluorescent switch is in the “off-state”, with a highly quenched fluorescence of PFP. While ATP exists, formation of the ATP-aptamer structure will partly destroy the part duplex and release the hemin aptamer. In this case, the hemin/aptamer complex forms and the fluorescent switch is on. The fluorescence of PFP is thus recovered by some degree. With the use of this method, fluorescence dependence to the 0.08-800 µM ATP (Figure 4B) is received, which is very sensitive. However, there is no linear range observed. Thus, only recognition can be realized. In the control experiments, GTP cannot lead to fluorescence enhancement, displaying the selectivity of the sensor (data not shown). CONCLUSION In conclusion, anionic PFP with high PL quantum efficiencies has been used to construct a fluorescent switch with hemin and its aptamers. Also, a series of label-free DNA-related sensing processes are realized based on this fluorescent switch, containing three DNA sensing strategies and one ATP sensing strategy. The sensors are simple and extend the applications of ACPs at the same time. One of the DNA detection strategies (strategy III) achieves the LOD of 0.6 nM and possesses high potential for single-mismatch discrimination. The ATP sensor is also effective. The LOD is as low as 0.08 µM, which is very sensitive among the reported fluorescent aptasenors. These results fully testify to the universality and practicability of our design. In addition, with the combination of previous works, until now, the PFP has been applied into several sensing systems with the targets ranging from Fe3+, methylviologen, TMPyP4, cytochrome C, hemin, ATP, and DNA. Furthermore, compared to the commonly used poly(9,9-bis(4′-sulfnoatobutyl) fluorenealt1,4-phenylene) sodium salt (PFS), PFP is more sensitive to hemin in the present condition. This illustrates its promising potential used as a signal producer. Finally, it should be noted that similar to a lot of label-free sensors, the sensing system developed in this work may have potential interferences in complex samples more than the labeled sensors. The most possible interferences in this system come from the materials affecting the PFP fluorescence, such as some proteins, high salts (e.g., KCl and NaCl), and Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

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Figure 4. (A) Concentration dependence of the C-AG4: CAG4 ) Chemin ) 59 nM, CPFP ) 2.5 × 10-4 mg/mL. I 0 represented the fluorescence of the original solution with only PFP. (B) Concentration dependence of ATP: CPFP ) 3.2 mg/mL, Chemin ) 59 nM, CMBA ) 59 nM, Cblocker ) 72 nM. I 0 represented the fluorescence of the original solution with only PFP. Imin represented the fluorescence of the original solution with PFP, hemin, and the duplex conformed by MBA and blocker.

Figure 5. (A) DNA sensing of strategy II: CT1 ) 0 (a), 9 (b), 26 (c), 53 (d), and 135 nM (e). (B) Concentration dependence of T1: I0max represented the fluorescence of the FPF, hemin, and G-quartet formed by S1 and S2. (C) Concentration dependence of T2: I 0 represented the fluorescence of the original solution only with FPF. (D) Single-mismatch discrimination experiment for strategy III: 30 (a) and 0.6 nM T2 (c) and 30 (b) and 0.6 nM single-mismatch strand (d). CPFP ) 2.5 × 10-4 mg/mL.

molecules which can quench the PFP fluorescence through FRET or ET. However, by further improvements, for example, sample pretreatment, purification, and dilution, the interferences will be much decreased or avoided. Meanwhile, experimental temperature, reaction time (for hemin-aptamer interaction, aptamer-target DNA interaction), and uniformity of the PFP solution are all proven very important for the sensing processes. To get reproducible data, these factors should be well controlled.

20735003, and the Chinese Academy of Sciences, Grant KJCX2YW-H11.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China, Grant Numbers 20575063, 20675076, and

Received for review January 16, 2009. Accepted March 11, 2009.

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SUPPORTING INFORMATION AVAILABLE Scheme for ATP recognition based on the fluorescent switch. This material is available free of charge via the Internet at http://pubs.acs.org.

AC900110A