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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 speciﬁc protein binding events. 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 ﬂuorescent detection of multiple DNA diagnostic tools. Here a graphene oxide (GO)-based multicolor ﬂuorescent 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 ﬂuorescent DNA probes posDNA molecules is reported. Because of the extraordinarily high quenching sesses several inherent advantages, such as efﬁciency of GO, the ﬂuorescent ssDNA probe exhibits minimal background operation convenience, rapid hybridizaﬂuorescence, while strong emission is observed when it forms a double helix tion kinetics, and potential compatibility with the speciﬁc 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 ﬂuorophoremultiple 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 ﬂuorescence 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 ﬂuorescent molecular beacons). More recently, in order to increase the signal-to-noise ratio of such DNA probes, 1. Introduction traditional organic ﬂuorophores and quenchers were replaced with highly bright quantum dots and organics with highly Biomolecular detection has found widespread applications efﬁcient nanoquenchers such as AuNPs and single-walled carbon in molecular diagnostics, industrial and environmental monitor[1,2] nanotubes (SWNTs), respectively.[20–23] 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.[3–7] 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.[26–28] However, biological optical, electronic, and catalytic properties. Signiﬁcantly, 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.[8–14] 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 [6,15–17] ﬂuorescent DNA analysis. Very recently, and during the nanoparticles (AuNPs). Exciton–plasmon 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 ﬂuorescence 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. 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 signiﬁcantly improved sensiShanghai 201800 (China) tivity and speed (Scheme 1), and explore the mechanism E-mail: firstname.lastname@example.org; email@example.com underlining GO–DNA interactions via both theoretical and experimental studies. DOI: 10.1002/adfm.200901639 453
Adv. Funct. Mater. 2010, 20, 453–459
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
tapping-mode atomic force microsﬂuorescence 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). 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.[31. 7. longer sequence led to slower kinetics (Fig. In the presence of T1 at ﬁvefold excess (50 nM). 2e). Results and Discussion mentary target T1 to form a duplex. S1b). GO showed the clearly visible honeycomb structure of graphene in high-resolution transmission electron microscopy (HRTEM. while both GO and DNA are negatively charged. Fluorescence spectra of mixture [31. P6. that is. 2). Funct. 2e). 2010. were almost nonﬂuorescent in the presence of GO (Fig. 20.32] we probes (P5. Consistent with Scheme 1. the their exfoliation.afm-journal. Also quenching of 6-ROX of 50 nM in the absence (black) and presence of GO with a series of of note.www. 30. 453–459 . The ﬂuorescence quenching ability of GO was evaluated via ﬂuorescent measurements of dyes in the presence of GO. 25 mg/mL). 15. As a FULL PAPER Table 1. see SI: Fig. T6 (red) and T7 (orange) with the previous theoretical prediction. 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 Hg2þ P9: Adenosine probe (Underlined part is the anti-adenosine aptamer sequence) Sequence 50 -FAM. P7) in the presence of different targets T5 (blue). which possibly arose from strong hydrophobic adsorption of these dyes at the GO surface and highly efﬁcient long-range energy transfer from the dye to GO. Importantly. with nearly 100% ﬂuorescence quenching Figure 1.de www. the FAM ﬂuorescence largely The chemically synthesized GO was readily water-dispersible due remained in the presence of GO (Fig.com $1 nm. Weinheim Adv. 35 mg/mL).32] Interestingly. and the lateral size of GO was fairly polydisperse. DNA sequences employed in this work. 1). Scheme for the GO-based multicolor DNA analysis. found that ﬂuorophores. when P1 was hybridized with its comple2. 20. the ﬂuorescence of the FAM-tagged ssDNA probe (P1. 643. 2c). 10.5. 15. We ﬁrst characterized as-prepared GO samples to conﬁrm rather weak. S1a). b) Fluorescence of GO in a DNA solution of 10 nM (Fig. Also. 20.5. The quenching kinetics were fairly fast. 10. and 587 nm. 25. Mater.MaterialsViews. on the length of the oligonucleotide. In direct contrast.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 -30 454 ß 2010 WILEY-VCH Verlag GmbH & Co. with the ﬂuorescence quenching dependent on the ratio between P1 and GO (Fig. see Table 1 for the sequence) was rapidly quenched by GO as well. ﬂuorescein) and ROX (6-carboxy-X-rhodamine). KGaA. such as FAM (carboxy the excitation wavelengths of 494. 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. characteristic of a fully exfoliated GO sheet (see Supporting Information (SI): Fig. 5. the quenching kinetics was dependent concentrations (top to bottom: 2.
the latter approach (‘‘premixing’’) is slow mix-and-detection of DNA. 453–459 ß 2010 WILEY-VCH Verlag GmbH & Co. respectively. that is. even when adsorption afﬁnity for ssDNA and dsDNA.MaterialsViews. This DNA sensor had a linear range of the previous report.com www.36] between the ring structures in the nucleobases and the hexagonal cells of the graphene. black). we computed the distance between the atoms of the nucleobases and the surface of graphene.. 50 pmol). to several hours) in the presence of excess T1 and in a which excels previously reported molecular beacons[4. As shown in Fig. 3c. all of the nucleobases lie nearly ﬂat at the GO surface. While hydroxy groups on GO might interact with phosphate groups of dsDNA.de in kinetics due to the presence of competition between P1/GO adsorption and the P1/T1 hybridization. We ﬁrst hybri(blue). 3b). which can be readily work[23. To characterize the degree of the adsorption. ssDNA binds to the whole assay time was reduced to only several minutes (in GO with signiﬁcantly higher afﬁnity than dsDNA. Fluorescence spectra of P1 in the absence (black) and presence of the complementary target T1 of 50 nM (red). Importantly. Also. which could not survive at room temperature and in aqueous solution (see Fig. 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. thus. sequence-speciﬁc able from the background.20] and concentration-dependent manner (Fig. 40. the ﬂuorescence was intensiﬁed along with the increase of comparison. we could obtain signals that were readily distinguishseparated under external competition (e. which makes it particularly suitable for hybridization). 34 bp (red). In contrast. 2010. We found that the ﬂuorescence of f) Kinetic study for the ﬂuorescence change of the GO-bound P1 in the presence of T1 of various unhybridized P1 was efﬁciently quenched by amounts (10. 20. These results imply that GO possesses signiﬁcantly different performance comparison in Table 2). the prequenched ﬂuorescence in P1/GO was slowly recovered (up the derived calibration curve (>3 standard deviations (SD)). 3a).afm-journal. a) Scheme for the ﬂuorescent DNA detection based on the ssDNA/dsDNA discrimiWe reason that this GO-based ﬂuorescence nation ability of GO. 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). there was a very sharp peak ˚ at $3. We ﬁrst 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. to which an aliquot of GO was added incubation with T1. 4a. 3b). we only observed minimal hydrogen bonding between them. We carried out a molecular dynamics (MD) simulation to interrogate the observed large discrepancy for interactions between ss. Weinheim .33]). we also performed a ‘‘premixing’’ protocol similar to the target concentration. observable ﬂuorescence that formed the basis of a ‘‘signal-on’’ DNA sensor. (postmixing). This suggests that most of the atoms in the nucleobases are directly adsorbed at GO. KGaA. 30. We attribute this strong adsorption to the p-stacking interaction[35. ssDNA contrast to hours using either SWNTs or graphene in the previous binds to GO in a noncovalent manner. Also of note. As shown in the snapshot.g. This is because nucleobases were effectively shielded within the densely negatively charged phosphate backbone of dsDNA.d.and dsDNA with GO. 20.www.5 A. FULL PAPER 455 Adv. As shown in Fig. quenching might serve as a sensing platform c) GO-based ﬂuorescence DNA assay.23.f). The excitation and the emission wavelengths are 494 and GO while the hybridized P1/T1 duplex led to 526 nm. Mater. and ssDNA were stably adsorbed by the GO (see Fig. Of note. we favor the use of a new ‘‘postmixing’’ strategy in the following DNA detection studies. dsDNA could not be stably adsorbed on GO in our MD simulation and retained its helical structure (shown in Fig.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. Funct. e) Kinetic study for the ﬂuorescence change of the FAM-tagged probes (20 nM) with different lengths in the presence of GO: 17 base pairs (bp. 2b. 51 bp (blue). suggesting that nanomaterials-based DNA detection[16. and random sequence R1 of 50 nM for quantitative DNA analysis. Figure 2. b) Scheme for the target hybridization-induced probe liberation from GO.
Given that the fully comtarget were designed to be in the middle (T2). in contrast. 2010. 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. the targets of different lengths (17 bp versus 47 bp). Probe mixture in the presence of different targets T5 (blue). and f) 587/609 nm/nm.de www. respectively). provided that the probe sequence is well designed. Mater. which was of the same length as P1. 456 ß 2010 WILEY-VCH Verlag GmbH & Co. and M3 (green). the complementary regions for P1 in the 47-bp mutated to T. The GO-based DNA detection exhibited high Figure 3. 2c).(T3). or the plementary duplex (P1/T1) and the mismatched duplexes (P1/M1. 20. and distinctly distinguishable from the background (Fig. a) Representative ﬂuorescence spectra for P1 (10 nM) in the presence of various concentrations of T1 (0. 6. 30 . Inset: calibration curve for the ﬂuorescence intensity versus P1 concentration. and from at least three independent experiments. The data are recoded with the excitation and emission wavelength of 494 and 526 nm. d–f) Fluorescence spectra for multicolor detection. M2 (blue). This position effect was attributed to the location of FAM and its interaction with GO. b) dsDNA does not adsorb at the surface of GO. we observed that the ﬂuorescence signal for T4 was signiﬁcantly larger than those of T2 and T3. FAM was more likely to be detached from the GO surface in the case of P1/T4. 453–459 . respectively. We observed that all these long targets led to smaller FULL PAPER Figure 4. T6 (red). at the 50 . or long targets T2 (cyan). Weinheim Adv. with the excitation/emission wavelengths of d) 494/526 e) 643/666.(T4) end.5. it is possible to employ the GO-based assay in practical DNA detection with long sequences. minimal ﬂuorescence increase in the presence The FAM. 15. As a result. green and cyan. and T4 (green). 4. and GO are shown in purple. In order to evaluate ﬂuorescence 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. 30. However. respectively. Consequently.www. this DNA sensor was of sufﬁcient selectivity to easily differentiate single mismatches. and 50 nM. Funct.MaterialsViews. c) Fluorescence spectra for P1 in the absence (black) and presence of the fully complementary target T1 (red). DNA.com ﬂuorescence signals than T1. T3 (blue). which offered the opportunity to allow single-nucleotide We then interrogated the hybridization between a probe and polymorphism (SNPs) analysis. Representative snapshots from MD simulation showing that a) FAM-tagged (red) sequence speciﬁcity. 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). More atoms in nucleobases at a distance from the GO surface. A or G in the middle). 4b).afm-journal. As shown in Fig. 0. and T7 (orange). KGaA. 4b. When FAM was tagged at the 50 end of P1. FAM was located at the interface of the ss and ds regions for T2/T3. c) Probability of the of excess noncognate DNA (Fig. importantly. it was located right at the duplex region after hybridization with T4. We only observed a ssDNA is tightly adsorbed at the surface of GO.
20. The selection of these three dyes avoided signiﬁcant dye-to-dye energy transfer. We employed three probes (P5.com Table 2. this high sequence speciﬁcity for single mismatches was fairly impressive. respectively. which were individually excited at 494. 453–459 ß 2010 WILEY-VCH Verlag GmbH & Co.38] For example. P6 and P7.de FULL PAPER Probe synthesis Difﬁcult (dual labeling) Difﬁcult (dual labeling) Difﬁcult (dual labeling) Convenient (single labeling) Convenient (single labeling) Convenient (single labeling) Convenient (single labeling) Ref. 100. P6. and ROX at the 50 end. and 587 nm. all other ions are of 1 mM). Cy5 (cyanine 5).[37. C. Performance comparison between homogeneous ﬂuorescent DNA sensors. As shown in Figure 4d–f. 10. respectively. this Figure 5. Funct... which motivated us to explore methods of simultaneous detection of multiple DNA targets (Scheme 1). 2010. d) Selectivity of the GO-based A sensor over G.5 h) Rapid (minutes) Multicolor analysis Yes Yes NR NR NR NR Yes www. while minimal emission of the other two colors. emitting blue (520 nm). 200. i. Simultaneous detection of multiple targets in a homogeneous solution brings about new opportunities for molecular diagnostics. The large planar surface of GO possesses the ability to interact with multiple DNA strands. Type Molecular beacons AuNP–DNA–dye conjugates (stem loop probe) AuNP–DNA–dye conjugates (linear probe) Unmodiﬁed 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.www. c) Fluorescence spectra of the anti-adenine aptamer (P9) in the presence of various concentrations of A (0. We attribute this high stringency to the competition between GO/ probe adsorption and probe/target hybridization. Inset: calibration curve for A detection. and P7) for three types of tumor-suppressor genes (exon segments of p16. and that sequences were not elaborately designed (e. 180 nM). Weinheim 457 . it is important to detect multiple tumorsuppressor genes in order to identify early-phase cancers in asymptomatic individuals.MaterialsViews. P1/M2. 80. [b] TW stands for ‘‘this work’’. KGaA. 50.37]      TW [b] [a] NC stands for ‘‘Not reported’’. the addition of the p21 target (T6) and the p53 gene produced the red (Cy5) and orange (ROX) colors. 1200. a) Fluorescence spectra of the MSO (P8) in the presence of various concentrations of Hg2þ (0. Inset: calibration curve for Hg2þ detection.afm-journal. 643. 800. 40. Mater. and P1/M3) have relatively small thermodynamic difference. the p16 target (T5) led to the speciﬁc emission of the blue color (FAM).e. The excitation wavelength was 495 nm. p21 and p53 genes) were labeled with FAM. Adv. 2000 mM). Therefore. and T (all of 100 mM). stem-loop structures with inherent stringency). red (670 nm) and orange (608 nm) colors. 60. respectively. we observed the emission from the corresponding wavelength.g. b) Selectivity of the GO-based Hg2þ sensor over a spectrum of interference metal ions (120 nM Hg2þ. Upon the addition of speciﬁc targets to the probe mixture containing P5. [4. Similarly. GO possibly competitively destabilizes mismatched duplexes.
only led to weak responses. 5 pmol of FAM-tagged P16 probe P5. MicroMasch). KGaA. The solution was further stirred for 2 h at 35 8C. Therefore. and distilled water (200 mL) was added. Unexfoliated graphite oxide was removed by centrifugation (3000 rpm.22 mm (Generay Biotech Co. The brown dispersion was extensively dialyzed to remove residual metal ions and acids. However. First. 2. Second. The reaction was stopped with the addition of a mixture of 560 mL of distilled water and 10 mL of H2O2 (30%). Conclusions In summary. Graphite powder was purchased from China National Pharmaceutical Group Corporation (Shanghai. to which GO of 20 mL (235 mg/mL) was added. Mater. China). All other conditions were similar to those in the single target detection. 453–459 . Brieﬂy. Based on the difference in ﬂuorescent intensity.[4. Other chemicals were of analytical grade. The product was washed with HCl (1:10).5 h (300 W). The as-prepared GO samples (0.com GO-based multicolor DNA analysis provides a promising approach for challenging applications such as allele discrimination. pH 7.1 M phosphate buffer of 980 mL (7. Similarly. cytidine (C). Experimental Materials: DNA oligonucleotides were synthesized and puriﬁed by TAKARA Biotechnology (Dalian. and then ﬁltered with a membrane of 0.1 M phosphate buffer of 980 mL for 30 min. and we expect that GO and other graphene materials will ﬁnd important and widespread applications in biology. After 1-min incubation. As a proof of concept. the FAM-labeled MSO (P8) was in the singlestranded state and not ﬂuorescent in the presence of GO. Instrumentation: The HRTEM was performed with a Philips CM-120 transmission electron microscope operating at 120 kV (Philips. Particularly. and then exfoliated via sonication for 1. 100 mM NaCl. The sequences of these oligonucleotides are shown in Table 1. All ﬂuorescence spectra were collected with a Hitachi F-4500 spectrophotometer equipped with a Xenon lamp excitation source. we designed a new homogeneous sensor for multiplex. 4. Also. the ﬂuorescence was largely quenched. This sensor had a linear range of 30–180 nM and a detection limit of 30 nM. When a FAM-tagged anti-adeonsine aptamer (P9) was incubated with GO. and then with water. A droplet of GO dispersion was cast onto a freshly cleaved mica surface. GO can be readily synthesized in large quantities.47 mg/mL) was then characterized with tapping-mode AFM [26. suggesting the high selectivity of this adenine sensor. and P2O5 (8 g). Adenine (A). the ﬂuorescence of MSO was dependent on the concentration of Hg2þ. and thymine (T). Japan). NaNO3 (2 g) was added to the mixture. In adenosine triphosphate (ATP) assays. The anti-adenine aptamer is an in vitro selected short oligonucleotides targeting the adenine molecule with antibodylike speciﬁcity and afﬁnity. 10 pmol of FAM-tagged mercury speciﬁc oligonucleotide (P8) was incubated with Hg2þ of a series of concentrations in a 3-N-morpholinopropanesulfonic acid (MOPS) buffer (10 mM with 50 mM NaNO3. and 20 pmol of (Cy 5)-tagged probe P7 were mixed in a solution. Fluorescence measurements were performed after 1 min. After 15 min. which compared favorably with previously reported Hg2þ ﬂuorescent sensors. analogous molecules. The observed small background ﬂuorescence might arise from the secondary structure of this sequence. Of note.MaterialsViews.2) for 10 min. and in contrast to dually labeled molecular beacons. Therefore it provides opportunities to develop low-cost and rapid molecular diagnostic tools. Funct. These pre-oxidized graphite powder (2 g) were added to 92 mL of cold H2SO4 (0 8C). the GO-based DNA detection improves the sensitivity by at least an order of magnitude as compared to conventional molecular beacons. In mercury assays.. China) and dried overnight at 60 8C. this is a homogeneous.and dsDNA using both theoretical calculations and experimental studies.de www. we could detect adenosine with a detection limit as low as 10 mM (Fig. 5 min) using Centrifuge himac-CF 16RX (Hitachi. Fluorescence measurements were 3. 2010. In multicolor DNA assays. Third. and the sample was maintained at room temperature for several minutes to allow water evaporation. AFM measurements were carried out on Nanoscope IIIa (Digital Instrument. Ltd. pH 7. and then suspended in distilled water.4) for 10 min..g. with a Pt/Ti coated tip (force constant of 4 N/m. this Hg2þ sensor was selective enough to distinguish a range of interference metal ions (Fig.. the introduction of Hg2þ led to the formation of the stem-loop structure that could not be quenched by GO. In the presence of adenosine. Also importantly. to which KMnO4 (12 g) was gradually added under continuous stirring in an ice-bath. we herein demonstrated the detection of mercuric ion (Hg2þ) and adenine (A) with a T-rich mercuryspeciﬁc oligonucleotide (MSO) and an anti-adenine aptamer. 20. guanine (G) and thymine (T) were purchased from Sigma. Samples for TEM were prepared by drop casting 20 mL of ˚ the as-prepared GO onto a standard carbon-coated (200–300 A) formvar ﬁlm on a copper grid. the ﬂuorescent probe P1 (10 pmol) was hybridized with the target in a 0. 10 pmol of ROX-tagged P21 probe P6.afm-journal. Given the convenience to perform calculations and modeling on GO. ﬂuorescence measurements were performed with a quartz cuvette to monitor the hybridization process. We employed different amounts of probes in order to optimize the detection performance.20] which reﬂects the superquenching ability of GO that minimizes the background ﬂuorescence. which reduces the cost for DNA assays. 5a). The resulting dark blue mixture was thermally isolated and slowly cooled to room temperature over a period of 6 h. we have demonstrated that GO possesses high ﬂuorescence quenching ability as well as different afﬁnities toward ss.www. The present study represents our ﬁrst attempt to connect the highly promising nanomaterial GO with DNA sensing. guanine (G). USA) under tapping mode. Weinheim Adv. the probe P9 was converted to a rigid tertiary structure. aptamers). Netherlands). Water was puriﬁed using a Millipore ﬁltration system. GO could not quench the ﬂuorescence of the rigid aptamer structure. the availability of large planar surfaces of GO makes it possible to detect multiple molecular targets in the same solution. but may ﬁnd applications in detecting a wide spectrum of analytes when complemented with the use of functional nucleic acid structures (e. A GO solution (235 mg/mL) of 20 mL was added to this mixture. sequence-speciﬁc DNA detection. the probe is only labeled with a single dye. The ability of GO to differentiate ss. the compatibility of GO with different DNA structures provides great versatility to develop sensors for a spectrum of analytes with relatively convenient design. such as cytosine (C). Preparation of GO: GO was synthesized from graphite powder based on the Hummer’s method .3 mM NaH2PO4. Fluorescent DNA Assays: In a typical DNA assay. This GO-based DNA sensor has several important advantages. Shanghai. it is also possible to predict and engineer GO/biomolecular interactions and further 458 ß 2010 WILEY-VCH Verlag GmbH & Co. Without Hg2þ. FULL PAPER optimize the sensor performance with computer-aided rational design. 10 pmol of FAMtagged anti-ATP aptamer probes (P8) was incubated with ATP in 0. The mixture was diluted to 300 mL. mix-and-detect assay method that can be ﬁnished within minutes. Based on these ﬁndings.29] and TEM .7 mM Na2HPO4.and dsDNA not only offers a new approach to detect DNA. China). The image was obtained at 20 8C at a humidity of 30%. 5b). graphite power (4 g) was oxidized in a hot solution (80 8C) of concentrated H2SO4 (24 mL) containing K2S2O8 (8 g).
Y. Hong. Wang. W. Porter. R. J. 393. 306. Bo Song. 14036. 86. J. Mod. J. J.3  and with the PME method  for full electrostatics with a cut-off of 1 nm. 2007. Liang. Dong. J. X. Lv. Y. C. Zimney. N. Mirkin. H. J. Berry. 2009 Revised: September 30. Muller. H. Kang. Z. F. A. 14. All simulations were carried out at a constant pressure 1 bar with a constant temperature (300 K) via Gromacs 3. W. J. Dikin.afm-journal. Nat. T. E. J. Chen. C. Taton. 4785. Dubertret. Gao. S. Maxwell. J. 2001. K. A. Zhang. Chem. Tan. A. J. Shao. G. A. Nat.and dsDNA. Sci. Z. J. S. J. B. Y. 303. A. Wang. T. 277. Nat. 2007. G. U. Huang. counter ions and water. Nat. G. E. J. I. 22. 442. Chen. 1355. W. C. Int. Wang. Hess. J. Int. First.www. Storhoff. X. T. Tan. 2007. Clin. Adv. Fan. Ellington. S. B. Kaner. D. Huang. Chem. S. Angew. 1996. V. Chem.-J. Sidransky. Wang. In the box. L. D. 49. J. Gong. J. Chem. X. Novoselov. Ed. H. C. Nanotechnol. W. 2009. Chem. S. 2005. Chem. 19. S. Wang. Nguyen. Dikin. J. K. 306. Fang. H. Li. The ﬁnal 10-ns simulations were employed for analysis. Barone. Song. W. Letsinger. A. X. Weinheim 459 . M. Zhang. Int. Mater. Sebastian. A. R. 1993. R. Taylor. 20. E. 1170. J. Chem. Lee. C. Fang. 1983. McGlennen. Muller. H. 60. Togashi. 4. 88. 104. Nie. Chem. Proc. Piner. the carbon atoms were modeled as uncharged Lennard–Jones particles with a distance parameter of s ¼ 0. 44. Phys. The TIP3P water model  was employed in our simulation. 2007AA06A406. we performed MD simulation for 1 ns for the system of the DNA segment on the GO to obtain the initial conformation. I. R. 1054. 291. Lucas. Int. Jiang. R. Nat. Piner. 6. Sorin. J. Am. Heller. FULL PAPER Acknowledgements Shijiang He. and a potential well depth of e ¼ 0. Biol. H. P. Sci. J. A. Kaner. Li. Stach. 3687. Zimney. 2001. K. Adv. Biol. Plaxco. J. Ed. 2472. Nanotechnol. Nat. C. 2. L. Swathi. Nat. Li. Jorgensen. Liu. H. 3085. Hill. Hernandez. 1547. Stankovich. X. Y. K. D. Mucic. Dommett.3598 kJ Á molÀ1. 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The conformation and the parameters of the FAM molecule were obtained from PRODRG . C. 135501. Zhang. H. M. Elghanian. Soc. Govorov. R. Chem. 102. X. B. Li. L. Opin. N. 818. J. Heeger. Kramer. Goodwin. S. J. Lu. J. L. Chem. 2001. 086101. R. Ruoff. 19. P. Z. R. T. Mirkin. A time step of 1 fs was employed and data were collected every 1 ps. N.46]. 278. L. Katz. 105. V. L. R. H. Mater. Ministry of Health (2009ZX10004-301). 6042. Yu. Chem. S. P. Swathi. R. L. C. C. S. Calame. Liu. Y. Shen. 98.. while the parameters for other components (e. D. 100. J. Bao. Strano. Mirkin. J. Soc. D. Tang. Zaric. 2005. A. J. E. Kramer. J. 2006. W. 101. A. Rev. A. Nature 2006. S. Liu. Welsher. 79. Annu. 129. J. D. C. Angew. Y. Tabakman. F. V. An. L. Chem. Shi. F. P. Dommett. 2010. Zhao. 1993. D. H. P. Science 2008. Ed. Ministry of Science and Technology (2006CB933000. Zhang. 4. J. Klein. D. K. E. 8351. 1856. P. M. 926. Y. Zhang. G. J. 246. K. Proc. 85. The carbon bond length of 0. A. Z. S.3400 nm. 282. Pan. Jin. R. McGaughey. 2. 2008. 16. Z. Z. 320. M. 5100.com performed after the addition of 20 mL of GO solution (235 mg/mL) for 1 min. Natl. D. 1339. Firsov. 2008. van der Spoel. a weak dihedral angle potential was applied to the bonded carbon atoms of GO [45. K. Natl. 295. S. Funct. A. J. X. W. 20873175. R. 6297. Nanotechnol. Gagne. G. Proc. Mol. Wan. 2888. S. D. Fan. Yao. S. 346. Nat. 43. USA 2003. J. Wang. 45. 2008. Chem. Morozov. Robinson. Sci. L. Acad. Natl. Madura. V. 80. F. Zhu. J. J. Nature 1990. T. Rappe. 2002. B. 2006. Protoc. D. 2004. L. respectively. H. B. Nano Res. Nature 2007. USA 2007. Park. Pedersen. D. Hummers. D 2004. Z. V. Mater. Prigodich. Li. S. 365. J. C. M. 130. S. 2. 130. 054703. Li. Sebastian. 9606. G. Mater. L. Willner. Zhang. A. Dai. E. Lindahl. Ye. Received: September 1. Soc. A. Ono. Garimella. G. Phys. 4469. ssDNA and hydroxy groups in GO) were directly obtained from the AMBER03 force ﬁeld . 2010CB934504). 2008.4 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 Â 10 nm Â 6 nm. 2009 Published online: January 4. Mohanty. S. Am. Yang. R. 2004. D. A. A. 2005. W. Hu. Ni. 1471. 1085. Zhang. Biophys. DiMagno. J. C. X. S. Sun. H. J.46]. Schuettelkopf. 1. 2005. 2010              M. 448. C. Zhu. D. A. R. Ed. D. K. Szostak. Fan. D. Wu. J. Chem. Phys. 3.MaterialsViews. S. Int. S. A. Fan. 10825520). 4300. Sun.de P. J. Science 1997. M. 19. Song. R. C. G. Mirkin. 101. Kotov. 203. Dubonos. Biomed. Science 2002. Chen. R. D. Gilje. 129. GO. Chen. Am. Angew. Rothberg. 48. D. This work was supported by National Natural Science Foundation (20725516. J. Lu. Chem. M. Nat. X. Curr. Li. H. Angew. W. R. Song. 2007. K. Lett. Am. Science 1997. Angew. H. Chem. Phys. Kohlhaas. Biotechnol. H. Acad. 457. 10089. Li. J. 1998. G. 666. D. 43. Angew. Nano Lett. Gong. S. Grigorieva. Supporting Information is available online from Wiley InterScience or from the author. Acad. 129. Bazan. 2009. In GO. Computational Methods: A piece of GO (7.                                       www. H. Ed. Heller.g. D. Welsher. H. 8676. Chem. D. Wallace. H. Acta Cryst. Y. Tang. U. Phys. 1. Adv. 15458. Nguyen. 1958. J. M. Eng. Z. there were approximately 10 700 water molecules and 16 or 32 sodium ions that neutralized the ss. N. Phys. E. 90913014. Chem. Mater. 1503. Baik. and Di Li contributed equally to this work. 7. Nat.14 nm and the bond angle of 1208 were maintained by harmonic potentials with spring constants of 393 and 960 kJ molÀ1 and 527 kJ molÀ1 radÀ2 before relaxation [45. Fan. Nano Res.
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