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A Graphene Nanoprobe for Rapid, Sensitive, and Multicolor Fluorescent DNA Analysis
By Shijiang He, Bo Song, Di Li, Changfeng Zhu, Wenpeng Qi, Yanqin Wen, Lihua Wang, Shiping Song, Haiping Fang,* and Chunhai Fan*

workers to monitor specic protein binding events.[18] In this work, we report a new Coupling nanomaterials with biomolecular recognition events represents a mix-and-detect strategy for simultaneous new direction in nanotechnology toward the development of novel molecular uorescent detection of multiple DNA diagnostic tools. Here a graphene oxide (GO)-based multicolor uorescent targets by exploring interactions between DNA nanoprobe that allows rapid, sensitive, and selective detection of DNA graphene and DNA oligonucleotides. targets in homogeneous solution by exploiting interactions between GO and Homogeneous detection of nucleic acids with uorescent DNA probes posDNA molecules is reported. Because of the extraordinarily high quenching sesses several inherent advantages, such as efciency of GO, the uorescent ssDNA probe exhibits minimal background operation convenience, rapid hybridizauorescence, while strong emission is observed when it forms a double helix tion kinetics, and potential compatibility with the specic targets, leading to a high signal-to-background ratio. with real-time polymerase chain reaction Importantly, the large planar surface of GO allows simultaneous quenching of (PCR) and in situ cellular imaging.[4,7,19] Such probes usually consist of uorophoremultiple DNA probes labeled with different dyes, leading to a multicolor quencher pairs and rely on Forster resosensor for the detection of multiple DNA targets in the same solution. It is nance energy transfer (FRET), for which also demonstrated that this GO-based sensing platform is suitable for the distance-dependent uorescence quenchdetection of a range of analytes when complemented with the use of ing is elaborately designed to be closely functional DNA structures. associated with DNA hybridization events (e.g., via stem-loop structures in uorescent molecular beacons). More recently, in order to increase the signal-to-noise ratio of such DNA probes, 1. Introduction traditional organic uorophores and quenchers were replaced with highly bright quantum dots and organics with highly Biomolecular detection has found widespread applications efcient nanoquenchers such as AuNPs and single-walled carbon in molecular diagnostics, industrial and environmental monitor[1,2] nanotubes (SWNTs), respectively.[2023] Graphene is an one-atoming, and civil defense, which has motivated intense interest thick 2D nanomaterial with extraordinary electronic, thermal, and in developing rapid, simple, and cost-effective biosensors for mechanical properties,[24,25] which, along with its water-soluble proteins, nucleic acids, and small molecules.[37] Nanomaterials derivative, graphene oxide (GO), has become extremely popular in have proven of particular utility in this regard due to their unique nanoelectronics and nanocomposites.[2628] However, biological optical, electronic, and catalytic properties. Signicantly, much applications of graphene and GO remain to be explored.[29,30] recent attention has been drawn toward the design of Of our particular interest, graphene was recently predicted nanomaterial-based biosensors based on interactions between through theoretical calculations to be a superquencher with the biomolecules and nanomaterials of different compositions, long-range nanoscale energy transfer property,[31,32] which, in dimensions, and shapes.[814] For example, Mirkin and others combination with the unique DNA/GO interactions, forms the developed a series of sensitive DNA assays by coupling DNA basis of a convenient and versatile strategy for multicolor hybridization events with unique plasmonic properties of gold [6,1517] uorescent DNA analysis. Very recently, and during the nanoparticles (AuNPs). Excitonplasmon interactions of preparation process of the current work, Lu et al. reported that different nanostructures were also exploited by Kotov and coGO could bind and quench a dye-labeled single-stranded DNA (ssDNA) probe; the uorescence was recovered when the probe [*] Prof. C. Fan, Prof. H. Fang, S. He, Dr. B. Song, Dr. D. Li, C. Zhu, W. Qi, formed a duplex with its target, which released the probe from Y. Wen, Dr. L. Wang, Prof. S. Song GO.[33] We herein report a new graphene-based strategy that Laboratory of Physical Biology allows multicolor DNA analysis in the same solution and a Shanghai Institute of Applied Physics Chinese Academy of Sciences mix-and-detect assay format with signicantly improved sensiShanghai 201800 (China) tivity and speed (Scheme 1), and explore the mechanism E-mail: fchh@sinap.ac.cn; fanghaiping@sinap.ac.cn underlining GODNA interactions via both theoretical and experimental studies. DOI: 10.1002/adfm.200901639 453

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$1 nm, characteristic of a fully exfoliated GO sheet (see Supporting Information (SI): Fig. S1a),[34] and the lateral size of GO was fairly polydisperse. Also, GO showed the clearly visible honeycomb structure of graphene in high-resolution transmission electron microscopy (HRTEM; see SI: Fig. S1b). The uorescence quenching ability of GO was evaluated via uorescent measurements of dyes in the presence of GO. Consistent with Scheme 1. Scheme for the GO-based multicolor DNA analysis. Fluorescence spectra of mixture [31,32] we probes (P5, P6, P7) in the presence of different targets T5 (blue), T6 (red) and T7 (orange) with the previous theoretical prediction, found that uorophores, such as FAM (carboxy the excitation wavelengths of 494, 643, and 587 nm. uorescein) and ROX (6-carboxy-X-rhodamine), were almost nonuorescent in the presence of GO (Fig. 1), which possibly arose from strong hydrophobic adsorption of these dyes at the GO surface and highly efcient long-range energy transfer from the dye to GO.[31,32] Interestingly, while both GO and DNA are negatively charged, the uorescence of the FAM-tagged ssDNA probe (P1, see Table 1 for the sequence) was rapidly quenched by GO as well, with the uorescence quenching dependent on the ratio between P1 and GO (Fig. 2). The quenching kinetics were fairly fast, with nearly 100% uorescence quenching Figure 1. a) Fluorescence quenching of FAM of 50 nM in the absence (black) and presence of GO within only several minutes after the addition with a series of concentrations (top to bottom: 5, 10, 15, 20, 25, 30, 35 mg/mL). b) Fluorescence of GO in a DNA solution of 10 nM (Fig. 2e). Also quenching of 6-ROX of 50 nM in the absence (black) and presence of GO with a series of of note, the quenching kinetics was dependent concentrations (top to bottom: 2.5, 5, 7.5, 10, 15, 20, 25 mg/mL). on the length of the oligonucleotide; that is, longer sequence led to slower kinetics (Fig. 2e). In direct contrast, when P1 was hybridized with its comple2. Results and Discussion mentary target T1 to form a duplex, the FAM uorescence largely The chemically synthesized GO was readily water-dispersible due remained in the presence of GO (Fig. 2c), which suggested that the to the presence of suspended hydroxyl and carboxylic groups at the interactions between double-stranded DNA (dsDNA) and GO were surface. We rst characterized as-prepared GO samples to conrm rather weak. In the presence of T1 at vefold excess (50 nM), the their exfoliation. Importantly, tapping-mode atomic force microsuorescence intensity was increased by approximately 50 times as copy (AFM) studies revealed that the thickness of GO was of compared to that of the ssDNA probe (post-mixing strategy). As a

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Table 1. DNA sequences employed in this work.

Oligonucleotide P1: FAM-tagged probe T1: target for P1 R1: random sequence M1: single-base mismatched target for P1 M2: single-base mismatched target for P1 M3: single-base mismatched target for P1 T2: long target for P1 (complementary part in the middle) T3: long target for P1 (complementary part at the 50 -end) T4: long target for P1 (complementary part at the 30 -end) P5: FAM-tagged P16 probe T5: target for P5 P6: Cy5-tagged P21 probe T6: target for P6 P7: ROX-tagged P53 proebe T7: target for P7 P8: MSO probe for Hg2 P9: Adenosine probe (Underlined part is the anti-adenosine aptamer sequence) Sequence 50 -FAM- TCGTTGGAGTTTGTCTG-30 50 -CAGACAAACTCCAACGA-30 50 -GCAGAGCCAGTTCCAAG-30 50 -CAGACA AATTCCAACGA-30 50 -CAGACA AAATCCAACGA-30 50 -CAGACA AAGTCCAACGA-30 50 -ATTTCACTGACAGTTCAGACAAACTCCAAC GACTAGCTACGTGCCGA-30 50 -CAGACAAACTCCAACGACTAGCTATG TGCCGAATTTCAAGGACAGTT-30 50 -CTAGCTATGTGCCGAATTTCAAGGACAGTTCAG ACAAACTCCAACGA-30 50 -FAM-CAGAGGCAGTAACCA-30 50 -TGGTTACTGCCTCTG-30 50 -Cy5-CCCTAATCCGCCCAC-30 50 -GTGGGCGGATTAGGG-30 50 -ROX-CCTGGTGCCGTAGAT-30 50 -ATCTACGGCACCAGG-30 50 -FAM-TTCTTTCTTCCCCTTGTTTGT T-30 50 -FAM-TAATTACCTGGGGGAGTATTGCGGAG GAAGGTTAT -30

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in kinetics due to the presence of competition between P1/GO adsorption and the P1/T1 hybridization; thus, we favor the use of a new postmixing strategy in the following DNA detection studies. We carried out a molecular dynamics (MD) simulation to interrogate the observed large discrepancy for interactions between ss- and dsDNA with GO. As shown in the snapshot, all of the nucleobases lie nearly at at the GO surface, and ssDNA were stably adsorbed by the GO (see Fig. 3a). We attribute this strong adsorption to the p-stacking interaction[35,36] between the ring structures in the nucleobases and the hexagonal cells of the graphene. To characterize the degree of the adsorption, we computed the distance between the atoms of the nucleobases and the surface of graphene. As shown in Fig. 3c, there was a very sharp peak at $3.5 A. This distance is fairly close to the van de Waals distance between a carbon atom in GO and a carbon/oxygen/nitrogen atom (the main elements of nucleobases). This suggests that most of the atoms in the nucleobases are directly adsorbed at GO. In contrast, dsDNA could not be stably adsorbed on GO in our MD simulation and retained its helical structure (shown in Fig. 3b). This is because nucleobases were effectively shielded within the densely negatively charged phosphate backbone of dsDNA. While hydroxy groups on GO might interact with phosphate groups of dsDNA, we only observed minimal hydrogen bonding between them, which could not survive at room temperature and in aqueous solution (see Fig. 3b). Figure 2. a) Scheme for the uorescent DNA detection based on the ssDNA/dsDNA discrimiWe reason that this GO-based uorescence nation ability of GO. b) Scheme for the target hybridization-induced probe liberation from GO. quenching might serve as a sensing platform c) GO-based uorescence DNA assay. Fluorescence spectra of P1 in the absence (black) and presence of the complementary target T1 of 50 nM (red), and random sequence R1 of 50 nM for quantitative DNA analysis. We rst hybri(blue). d) Fluorescence spectra for P1 in the presence of GO before (black) and after (red) dized P1 with T1 of various concentrations for 10 min, to which an aliquot of GO was added incubation with T1. e) Kinetic study for the uorescence change of the FAM-tagged probes (20 nM) with different lengths in the presence of GO: 17 base pairs (bp; black); 34 bp (red); 51 bp (blue). (postmixing). We found that the uorescence of f) Kinetic study for the uorescence change of the GO-bound P1 in the presence of T1 of various unhybridized P1 was efciently quenched by amounts (10, 20, 30, 40, 50 pmol). The excitation and the emission wavelengths are 494 and GO while the hybridized P1/T1 duplex led to 526 nm, respectively. observable uorescence that formed the basis of a signal-on DNA sensor. As shown in Fig. 4a, the uorescence was intensied along with the increase of comparison, we also performed a premixing protocol similar to the target concentration. This DNA sensor had a linear range of the previous report.[33] We rst mixed P1 with GO and then 0$25 nM, with a detection limit of 100 pM DNA as estimated from challenged the P1/GO complex with T1. Importantly, the prequenched uorescence in P1/GO was slowly recovered (up the derived calibration curve (>3 standard deviations (SD)), to several hours) in the presence of excess T1 and in a which excels previously reported molecular beacons[4,20] and concentration-dependent manner (Fig. 2b,d,f), suggesting that nanomaterials-based DNA detection[16,23,33] (typically in the P1 was liberated from the GO surface during its hybridization with nanomolar range) by at least an order of magnitude (see T1. These results imply that GO possesses signicantly different performance comparison in Table 2). Also of note, even when adsorption afnity for ssDNA and dsDNA; that is, ssDNA binds to the whole assay time was reduced to only several minutes (in GO with signicantly higher afnity than dsDNA. Also, ssDNA contrast to hours using either SWNTs or graphene in the previous binds to GO in a noncovalent manner, which can be readily work[23,33]), we could obtain signals that were readily distinguishseparated under external competition (e.g., sequence-specic able from the background, which makes it particularly suitable for hybridization). Of note, the latter approach (premixing) is slow mix-and-detection of DNA.

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uorescence signals than T1, which was of the same length as P1. However, we observed that the uorescence signal for T4 was signicantly larger than those of T2 and T3, and distinctly distinguishable from the background (Fig. 4b). This position effect was attributed to the location of FAM and its interaction with GO. When FAM was tagged at the 50 end of P1, it was located right at the duplex region after hybridization with T4; in contrast, FAM was located at the interface of the ss and ds regions for T2/T3. As a result, FAM was more likely to be detached from the GO surface in the case of P1/T4, while it tended to be partially adsorbed due to the presence of ss regions in P1/T2 and P1/T3 at the surface of GO. Consequently, it is possible to employ the GO-based assay in practical DNA detection with long sequences, provided that the probe sequence is well designed. The GO-based DNA detection exhibited high Figure 3. Representative snapshots from MD simulation showing that a) FAM-tagged (red) sequence specicity. We only observed a ssDNA is tightly adsorbed at the surface of GO; b) dsDNA does not adsorb at the surface of GO. minimal uorescence increase in the presence The FAM, DNA, and GO are shown in purple, green and cyan, respectively. c) Probability of the of excess noncognate DNA (Fig. 2c). More atoms in nucleobases at a distance from the GO surface. importantly, this DNA sensor was of sufcient selectivity to easily differentiate single mismatches, which offered the opportunity to allow single-nucleotide We then interrogated the hybridization between a probe and polymorphism (SNPs) analysis. As shown in Fig. 4b, the targets of different lengths (17 bp versus 47 bp). In order to evaluate uorescence signal for that target T1 was approximately three the perturbation of the unpaired region of the target that might times higher than those for the 1-base mismatched targets (C is interact with GO, the complementary regions for P1 in the 47-bp mutated to T, A or G in the middle). Given that the fully comtarget were designed to be in the middle (T2), at the 50 - (T3), or the plementary duplex (P1/T1) and the mismatched duplexes (P1/M1, 30 - (T4) end. We observed that all these long targets led to smaller

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Figure 4. a) Representative uorescence spectra for P1 (10 nM) in the presence of various concentrations of T1 (0, 0.5, 4, 6, 15, 30, and 50 nM, respectively). Inset: calibration curve for the uorescence intensity versus P1 concentration. The data are recoded with the excitation and emission wavelength of 494 and 526 nm, respectively, and from at least three independent experiments. b) Fluorescence spectra for P1 (10 nM) in the presence of 10 nM of the fully complementary target T1 (black) and single-base mismatched target M1 (red), M2 (blue), and M3 (green). c) Fluorescence spectra for P1 in the absence (black) and presence of the fully complementary target T1 (red), or long targets T2 (cyan), T3 (blue), and T4 (green). df) Fluorescence spectra for multicolor detection. Probe mixture in the presence of different targets T5 (blue), T6 (red), and T7 (orange), with the excitation/emission wavelengths of d) 494/526 e) 643/666, and f) 587/609 nm/nm.

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www.MaterialsViews.com Table 2. Performance comparison between homogeneous uorescent DNA sensors.

Type Molecular beacons AuNPDNAdye conjugates (stem loop probe) AuNPDNAdye conjugates (linear probe) Unmodied AuNP-based DNA detection SWNT-based DNA detection GO-based DNA detection (premixing) GO-based DNA detection (postmixing) Sensitivity nM nM NR [a] $10 nM 4 nM $10 nM 100 pM Assay time Rapid (minutes) Rapid (minutes) Slow ($1 h) Rapid (minutes) Slow (several hours) Slow (0.5 h) Rapid (minutes) Multicolor analysis Yes Yes NR NR NR NR Yes

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Probe synthesis Difcult (dual labeling) Difcult (dual labeling) Difcult (dual labeling) Convenient (single labeling) Convenient (single labeling) Convenient (single labeling) Convenient (single labeling)

Ref. [4,37] [20] [22] [16] [23] [33] TW [b]

[a] NC stands for Not reported. [b] TW stands for this work.

P1/M2, and P1/M3) have relatively small thermodynamic difference, and that sequences were not elaborately designed (e.g., stem-loop structures with inherent stringency), this high sequence specicity for single mismatches was fairly impressive. We attribute this high stringency to the competition between GO/ probe adsorption and probe/target hybridization; i.e., GO possibly competitively destabilizes mismatched duplexes. Simultaneous detection of multiple targets in a homogeneous solution brings about new opportunities for molecular diagnostics.[37,38] For example, it is important to detect multiple tumorsuppressor genes in order to identify early-phase cancers in asymptomatic individuals.[39] The large planar surface of GO possesses the ability to interact with multiple DNA strands, which motivated us to explore methods of simultaneous detection of

multiple DNA targets (Scheme 1). We employed three probes (P5, P6, and P7) for three types of tumor-suppressor genes (exon segments of p16, p21 and p53 genes) were labeled with FAM, Cy5 (cyanine 5), and ROX at the 50 end, respectively. The selection of these three dyes avoided signicant dye-to-dye energy transfer, which were individually excited at 494, 643, and 587 nm, emitting blue (520 nm), red (670 nm) and orange (608 nm) colors, respectively. Upon the addition of specic targets to the probe mixture containing P5, P6 and P7, we observed the emission from the corresponding wavelength. As shown in Figure 4df, the p16 target (T5) led to the specic emission of the blue color (FAM), while minimal emission of the other two colors. Similarly, the addition of the p21 target (T6) and the p53 gene produced the red (Cy5) and orange (ROX) colors, respectively. Therefore, this

Figure 5. a) Fluorescence spectra of the MSO (P8) in the presence of various concentrations of Hg2 (0, 40, 60, 80, 100, 180 nM). Inset: calibration curve for Hg2 detection. b) Selectivity of the GO-based Hg2 sensor over a spectrum of interference metal ions (120 nM Hg2, all other ions are of 1 mM). c) Fluorescence spectra of the anti-adenine aptamer (P9) in the presence of various concentrations of A (0, 10, 50, 200, 800, 1200, 2000 mM). Inset: calibration curve for A detection. d) Selectivity of the GO-based A sensor over G, C, and T (all of 100 mM). The excitation wavelength was 495 nm.

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GO-based multicolor DNA analysis provides a promising approach for challenging applications such as allele discrimination. The ability of GO to differentiate ss- and dsDNA not only offers a new approach to detect DNA, but may nd applications in detecting a wide spectrum of analytes when complemented with the use of functional nucleic acid structures (e.g., aptamers). As a proof of concept, we herein demonstrated the detection of mercuric ion (Hg2) and adenine (A) with a T-rich mercuryspecic oligonucleotide (MSO) and an anti-adenine aptamer.[40] Without Hg2, the FAM-labeled MSO (P8) was in the singlestranded state and not uorescent in the presence of GO. However, the introduction of Hg2 led to the formation of the stem-loop structure that could not be quenched by GO. Therefore, the uorescence of MSO was dependent on the concentration of Hg2. This sensor had a linear range of 30180 nM and a detection limit of 30 nM, which compared favorably with previously reported Hg2 uorescent sensors.[41] Also importantly, this Hg2 sensor was selective enough to distinguish a range of interference metal ions (Fig. 5a). The anti-adenine aptamer is an in vitro selected short oligonucleotides targeting the adenine molecule with antibodylike specicity and afnity.[42] When a FAM-tagged anti-adeonsine aptamer (P9) was incubated with GO, the uorescence was largely quenched. The observed small background uorescence might arise from the secondary structure of this sequence. In the presence of adenosine, the probe P9 was converted to a rigid tertiary structure. Similarly, GO could not quench the uorescence of the rigid aptamer structure. Based on the difference in uorescent intensity, we could detect adenosine with a detection limit as low as 10 mM (Fig. 5b). Of note, analogous molecules, such as cytosine (C), guanine (G), and thymine (T), only led to weak responses, suggesting the high selectivity of this adenine sensor.

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optimize the sensor performance with computer-aided rational design. The present study represents our rst attempt to connect the highly promising nanomaterial GO with DNA sensing, and we expect that GO and other graphene materials will nd important and widespread applications in biology.

4. Experimental
Materials: DNA oligonucleotides were synthesized and puried by TAKARA Biotechnology (Dalian, China). The sequences of these oligonucleotides are shown in Table 1. Adenine (A), cytidine (C), guanine (G) and thymine (T) were purchased from Sigma. Graphite powder was purchased from China National Pharmaceutical Group Corporation (Shanghai, China). Other chemicals were of analytical grade. Water was puried using a Millipore ltration system. Preparation of GO: GO was synthesized from graphite powder based on the Hummers method [43]. Briey, graphite power (4 g) was oxidized in a hot solution (80 8C) of concentrated H2SO4 (24 mL) containing K2S2O8 (8 g), and P2O5 (8 g). The resulting dark blue mixture was thermally isolated and slowly cooled to room temperature over a period of 6 h. The mixture was diluted to 300 mL, and then ltered with a membrane of 0.22 mm (Generay Biotech Co., Ltd., Shanghai, China) and dried overnight at 60 8C. These pre-oxidized graphite powder (2 g) were added to 92 mL of cold H2SO4 (0 8C), to which KMnO4 (12 g) was gradually added under continuous stirring in an ice-bath. After 15 min, NaNO3 (2 g) was added to the mixture. The solution was further stirred for 2 h at 35 8C, and distilled water (200 mL) was added. The reaction was stopped with the addition of a mixture of 560 mL of distilled water and 10 mL of H2O2 (30%). The product was washed with HCl (1:10), and then with water, and then suspended in distilled water. The brown dispersion was extensively dialyzed to remove residual metal ions and acids, and then exfoliated via sonication for 1.5 h (300 W). Unexfoliated graphite oxide was removed by centrifugation (3000 rpm, 5 min) using Centrifuge himac-CF 16RX (Hitachi, Japan). The as-prepared GO samples (0.47 mg/mL) was then characterized with tapping-mode AFM [26,29] and TEM [44]. Instrumentation: The HRTEM was performed with a Philips CM-120 transmission electron microscope operating at 120 kV (Philips, Netherlands). Samples for TEM were prepared by drop casting 20 mL of the as-prepared GO onto a standard carbon-coated (200300 A) formvar lm on a copper grid. AFM measurements were carried out on Nanoscope IIIa (Digital Instrument, USA) under tapping mode, with a Pt/Ti coated tip (force constant of 4 N/m, MicroMasch). A droplet of GO dispersion was cast onto a freshly cleaved mica surface, and the sample was maintained at room temperature for several minutes to allow water evaporation. The image was obtained at 20 8C at a humidity of 30%. All uorescence spectra were collected with a Hitachi F-4500 spectrophotometer equipped with a Xenon lamp excitation source. Fluorescent DNA Assays: In a typical DNA assay, the uorescent probe P1 (10 pmol) was hybridized with the target in a 0.1 M phosphate buffer of 980 mL (7.7 mM Na2HPO4, 2.3 mM NaH2PO4, 100 mM NaCl, pH 7.4) for 10 min, to which GO of 20 mL (235 mg/mL) was added. After 1-min incubation, uorescence measurements were performed with a quartz cuvette to monitor the hybridization process. In multicolor DNA assays, 5 pmol of FAM-tagged P16 probe P5, 10 pmol of ROX-tagged P21 probe P6, and 20 pmol of (Cy 5)-tagged probe P7 were mixed in a solution. We employed different amounts of probes in order to optimize the detection performance. All other conditions were similar to those in the single target detection. In mercury assays, 10 pmol of FAM-tagged mercury specic oligonucleotide (P8) was incubated with Hg2 of a series of concentrations in a 3-N-morpholinopropanesulfonic acid (MOPS) buffer (10 mM with 50 mM NaNO3, pH 7.2) for 10 min. A GO solution (235 mg/mL) of 20 mL was added to this mixture. Fluorescence measurements were performed after 1 min. In adenosine triphosphate (ATP) assays, 10 pmol of FAMtagged anti-ATP aptamer probes (P8) was incubated with ATP in 0.1 M phosphate buffer of 980 mL for 30 min. Fluorescence measurements were

3. Conclusions
In summary, we have demonstrated that GO possesses high uorescence quenching ability as well as different afnities toward ss- and dsDNA using both theoretical calculations and experimental studies. Based on these ndings, we designed a new homogeneous sensor for multiplex, sequence-specic DNA detection. This GO-based DNA sensor has several important advantages. First, GO can be readily synthesized in large quantities, and in contrast to dually labeled molecular beacons, the probe is only labeled with a single dye, which reduces the cost for DNA assays. Also, this is a homogeneous, mix-and-detect assay method that can be nished within minutes. Therefore it provides opportunities to develop low-cost and rapid molecular diagnostic tools. Second, the availability of large planar surfaces of GO makes it possible to detect multiple molecular targets in the same solution. Particularly, the compatibility of GO with different DNA structures provides great versatility to develop sensors for a spectrum of analytes with relatively convenient design. Third, the GO-based DNA detection improves the sensitivity by at least an order of magnitude as compared to conventional molecular beacons,[4,20] which reects the superquenching ability of GO that minimizes the background uorescence. Given the convenience to perform calculations and modeling on GO, it is also possible to predict and engineer GO/biomolecular interactions and further

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www.MaterialsViews.com performed after the addition of 20 mL of GO solution (235 mg/mL) for 1 min. Computational Methods: A piece of GO (7.3 nm 8.4 nm) with 2400 carbon atoms and an ssDNA segment (with the sequence of P1) labeled with a FAM molecule were immersed in a periodic water box with dimensions of 10 nm 10 nm 6 nm. In the box, there were approximately 10 700 water molecules and 16 or 32 sodium ions that neutralized the ss- and dsDNA, respectively. In GO, the carbon atoms were modeled as uncharged LennardJones particles with a distance parameter of s 0.3400 nm, and a potential well depth of e 0.3598 kJ mol1. The carbon bond length of 0.14 nm and the bond angle of 1208 were maintained by harmonic potentials with spring constants of 393 and 960 kJ mol1 and 527 kJ mol1 rad2 before relaxation [45,46]. In addition, a weak dihedral angle potential was applied to the bonded carbon atoms of GO [45,46]. The conformation and the parameters of the FAM molecule were obtained from PRODRG [47], while the parameters for other components (e.g., ssDNA and hydroxy groups in GO) were directly obtained from the AMBER03 force eld [48]. The TIP3P water model [49] was employed in our simulation. All simulations were carried out at a constant pressure 1 bar with a constant temperature (300 K) via Gromacs 3.3 [50] and with the PME method [51] for full electrostatics with a cut-off of 1 nm. A time step of 1 fs was employed and data were collected every 1 ps. First, we performed MD simulation for 1 ns for the system of the DNA segment on the GO to obtain the initial conformation. Then a 20-ns MD simulation were done for the whole system including DNA, GO, counter ions and water. The nal 10-ns simulations were employed for analysis.
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Acknowledgements
Shijiang He, Bo Song, and Di Li contributed equally to this work. This work was supported by National Natural Science Foundation (20725516, 20873175, 20805055, 90913014, 10825520), Ministry of Science and Technology (2006CB933000, 2007CB936000, 2007AA06A406, 2010CB934504), Ministry of Health (2009ZX10004-301), Shanghai Municipal Commission for Science and Technology (0952nm04600) and Chinese Academy of Sciences. Supporting Information is available online from Wiley InterScience or from the author. Received: September 1, 2009 Revised: September 30, 2009 Published online: January 4, 2010
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