Amplified Fluorescent Sensing of DNA Using Graphene Oxide and a

Article pubs.acs.org/Biomac

Amplified Fluorescent Sensing of DNA Using Graphene Oxide and a Conjugated Cationic Polymer Xiao-Jing Xing, Xue-Guo Liu, Yue He, Yi Lin, Cui-Ling Zhang, Hong-Wu Tang,* and Dai-Wen Pang Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Research Center for Nanobiology and Nanomedicine (MOE 985 Innovative Platform), Wuhan Institute of Biotechnology, and State Key Laboratory of Virology, Wuhan University, Wuhan 430072, China S Supporting Information *

ABSTRACT: We explore the interactions between a fluorescein (FAM)-labeled single-stranded DNA (P), graphene oxide (GO), and a cationic conjugated polymer, poly [(9,9bis(6′-N,N,N-trimethylammonium)hexyl)-fluorenylene phenylene dibromide] (PFP). It is found that the fluorescence change of P-GO-PFP system is dependent on the addition order of P and PFP. When adding PFP into P/GO complex, the fluorescence resonance energy transfer (FRET) from PFP to P is inefficient. If P is added to PFP/GO complex, efficient FRET is obtained. This may be attributed to the equal binding ability for P and PFP to GO. The results of time-resolved fluorescence and fluorescence anisotropy support the different fluorescent response under different addition order of P and PFP to GO. Based on the above phenomenon, we demonstrate a method to reduce the high background signal of a traditional PFP-based DNA sensor by introducing GO. In comparison to the use of single PFP, the combination of PFP with GO-based method shows enhanced sensitivity with a detection limit as low as 40 pM for target DNA detection.

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intercalator of double-stranded DNA.12 However, the energy transfer from CCP to the intercalated EB is not efficient due to a nonoptimal (orthogonal) orientation between the transition moment of the optically active polymer backbone and that of the intercalated EB. Then, they introduced DNA probe to act as a FRET gate to relay the excitation energy and obtained efficient energy transfer for fluorescence enhancement.13 Nevertheless, the intrinsic toxicity of EB limits its practical biomedical applications. Further study by Fan et al. described a new strategy for CCP-based DNA detection by introducing magnetic microparticles.14 Though a “signal-off” in the unhybridized state is obtained, the stringent washing step and rehybridization make the detection complex and timeconsuming. Therefore, the development of simple and effective approaches which are capable of reducing the background signal of CCP-based platform still pose a significant challenge. Very recently, graphene oxide (GO), a single-atom-thick, two-dimensional nanosheet prepared by acid exfoliation of graphite,15 has led to intense interest in biological applications owing to its unique characteristics such as good watersolubility,16 versatile surface modification and superior fluorescence quenching ability.17 Based on these properties, several GO-based sensors have been developed for the detection of DNA,18 proteins,19 enzyme activity,20 and other small molecules21 by using dye-labeled complementary oligonucleotides or aptamers as recognition units. Meanwhile,

INTRODUCTION The design of amplified nucleic acids sensors has gained much interest because of its significant role in medical diagnosis, gene expression analysis, biomedical studies, and so on.1−3 Within the past decade, various approaches to amplify the detection of nucleic acids have been established, including the polymerase chain reaction (PCR), rolling circle amplification process (RCA),4 ligase chain reaction,5 hybridization chain reaction,6 and isothermal DNA replication machinaries.7 Although amplified optical response is obtained in these methods, they are either laborious, time-consuming, or in need of strict laboratory controls and highly trained personnel, This highly motivates the development of simple, reliable, and amplifying nucleic acid detection systems. Conjugated polyelectrolytes (CP) provide an effectively amplified platform for chemical and biological sensors by virtue of their light harvesting properties.8,9 Among the analytical applications, the homogeneous detection in relation with DNA which relies on electrostatic interactions between DNA and cationic conjugated polymer (CCP) has always attracted a great deal of research attention. Bazan and Heeger research groups conducted their pioneering investigations using CCP to detect DNA with peptide nucleic acid (PNA) probes based on the fluorescence resonance energy transfer (FRET) mechanism.8a,10 Subsequent work to replace PNA probe with low cost DNA probe11 was restricted by high background fluorescence stemming from the nonspecific electrostatic interactions between the DNA probe and CCP. In this regard, enormous efforts have been made to circumvent this limitation. Wang et al. substituted DNA probe with ethidium bromide (EB), an © XXXX American Chemical Society

Received: September 19, 2012 Revised: December 1, 2012

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Scheme 1. Schematic Representation of GO-Based Low Background-Signal Platform for the Detection of Target DNA

some label-free GO sensors are constructed utilizing the strong adsorption between GO and some pyrene derivatives with πelectron-rich frameworks. Loh et al. reported a label-free optical sensor for DNA assay based on GO and a pyrene derivative, 4(1-pyrenylvinyl)-N-butylpyridinium bromide (PNPB).22 They found that efficient fluorescence quenching of PNPB by GO occurred and the PNP+GO− complex was selective for dsDNA relative to that for RNA, proteins, and mono/polysaccharides, attributed to the higher energy of ion-complex formation of PNP+DNA− compared with PNP+GO−. We investigate the interaction between a fluorescein (FAM)labeled single-stranded DNA (P), GO, and a cationic conjugated polymer, poly[(9,9-bis(6′-N,N,Ntrimethylammonium)hexyl)-fluorenylene phenylene dibromide] (PFP). We found that FRET from PFP to P adsorbed on GO surface is inefficient, implying that the addition of PFP cannot release P from GO surface. Based on this finding, a low background signal platform for amplified DNA detection using GO and PFP is constructed (Scheme 1). Compared with the reported strategies based on PFP, this approach effectively decreases the high background signal to obtain high sensitivity by simply introducing low amount of GO.

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Target DNA: 5′-TAGCTTATCAGACTGATGTTGA-3′ DNA1NC: 5′-TAGCTTATGAGACTGATGTTGA-3′ DNA2NC: 5′-TAGCTTATGAGACTGCTGTTGA-3′ DNANC: 5′-CGTGATGAACGTATGAGCGTAT-3′ Apparatus. A Hitachi F-4600 fluorescence spectrophotometer (Hitachi Company, Tokyo, Japan) was used to record the fluorescence spectra and measure the fluorescence intensity. UV−vis absorption spectra data were recorded by a UV-2550 spectrophotometer (Shimadzu, Tokyo, Japan). The 1H NMR spectra were recorded on Mercury VX 300 MHz spectrometer (Varian, U.S.A.). The chemical structure of PFP was confirmed using a Fourier transform infrared microspectrometer (FT-IR; Nicolet-5700, U.S.A.). The number and molecular weight of polymer were characterized by gel permeation chromatography (GPC) equipped with a Waters 2690D separations module and Waters 2410 refractive index detector. Tetrahydrofuran (THF) was used as eluent and polystyrene standards as calibration. Fluorescence intensity decay curves were measured on a FELIX32 system (Photon Technology International). Synthesis of Graphene Oxide. Graphite oxide was synthesized from natural graphitic powder according to Hummer’s method with some modification.23 Briefly, a mixture of natural graphite (3.0 g), K2S2O8 (2.5 g), P2O5 (2.5 g), and H2SO4 (12 mL) was vigorously stirred at 80 °C and then diluted with deionized water (0.5 L). After treating the above mixture via filtering and washing, the initial product was redispersed into concentrated H2SO4 (120 mL). Then, KMnO4 (15 g) was slowly added at 0 °C. Successively, the mixed solution was stirred at 35 °C for 2 h, followed by dilution with deionized water (250 mL). After continuously stirring for another 2 h, additional deionized water (0.7 L) and 30% H2O2 (20 mL) were added to the mixture drop by drop. The resulting mixture was filtered and washed with 10% HCl aqueous solution and deionized water. Finally, the as-synthesized product was purified by dialysis for one week, dried in a desiccator, and then dispersed in water under sonication for 5 h. Synthesis of Poly[(9,9-bis(6′-N,N,N-trimethylammonium)hexyl)-fluorenylene Phenylene Dibromide] (PFP). Poly[(9,9bis(6′-N,N,N-trimethylammonium)hexyl)-fluorenylene phenylene dibromide] was synthesized using a previously reported method via the Suzuki reaction.24 A mixture of 2,7-dibromo-9,9-bis(6′-bromo-hexyl)fluorene (325 mg, 0.5 mmol), 1,4-phenyldiboronic acid (82.9 mg, 0.5 mmol), Pd(dppf)Cl2 (7 mg), and potassium carbonate (830 mg, 6

EXPERIMENTAL SECTION

Materials and Reagents. Graphite powder, sulfuric acid, potassium persulfate, phosphorus pentoxide, hydrogen peroxide, and potassium permanganate were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2,7-Dibromo-9,9-bis(6′bromohexyl)fluorene, 1,4-phenyldiboronic acid, and Pd(dppf)Cl2 were purchased from Synwit Technology Co., Ltd. (Beijing, China). All chemicals were of analytical grade or of the highest purity available. The oligonucleotides were purchased from Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China) and purified using high performance liquid chromatography. The sequences of the nucleic acids used in the study are as follows: Probe DNA: 5′-6-FAM-TCAACATCAGTCTGATAAGCTA3′ B

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mmol) in water (3 mL) and THF (6 mL) was vigorously stirred at 85 °C for 24 h under a nitrogen atmosphere and cooled, and methanol was added; the resulting precipitate was filtered and washed with methanol and acetone and then dried under vacuum to afford the neutral polymer as a yellow solid. Yield: 70.9% (200 mg). 1H NMR (300 MHz, CDCl3, ppm): δ7.8 (m, 5H), 7.7−7.6 (m, 4H), 7.5 (m, 1H), 3.3 (t, 4H), 2.1 (m, 4H), 1.7 (m, 4H), 1.3−1.2(m, 8H), 0.8 (m, 4H). FT-IR (KBr disk, cm−1): 3448, 3025, 2961, 2929, 2853, 1461, 1261, 1096, 1022, 809, 758, 698, 642, 561. GPC (THF, polystyrene standard), Mw: 37449 kg/mol; Mn: 12833 kg/mol; PDI: 2.9. The final water solution of PFP (polymer 2, Figure S1) was obtained by treating the neutral polymer with condensed trimethylamine. Briefly, 30% trimethylamine (2 mL) was added dropwise to a solution of polymer 1 (60 mg) in THF (10 mL) at −78 °C and then the mixture was allowed to warm to room temperature. The precipitate was redissolved by the addition of water (10 mL). After the mixture was cooled down to −78 °C, extra 30% trimethylamine (2 mL) was added, and the mixture was stirred at room temperature for 24 h. After removing the solvent, acetone was added to precipitate PFP as a yellow powder. Yield: 74.2% (60 mg). 1H NMR (300 MHz, CD3OD, ppm): δ7.9−7.8 (m, 10H), 3.2 (t, 4H), 3.0 (s, 18H), 2.3 (br, 4H), 1.6 (br, 4H), 1.2 (br, 8H), 0.8 (br, 4H). FT-IR (KBr disk, cm−1): 3409, 3022, 2928, 2856, 1606, 1462, 1259, 1095, 965, 908, 817, 744, and 599. DNA Hybridization Assay. All experiments were performed in 10 mM PBS (pH 7.4) containing 100 mM NaCl. For DNA hybridization assay, a solution containing 20 nM of P and different concentrations of target DNA was quickly heat-annealed (80 °C for 5 min and cooled to room temperature for 1 h), then 5 μg/mL GO was added into the solution. After the mixture was incubated for 20 min at room temperature, 1.57 μM PFP in repeat units was added and incubated for another 20 min at 4 °C. Then the fluorescence emission spectra were recorded immediately. The same procedures were repeated in the presence of one base, two bases mismatch, and noncomplementary strand instead of complementary strand to assess the selectivity.

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RESULTS AND DISCUSSION Working Principle. The important insight in our design comes from a key finding that the addition of CCP cannot release P from GO surface (curve c, Figure 1A), which we will defer discussion until later. Scheme 1 illustrates the principle of this strategy. Energy transfer experiment is carried out by using PFP as the donor due to the high fluorescence quantum yield and blue emission being suitable for use in FRET. FAM, with maximum absorption at 488 nm and emission at 518 nm, is chosen for labeling the probe DNA since its absorption overlaps with the characteristic emission of PFP (420 nm; Figure S2). In the absence of target DNA, the fluorescence intensity of P is greatly quenched after the addition of GO, originating from the strong adsorption of ssDNA on GO surface and the super fluorescence quenching ability of GO.18c When the PFP is added to the P/GO complex, less FRETinduced fluorescence signal at around 525 nm is observed. On the other hand, in the presence of target DNA, P forms a dsDNA with target DNA, making the P far away from GO due to the weak adsorption between dsDNA and GO. In this case, the addition of PFP induces obvious FRET signal as a result of the strong electrostatic interaction between PFP and dsDNA.11 Therefore, a low background and amplified fluorescent detection of DNA could be easily obtained by monitoring the fluorescence signal change. GO Based Low Background Signal Platform for Amplified DNA Detection. To demonstrate the feasibility of this strategy, the fluorescence intensity of the assay system under different conditions was measured. As shown in Figure 1A, the initial fluorescence from P (curve a) was greatly

Figure 1. (A) Normalized fluorescence spectra of the assay system under different conditions: (a) P in PBS buffer; (b) P + GO; (c) P + GO + PFP; (d) target DNA + P + GO + PFP. (B) Normalized fluorescence spectra without (black) and with (red) target DNA in the absence of GO, by exciting at 370 nm. (C) Normalized fluorescence spectra without (black) and with (red) target DNA in the presence of GO, by exciting at 370 nm.

quenched to 2.5% in the presence of 5 μg/mL GO (curve b), resulting from the strong binding of P with GO and the high quenching ability of GO. After the introduction of PFP, no FRET signal from PFP to P was observed (curve c), indicating that PFP cannot bind with P adsorbed on GO surface. This may be attributed to the high binding affinity between P and GO. When P hybridized with its target DNA, a strong FRET signal occurred upon the addition of PFP (curve d), following a red-shift of the emission from 518 to 525 nm. The enhanced fluorescence and the red-shift are due to the formation of dsDNA, which separates P from GO surface and makes it close C

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Figure 2. (A) Effect of NaCl concentration on the quenching efficiency of GO. The concentration of NaCl range from 0.002 to 0.102 M with a = 0.002 M, b = 0.004 M, c = 0.012 M, d = 0.102 M by exciting at 480 nm. (B) Effect of NaCl concentration on the energy transfer between target DNA/P complex and PFP. The concentration of NaCl range from 0.002 to 0.162 M with a = 0.002 M, b = 0.042 M, c = 0.082 M, d = 0.102 M, e = 0.162 M by exciting at 370 nm. The spectra are normalized with respect to the emission of PFP. (C) Effect of GO concentration on the fluorescence intensity of P without (blue) and with target DNA (red) by exciting at 480 nm. (D) Effect of PFP concentration on the FRET-induced fluorescence intensity at 525 nm without (blue) and with target DNA (red) by exciting at 370 nm.

strength, the concentrations of GO and PFP, and the reaction time between target DNA-bound P and GO were investigated. The ionic strength of the buffer solution is a crucial parameter in our work. From previous reports, the presence of metal ions can minimize the electrostatic repulsion during DNA hybridization process, and are needed to bring ssDNA close to the GO surface.25 However, Bazan et al. reported that the attraction between the PFP and DNA decrease by increasing the concentration of NaCl and result in a drop in efficient energy transfer.11 Thus, the concentration of NaCl was first investigated. As shown in Figure 2A, an increasing [NaCl] caused gradual fluorescence quenching, and the quenching closed to 100% with 0.102 M NaCl. While Figure 2B depicts a noticeable decrease in energy transfer from PFP to P with increasing [NaCl], which contributes to the electrostatic screening caused by NaCl. According to the above results and the previous report,11 0.1 M NaCl was used in the following experiment. In addition, the concentration of GO and the reaction time between target DNA-bound P and GO were optimized. Figure 2C reflects the effect of GO concentration on the fluorescence intensity of P in the absence (curve a) and presence of 20 nM target DNA (curve b) at different concentrations of GO. The fluorescence intensity of P was dramatically decreased on the incremental addition of GO, and the highest fluorescence turnon ratio (F/F0, where F0 and F are the fluorescence intensities

proximity to PFP to favor FRET. These observations confirm that this GO-based platform can be used for the assay of DNA under a low background. Our strategy was also identified by comparing the FRETinduced fluorescence intensity in the absence and presence of GO. First, for both the traditional PFP-based platform and this approach, the FRET-induced fluorescence intensity in the absence of target DNA as functions of PFP concentrations was studied (Figure S3). In the absence of GO, the FRET signal increased gradually with increasing [PFP] by forming the electrostatic complex, PFP/P. In contrast, when GO was introduced, less FRET signal was observed increasing [PFP], indicating that the presence of GO prevents the binding of P with PFP partly. Furthermore, the fluorescence turn-on ratio (signal-to-background ratio, S/B, where S and B are the fluorescence intensities at 525 nm with and without target DNA, respectively) in the absence (Figure 1B) and presence of GO (Figure 1C) was compared. It can be found that under the same experimental conditions, the fluorescence turn-on ratio is 7.60 in the presence of GO, which is significantly higher than that observed from the traditional PFP-based system (S/B = 1.20). These results clearly indicate that enhanced sensitivity and selectivity can be achieved by the introduction of GO. Optimization of Experiment Conditions for DNA Detection. In an attempt to obtain an optimal assay condition and achieve a high fluorescence turn-on ratio, the effect of ionic D

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of P in the absence and presence of the target DNA, respectively) was obtained at [GO] = 5 μg/mL (Figure S4). The kinetic behavior of target DNA-bound P complex in the presence of GO results reveal that at least 20 min is needed for the reaction between target DNA-bound P complex and GO (Figure S5). On the basis of these results, a concentration of GO at 5 μg/mL and a reaction time of 20 min were chosen for the subsequent experiments. The concentration of PFP is another important aspect involved in the detection system. Higher concentrations of PFP could result in higher fluorescence intensity at around 525 nm via FRET partly and, thus, increases the sensitivity. However, higher background signal was present at higher concentrations of PFP (red curve, Figure S3), which may be contributed to the binding between PFP and a lesser amount of free P that is not adsorbed on GO surface. Figure 2D shows the fluorescence enhancement at 525 nm in the presence of different concentrations of PFP. It is observed that by increasing the concentration of PFP, the FRET signal at 525 nm is dramatically enhanced in the presence of target DNA (curve b, Figure 4D) compared with that in the absence of target DNA (curve a). The best F/F0 (where F0 and F are the fluorescence intensities at 525 nm in the absence and presence of target DNA, respectively) was acquired when 0.26 μM of PFP in repeat units is added (Figure S6). However, upon adding 0.26 μM PFP, the fluorescence intensity at 525 nm is nearly equivalent to that without PFP (Figure 4D), which does not provide amplified signal. Accordingly, the ultimate concentration of PFP was chosen to be 1.57 μM in repeat units (Figure S6). Sensitivity and Selectivity of the Detection System. The quantitative detection of target DNA using this method under the optimized experimental conditions was evaluated by monitoring FRET ratio (I525/I425) at the different concentrations of target DNA. As shown in Figure 3A, with increasing the concentration of target DNA, the FRET ratio dramatically increased, implying the increase of the amount of dsDNA. Furthermore, the FRET ratio showed a clear linear dependence on the target DNA concentration (R = 0.9894) in the range of 0−20 nM (Figure 3B). Notably, the FRET signal can still be clearly identified from the background even for 40 pM target DNA (inset of Figure 3B). Thus, we estimate that the limit of DNA detection is as low as 40 pM, which is more than 1 order of magnitude lower than that of previously reported DNA detection methods using PFP12,14,26 and is comparable to the result obtained from a dual-amplification platform based on PFP and exonuclease III.27 The substantial improvement of our method in sensitivity is mainly attributed to the low background signal induced by GO. To further prove the high sensitivity of our design, the fluorescent response of GO-based platform18b,c in the absence of PFP was also investigated. As can be seen from Table S1, only slight fluorescence change was obtained especially with very low concentrations of target DNA. In contrast, the assay system showed a dramatically higher fluorescence change at the same concentrations of PFP, demonstrating that PFP does provide the advantage of collective response and fluorescence amplification in this system. These results reveal that GO-PFPbased system enables highly sensitive DNA quantification. To investigate the selectivity of our design for DNA assay, the fluorescence responses of the sensor to one-base mismatched DNA1NC, two-base mismatched DNA2NC, and noncomplementary DNANC were tested with the procedures of

Figure 3. (A) Normalized FRET-induced PL spectra of P-GO-PFP system in the presence of different concentrations of DNA (bottom to top: 0−20 nM), by exciting at 370 nm. (B) Linear relationship between the FRET ratio and DNA concentrations. Inset: the ratio values of I525/I425 at 0, 40, and 200 pM of DNA, respectively.

complementary DNAC, respectively. The results are shown in Figure 4. It was found that one-base mismatched DNA1NC

Figure 4. Normalized fluorescence intensity at around 525 nm in the presence of 20 nM DNAC, DNA1NC, DNA2NC, DNANC, and blank, respectively.

increased the fluorescence intensity by 65.1% compared with that of complementary DNAC, and there was almost no change in the fluorescence intensity ratios for two-base mismatched DNA2NC and noncomplementary DNANC compared with the blank solution. These results indicate that the proposed strategy E

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has good selectivity for target DNA detection and could be used to probe single-base mismatch. Interactions between P and GO and between PFP and GO. Loh et al. demonstrated that the electrostatic binding between PNP+ and dsDNA was stronger than that of PNP+ and GO,22 but the detailed interplay between PNP+GO− and singlestranded DNA was not studied. Moreover, unlike PFP, PNP+ is a small molecule that contains no repeated absorbing units. Considering this, the relative binding strength between P and GO and between GO and PFP was examined via measuring the fluorescence intensity of P-GO-PFP system at a different mixing order. It is worth noting that an inefficient FRET between P and PFP took place (Figure 5a) when adding PFP to Figure 6. Fluorescence decay of the P-GO-PFP system under different addition sequence: addition of PFP into P/GO complex (black) and addition of P into PFP/GO complex (red) by exciting at 481 nm.

GO complex upon adding of P, a fluorescence lifetime of 2.36 ns, close to the free FAM, is obtained, indicating an absence of energy transfer. Fluorescence anisotropy, an effective tool for investigating molecular interaction,28 provides further information on this finding. As shown in Figure S7, the fluorescence anisotropy of the system is 0.37 upon adding PFP into P/GO mixture, almost the same as that of PFP (0.36). This supports the inefficient energy transfer from PFP to P observed in Figure 5a. Nevertheless, upon the addition of P to the complex of PFP/ GO, a visible increase of fluorescence anisotropy (0.42) is obtained, indicating the presence of the energy-transfer processes resulted from the formation of P/PFP complex (curve b, Figure 5).

Figure 5. Normalized FRET-induced fluorescence spectra of P-GOPFP system under different addition order: (a) addition of PFP into P/GO complex and (b) addition of P into PFP/GO complex by exciting at 370 nm.

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P/GO solution, which indicates that P was still adsorbed on GO surface after introducing PFP. Above results might be attributed to the stronger binding between P and GO compared with that between PFP and GO. If GO displays higher affinity to P compared with PFP, upon the addition of P to the PFP/GO complex, P will replace PFP and be adsorbed on GO surface, resulting in no observed FRET signal. While Figure 5b showed efficient FRET from PFP to P after the addition of P to the PFP/GO complex. Therefore, we deduce that the binding between P and GO equals to that between PFP and GO. Specifically, the addition of PFP cannot release the single-stranded DNA from GO surface and that the introduction of single-stranded DNA has little effect on the adsorption of PFP on GO surface. This consequence paves the way for the amplified fluorescence detection of DNA in this study. Time-resolved fluorescence, having been used to explore the energy-transfer process, was performed to study the interaction between P, PFP, and GO. Considering that the fluorescence lifetime of PFP is short and any shortening of its lifetime due to FRET could not be measured accurately, we focused on the fluorescence decay of FAM upon excitation of P-GO-PFP system at 481 nm, where direct excitation of PFP does not result in FRET emission. As shown in Figure 6, a monoexponential decay with a lifetime of 0.36 ns is obtained for the P/GO complex after the addition of PFP, which is much shorter than the fluorescence lifetime of FAM (≈2 ns).10 This shortening is due to the bound complex between P and GO, resulting in the energy or electron transfer. In the case of PFP/

CONCLUSION In summary, we have proposed a simple strategy for reducing the background signal of traditional PFP-based DNA detection platform by introducing GO. The design mechanism is based on key finding that the probe DNA cannot be released from GO surface in the presence of PFP. This was confirmed by time-resolved fluorescence and fluorescence anisotropy. Under the optimized conditions, as low as 40 pM of target DNA could be detected, which is more than 1 order of magnitude lower than that of previously reported DNA detection methods using PFP. The enhanced sensitivity may be ascribed not only to the light harvesting properties of CCP but also to the minimized background signal induced by the addition of GO. In addition, compared with the previously reported strategies such as the introduction of EB and magnetic microparticles, this approach is nontoxic, simple, and highly sensitive, which would also and promote the further application of CCP in biomedical analysis.

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ASSOCIATED CONTENT

S Supporting Information *

Synthetic routes of PFP and other supplementary data as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 86-27-68756759. Fax: 86-27-68754685. E-mail: [email protected]. F

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Notes

(19) Chang, H. X.; Tang, L. H.; Wang, Y.; Jiang, J. H.; Li, J. H. Anal. Chem. 2010, 82, 2341−2346. (20) Jang, H.; Kim, Y. K.; Kwon, H. M.; Yeo, W. S.; Kim, D. E.; Min, D. H. Angew. Chem., Int. Ed. 2010, 49, 5703−5707. (21) Lu, C. H.; Li, J. A.; Lin, M. H.; Wang, Y. W.; Yang, H. H.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2010, 49, 8454−8457. (22) Balapanuru, J. H.; Yang, J. H.; Xiao, S.; Bao, Q. L.; Jahan, M.; Polavarapu, L.; Wei, J.; Xu, Q. H.; Loh, K. P. Angew. Chem., Int. Ed. 2010, 49, 6549−6553. (23) (a) Hummers, W. S.; Offeman, R. E. J. Am. Chem. Soc. 1958, 80, 1339−1339. (b) Xu, Y. X.; Bai, H.; Lu, G. W.; Li, C.; Shi, G. Q. J. Am. Chem. Soc. 2008, 130, 5856−5857. (24) Stork, M.; Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Adv. Mater. 2002, 14, 361−366. (25) Wu, M.; Kempaiah, R.; Huang, P. J. J.; Maheshwari, V.; Liu, J. W. Langmuir 2011, 27, 2731−2738. (26) He, F.; Feng, F.; Duan, X.; Wang, S.; Li, Y.; Zhu, D. Anal. Chem. 2008, 80, 2239−2243. (27) Feng, X. L.; Liu, L. B.; Yang, Q.; Wang., S. Chem. Commun. 2011, 47, 5783−5785. (28) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: New York, 1999.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the support of the National Basic Research Program of China (973 Program, Nos. 2011CB933600 and 2006CB933100), the Science Fund for Creative Research Groups of NSFC (Nos. 20621502 and 20921062), the National Natural Science Foundation of China (21275110; 20833006; 20875071; 21005056), and the Fundamental Research Funds for the Central Universities (20100141110015). We also thank Prof. Zhike He’s group for their technical assistance in fluorescence lifetime measurements.

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dx.doi.org/10.1021/bm301469q | Biomacromolecules XXXX, XXX, XXX−XXX