DOI: 10.1002/chem.201404730
Communication
& Nanotechnology | Hot Paper|
Simultaneous Detection of Multiple DNA Targets by Integrating Dual-Color Graphene Quantum Dot Nanoprobes and Carbon Nanotubes Zhaosheng Qian, Xiaoyue Shan, Lujing Chai, Jianrong Chen, and Hui Feng*[a] Abstract: Simultaneous detection of multiple DNA targets was achieved based on a biocompatible graphene quantum dots (GQDs) and carbon nanotubes (CNTs) platform through spontaneous assembly between dual-color GQDbased probes and CNTs and subsequently self-recognition between DNA probes and targets.
Developments in biomarker sensing methodologies are of great importance owing to their versatile potential applications in gene expression profiling for early diagnosis and treatment.[1] Notably, many diseases are associated with multiple biomarkers and the detection of a single biomarker may cause false diagnosis.[2] Simultaneous detection of multiple targets brings new opportunities for improving the accuracy of early disease detection over the single-marker assay. Moreover, simultaneous determination of multiple biomarkers within a single sample has many advantages over monoplex assays such as less sample requirement, lower cost per test, shorter analysis time, and fewer repetitions of tedious procedures. However, up to date, convenient and simultaneous detection of multiple biomarkers such as DNA and proteins with biocompatible materials and good analytical performance still remains a challenge. Several nanosensors have been reported to achieve separate detection of different targets. Most proceeding research focused on respective detection of multiplex targets or species with dye-labeled probes.[3–7] However, organic dyes as fluorophores in the probes have non-negligible disadvantages such as poor photostability, easy photobleaching, and small Stokes shifts. To overcome these problems, novel fluorescence platforms based on semiconductor QDs and AgNPs were recently established.[8–11] Nevertheless, the drawbacks of semiconductor QDs[12] and AgNPs[13] including high toxicity, relatively high cost and hard manipulation still do not enable them as excellent fluorophores of probes for biomolecules, which urges scientists to discover and exploit alternative fluorescent materials with good biocompatibility because the safety and biocompatibility [a] Dr. Z. Qian, X. Shan, L. Chai, Prof. J. Chen, Dr. H. Feng Department of Chemistry, Zhejiang Normal University 688 Yingbin Road, Jinhua, Zhejiang Province (P.R. China) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404730. Chem. Eur. J. 2014, 20, 16065 – 16069
of used materials are also of great significance for the further application of constructed biosensors in cellular tissue or in vivo.[14] Graphene quantum dots (GQDs) have been used in photovoltaic devices, photocatalysis and biological imaging because of their distinctive optical and electronic properties.[15] With respect to conventional dye molecules and semiconductor QDs, GQDs have many superiorities such as stable light emitting, high quantum yield, good photostability, easy modulation, and excellent biocompatibility. To date, the synthesis and optical properties of GQDs have been extensively investigated and great progress was made in the development of facile preparations of GQDs with high and controllable fluorescence.[16] Our previous work showed that modified-GQDs with small organic compounds and heteroatom-doped GQDs possess strong fluorescence, low toxicity, and excellent biolabeling to human Hela cells.[17] Based on these doped GQDs, a multifunctional fluorescence sensing platform for selective detection of AgI, FeIII and hydrogen peroxide, glucose, and melamine were constructed. Recently, we employed GQDs as the fluorophore in the probe and graphene oxide as the platform to achieve highly sensitive and selective detection of a single DNA target for the first time.[18] In this communication, we report simultaneous detection of multiplex DNA targets with high sensitivity and selectivity based on a biocompatible GQDs and CNTs platform by taking advantage of intense and dual-color fluorescence of GQDs, efficient quenching capability of CNTs, specific recognition between probes and targets, and unique self-assembly between GQDs and CNTs. Dual-color GQDs were employed to synthesize blue probe (P1) and green probe (P2) for recognition of two DNA targets respectively. To realize simultaneous determination, two GQD-labeled probes were assembled on the CNTs surface at the same time. By the integration of their specific interactions, the dual-functional nanosensor assembled with CNTs and two probes succeeded for convenient and simultaneous detection of multiplex DNA through two cycles of fluorescence on-off-on for the first time. Scheme 1 displays the schematic illustration of simultaneous detection strategy for multiplex DNA targets through the simultaneous assembly of two probes on the CNTs surface and synchronous recognition between probes and targets. Simultaneous detections of two DNA targets are accomplished in the following procedure: In the first step, two probes are mixed with CNTs so that the probes can adsorb on the surface of CNTs through electrostatic attraction and p–p stacking interactions, where assembly of (P1 and P2)/CNTs occurs. The formation of the corresponding
16065
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
Figure 1. TEM (A) and HRTEM (B) images of ssDNA-eGQDs (P1) and ssDNApGQDs (P2) adsorbed on CNTs.
Scheme 1. Schematic illustration of the proposed detection strategy for simultaneous detection of multiplex DNA targets. P1: ssDNA-eGQDs (DNA probe 1), P2: ssDNA-pGQDs (DNA probe 2), T1: DNA target 1, T2: DNA target 2, P1/T1: dsDNA-eGQDs, P2/T2: dsDNA-pGQDs.
assembly leads to substantial fluorescence quenching of the original probe through static quenching between probes and CNTs. It has been proven that efficient static quenching between GQDs and CNTs occurs and subsequently leads to effective fluorescence quenching of GQDs.[19] In the second step, upon the addition of DNA target 1 (T1) and DNA target 2 (T2), T1 and T2 can hybridize with P1 and P2 in the assembly (P1 + P2)/CNTs to produce double-stranded DNA assembly P1/T1 and P2/T2 through their corresponding base pairing. The formation of double-stranded DNA assembly breaks up the electrostatic attraction and p–p stacking interaction between the probes and CNTs, and thus liberates double-stranded DNA assemblies from CNTs. The free double-stranded DNA assemblies P1/T1 and P2/T2 re-emit blue and green fluorescence, respectively, and lead to gradual recovery of the two fluorescences upon addition of two DNA targets. Highly fluorescent 1,2-ethylenediamine-functionalized GQDs (eGQDs) and pristine GQDs (pGQDs) were prepared and well characterized in our previous paper.[17] eGQDs and pGQDs exhibit blue and green fluorescence, respectively, and thus they are used as fluorophores for P1 and P2 probes, respectively. eGQDs possess a great deal of NH2 groups, while pGQDs are rich in the COOH group.[17] Through the condensation reaction with the same reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), eGQDs react with COOH-functionalized DNA (ssDNA) to form a ssDNA-eGQDs probe (P1) bonded with a CO NH group, whereas pGQDs bind with NH2-functionalized DNA (ssDNA) to produce ssDNA-pGQDs probe (P2) through CO NH group. TEM images in Figure 1 clearly show that the two probes are adsorbed on the surface of multiwalled carbon nanotubes (MWCNTs), and their size distribution falls in the range of 3–5 nm. P1 and P2 present blue and green fluorescence under UV lamp as shown in Figure 2, inset. Normalized fluorescence spectra illustrate that P1 shows intense fluorescence emission centered at l = 414 nm, whereas the P2 probe Chem. Eur. J. 2014, 20, 16065 – 16069
www.chemeurj.org
Figure 2. Fluorescence spectra of eGQDs, ssDNA-eGQDs (P1), dsDNA-eGQDs (P1/T1), pGQDs, ssDNA-pGQDs (P2), and dsDNA-pGQDs (P2/T2). Inset: the fluorescence images of ssDNA-eGQDs (P1) and ssDNA-pGQDs probes (P2).
strongly emits yellowlish–green light at around l = 514 nm when they are excited by their own optimal excitation light. The large gaps in fluorescence emission between the probes and their corresponding GQDs provide solid evidence for successful binding link between GQDs and DNA sequence, that is, successful preparation of the probes. It is worth noting that eGQD-labeled probe P1 separates well in fluorescence emission from the pGQD-labeled probe P2. This good separation in fluorescence emission enables the subsequent accomplishment of simultaneous detection of two DNA targets. After addition of DNA target 1 (T1), the fluorescence emission is shifted to l = 438 nm, which is from the assembly P1/T1 due to their self-recognition through base paring. However, no apparent shift in fluorescence emission can be observed for the P2/T2 assembly, which is consistent with the observation in our previous report. The large difference in the time-resolved decay curve between P2 and P2/T2 in Figure S1 (Supporting Information) offers the proof for the formation of P2/T2 assembly with addition of T2 into P2 solution. Moreover, it should be noted that the high quantum yields of probes shown in Table S1 (Supporting Information) has been attained due to high emission efficiency from eGQDs or pGQDs, which endows the probes with a high sensitivity of the assembled sensors.
16066
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication A dual-functional biosensor based on two-colored probes and CNTs was further constructed to explore simultaneous detection of two DNA targets. Here we used synchronous fluorescence spectroscopy to differentiate the respective emission of the mixed probes because of the broad emission band of the probes. The two probes show an apparently distinct fluorescence emission peaks at l = 364 and 440 nm, respectively, with a Dl of 60 nm in the synchronous spectra, which enables simultaneous detection of two DNA targets. After the addition of 3.48 mg mL 1 of CNTs into the mixed probes system, the two probes were assembled onto the surface of CNTs at the same time. The formation of the assembly (P1 and P2)/CNTs resulted in substantial fluorescence quenching due to static quenching between probes and CNTs. Figure 3 shows a similarly strong quenching effect of CNTs to both probes, and the presence of 3.48 mg mL 1 of CNTs leads to a reduction of more than 60 % in intensity. The formation of the assembly (P1 + P2)/CNTs was also verified by the TEM images shown in Figure 1, which demonstrates that a great deal of GQDs-labeled probes are adsorbed on the surface of CNTs as the assembly. Condition optimization including amounts of added CNTs, the incubation time for fluorescence quenching and recovery were carried out. According to Figure 3A and Figures S3 and S4 (Supporting Information), 3.48 mg mL 1 of CNTs and 2 and 40 min mixing for quenching and recovery were chosen to assess the performance of the dual-functional biosensor in quantitative analysis of T1 and T2 at the same time. Figure 3B
shows a gradual increase in fluorescence intensity of two types of dsDNA-GQDs with the concentration of both T1 and T2 up to 140.0 nm. The fluorescence enhancement can be attributed to the increase of fluorescent P1/T1 and P2/T2 in amount due to hybridization between probe DNA and target DNA. Figure 3C displays the linear relationship of fluorescence intensity to concentration of T1 from 6.9 to 80.0 nm, and this equation can be expressed as y = 1.23x + 215.2, in which R2 = 0.994. Its detection limit is estimated as 4.2 nm from derived calibration curve ( 3 standard deviations). The method for T2 in Figure 3D has a broad detection range of 8.2–100 nm with a detection limit of 3.6 nm, and this equation can be expressed as y = 0.80x + 191.2, in which R2 = 0.999. The detection limits for T1 and T2 are comparable to those of DNA sensors for single detection (0.1–1.0 nm),[5,6,9,18] which indicates that this dualfunctional biosensor does not lose its high sensitivity while largely expanding its detection scope. Furthermore, the selectivity of this nanosensor was also assessed with target DNA and mismatched DNA sequences as shown in Figure 4. The presence of T1 and T2 leads to respective fluorescence recovery of P1 and P2, but does not have an influence on each other. The mismatched DNA with one, two, and three bases with respect to T1 shows nearly no fluorescence response to this sensor, whereas the fluorescence intensity doubles relative to the blank with the presence of T1. The high selectivity is consistent with our previous results.[18] To demonstrate the feasibility of the practical application of the proposed method, we detected the target DNA in real samples (Bovine serum). Before analysis, the 100-fold diluted serum sample was spiked with 2.0 mL of target DNA (T1) and target DNA (T2), and then added into the ssDNA-eGQDs and ssDNA-pGQDs probe solution which has been quenched by CNTs, respectively. After incubation for 60 min at room temperature, the fluorescence intensity of the resulting solutions was measured. As shown in Figure S5 (Supporting Information), comparable responses were obtained for the detection of target DNA in real samples with those results in buffer, which indicated the potentiality of the proposed method for real biological sample analysis. In summary, the present study has introduced biocompatible carbon-based materials to assemble multifunctional biosenFigure 3. A) Fluorescence quenching spectra of P1 and P2 with various concentrations of CNTs (0, 0.29, 0.58, 0.87, sors for the simultaneous detec1 1.16, 1.45, 1.74, 2.32, 2.90, 3.48, 4.06, 5.22 mg mL ). B) Fluorescence recovery of the (P1 + P2)/CNTs system after intion of multiplex DNA. Benign cubation with various concentrations of DNA targets (T1 and T2) (0, 20.0, 40.0, 60.0, 80.0, 100.0, 120.0, 140.0 nm). GQDs and CNTs were used as C,D) Linear response in fluorescence to DNA target 1 and 2. Chem. Eur. J. 2014, 20, 16065 – 16069
www.chemeurj.org
16067
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication Synthesis of 1,2-ethylenediamine functionalized graphene quantum dots (eGQDs)
Figure 4. Fluorescence intensity changes (F/F0) of the aptasensor in the presence of T1, T2, mT1, mT2, and mT3 (100.0 nm) in buffer.
fluorophores in two-colored probes and platforms respectively. High emission efficiency of GQDs guarantees high sensitivity of the constructed biosensors, and good biocompatibility provides promising opportunities for further utilization of the biosensors in vivo. This dual-functional biosensor assembled with two GQDs-labeled probes and CNTs platform possesses the capability to simultaneous and quantitative determination of two DNA targets with a broad linear range and low detection limit, which can serve as a general detection model for synchronous detection of multiple species in complex systems.
Experimental Section Materials and reagents Triplex distilled water was used in the whole experimental process and to prepare PBS buffers. Graphite powder, sodium borohydride, tetrahydrofuran, 1,2-ethylenediamine, thionyl dichloride, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and N-hydroxylsuccinimide sodium salt (NHS) were purchased from Aladdin Ltd. (Shanghai, China). Phosphate buffer solution (PBS) was prepared by mixing stock solutions of NaH2PO4 and Na2HPO4. All reagents were of analytical grade and without any further purification. DNA oligonucleotides were purchased from Shanghai Sangon Biotechnology (Shanghai, China). Their sequences are listed as follows: Capturing ssDNA sequence in probes: P1 = 5’COOH-CTG ATT ACT ATT GCA TGA GGC CTT-3’, P2 = 5’-NH2-TTG GTG AAG CTA ACG TTG AGG-3’; target ssDNA sequence (tDNA: perfectly matched with capturing ssDNA) T1 = 5’-AAG GCC TCA TGC AAT AGT AAT CAG-3’, T2 = 5’-CCT CAA CGT TAG CTT CAC CAA3’. Single-base mismatched ssDNA sequence (mT1): mT1 = 5’-CCT CAA CGT TCG CTT CAC CAA-3’; two-base mismatched ssDNA sequence (mT2): mT2 = 5’-CCT CAT CGT TCG CTT CAC CAA-3’; threebase mismatched ssDNA sequence (mT3): mT3: 5’-CCT CAT CGT TCG CTT CTC CAA-3’.
Synthesis of oxidized carbon nanotubes (CNTs) Briefly, MWNTs (1.0 g) were oxidized with HNO3/H2SO4 (100 mL, 1:3, v/v) in an ultrasonic bath for 24 h. After repetitive washing with water to neutral pH, the resulted CNTs were dried in the vacuum drying oven at 60 8C for 24 h. Chem. Eur. J. 2014, 20, 16065 – 16069
www.chemeurj.org
The preparation of eGQDs was described in our previous paper in detail.[17] Graphite powder (0.3 g) was added into a mixture of concentrated sulfuric acid (180 mL) and nitric acid (60 mL). The solution was sonicated for 2 h and heated at 80 8C for 24 h. The mixture was cooled and diluted with deionized water (800 mL). The dark-brown GQDs solution was neutralized with sodium carbonate. The final product solution was further dialyzed in a dialysis bag (1000 Da) for 3 days. A mixture of as-prepared GQDs (0.1 g) and SOCl2 (20 mL) reacted for 2 h at 80 8C, and then vacuum-distillation was carried out to remove excessive SOCl2. 1,2-Ethylenediamine (20 mL) was added into the chlorinated GQDs and the mixture was heated at 100 8C for 4 h. The excessive 1,2-ethylenediamine of the resulting mixture was removed by vacuum-distillation, and the residue was washed with ethanol several times.
Synthesis of pristine graphene quantum dots (pGQDs) Carbon fiber (1.0 g) was added into a mixture of concentrated sulfuric acid (180 mL) and nitric acid (60 mL). The solution was sonicated for 2 h and heated at 120 8C for 24 h. The mixture was cooled and diluted with deionized water (800 mL). The dark-brown GQDs solution was neutralized with sodium carbonate. The final product solution was further dialyzed in a dialysis bag (1000 Da) for 3 days.
Preparation of the ssDNA-eGQDs probe (P1) and ssDNA– pGQDs probe (P2) The obtained eGQDs were first dissolved in PBS solution (10.0 mL, 10 mm, pH 7.4), and then EDC (80.0 mg) and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) (80.0 mg) were added to the proceeding solution at room temperature with continuous stirring. After 30 min activation, capturing ssDNA (DNA 1 in P1) (15 mL, 100.0 mm) was added into the proceeding solution. The condensation reaction was allowed for 24 h at room temperature with stirring. The DNA probes were obtained by the following centrifugation and washing with PBS buffer. For the preparation of ssDNApGQDs probe (P2), as-prepared pGQDs were first dissolved in phosphate buffer (5.0 mL, 10 mm, pH 7.0), and then the solution was adjusted to pH 5 for protonation of carboxyl groups of pGQDs with dilute hydrochloric acid. After that, EDC (80.0 mg) and sulfoNHS (80.0 mg) were added into the proceeding solution to activate the carboxylic group of pGQDs for 30 min at room temperature with stirring. Capturing ssDNA (DNA 2 in P2) (20.0 mL, 100.0 mm) was subsequently added into the above solution, and the reaction was allowed to process for 24 h. Finally, the ssDNA-pGQDs probe was attained by centrifugation and washing with PBS buffer.
Simultaneous detection of two DNA targets ssDNA-eGQDs probe (1.5 mL) and the ssDNA-pGQDs probe (400.0 mL) were mixed, and then the mixture was diluted with PBS to a volume of 500.0 mL. CNTs (6.0 mL, 0.29 mg mL 1) was introduced into the resultant mixture at room temperature to quench fluorescence with 2 min of incubation. Then, different concentrations of DNA target 1 (T1) (0, 20, 40, 60, 80, 100, 120, 140 nm) and DNA target 2 (T2) (0, 20, 40, 70, 100, 130, 160, 190 nm) were added into the above solution to initiate the fluorescence recovery. After incubation for 40 min at room temperature, the fluorescence intensity of resulting solutions was measured by using synchronous
16068
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication fluorescence ranging from 260 to 600 nm with the constant value of the Dl = 60 nm.
Target DNA detection in bovine serum sample Bovine serum sample was diluted 100-fold with PBS solution (10 mm, pH 7.4) before analysis. ssDNA-eGQDs probe (30.0 mL) or the ssDNA-pGQDs probe (200.0 mL) was diluted with the 100-fold diluted serum sample to a volume of 1.0 mL. CNTs (2.0 mL, 0.29 mg mL 1) was introduced into the resultant mixture at room temperature to quench fluorescence with 2 min of incubation. Then, target DNA (T1) (2.0 mL) was added into the above ssDNAeGQDs probe solution and target DNA (T2) (2.0 mL) was added into the above ssDNA–pGQDs solution to initiate the fluorescence recovery, respectively. After incubation for 60 min at room temperature, the fluorescence intensity of resulting solutions was measured using fluorescence spectrometer.
Characterization methods The morphologies of all samples were characterized by TEM, which was performed on a JEOL-2100F instrument with an accelerating voltage of 200 kV. Samples were prepared by dropping aqueous suspensions of the separated fractions of samples onto Cu TEM grids coated with a holey amorphous carbon film and following solvent evaporation in a dust protected atmosphere. The UV/Vis spectra were recorded on a Perkin–Elmer Lambda 950 spectrometer, in which the sample was dispersed in water after ultrasonication for 30 min. The photoluminescence spectra were conducted on a Perkin–Elmer LS-55 fluorescence spectrometer, and lifetimes were determined by using a FLS920 fluorescence spectrophotometer.
Acknowledgements We are thankful for the support by the National Natural Science Foundation of China (No. 21405142, 21005073 and 21275131) and Zhejiang Province (No. LY13B050001, LQ13B050002 and ZJHX201403).
[2] D. Sidransky, Science 1997, 278, 1054 – 1058. [3] R. H. Yang, Z. W. Tang, J. L. Yan, H. Z. Kang, Y. M. Kim, Z. Zhu, W. H. Tan, Anal. Chem. 2008, 80, 7408 – 7413. [4] J. Chen, Y. Huang, M. Shi, S. L. Zhao, Y. C. Zhao, Talanta 2013, 109, 160 – 166. [5] S. J. He, B. Song, D. Li, C. F. Zhu, W. P. Qi, Y. Q. Wen, L. H. Wang, S. P. Song, H. P. Fang, C. H. Fan, Adv. Funct. Mater. 2010, 20, 453 – 459. [6] M. Zhang, B. C. Yin, W. H. Tan, B. C. Ye, Biosens. Bioelectron. 2011, 26, 3260 – 3265. [7] N. Li, C. Y. Chang, W. Pan, B. Tang, Angew. Chem. Int. Ed. 2012, 51, 7426 – 7430; Angew. Chem. 2012, 124, 7544 – 7548. [8] Q. S. Mei, Z. P. Zhang, Angew. Chem. Int. Ed. 2012, 51, 5602 – 5606; Angew. Chem. 2012, 124, 5700 – 5704. [9] X. Q. Liu, F. Wang, R. Aizen, O. Yehezkeli, I. Willner, J. Am. Chem. Soc. 2013, 135, 11832 – 11839. [10] L. Chen, X. W. Zhang, G. H. Zhou, X. Xiang, X. H. Ji, Z. H. Zheng, Z. K. He, H. Z. Wang, Anal. Chem. 2012, 84, 3200 – 3207. [11] H. Zhang, L. Zhang, R. P. Liang, J. Huang, J. D. Qiu, Anal. Chem. 2013, 85, 10969 – 10976. [12] A. Nagy, A. Steinbrck, J. Gao, N. Doggett, J. A. Hollingsworth, R. Lyer, ACS Nano 2012, 6, 4748 – 4762. [13] S. Choi, R. M. Dickson, J. H. Yu, Chem. Soc. Rev. 2012, 41, 1867 – 1891. [14] H. Xu, Q. Li, L. H. Wang, Y. He, J. Y. Shi, B. Tang, C. H. Fan, Chem. Soc. Rev. 2014, 43, 2650 – 2661. [15] a) S. N. Baker, G. A. Baker, Angew. Chem. Int. Ed. 2010, 49, 6726 – 6744; Angew. Chem. 2010, 122, 6876 – 6896; b) J. H. Shen, Y. H. Zhu, X. L. Yang, C. Z. Li, Chem. Commun. 2012, 48, 3686 – 3699; c) H. T. Li, Z. K. Kang, Y. Liu, S.-T. Lee, J. Mater. Chem. 2012, 22, 24230 – 24253. [16] a) L. L. Li, J. Ji, R. Fei, C. Z. Wang, Q. Lu, J. R. Zhang, L. P. Jiang, J. J. Zhu, Adv. Funct. Mater. 2012, 22, 2971 – 2979; b) J. Peng, W. Gao, B. K. Gupta, Z. Liu, R. Romero-Aburto, L. Ge, L. Song, L. B. Alemany, X. Zhan, G. Gao, S. A. Vithayathil, B. A. Kaipparettu, A. A. Marti, T. Hayashi, J. J. Zhu, P. M. Ajayan, Nano Lett. 2012, 12, 844 – 849. [17] a) Z. S. Qian, J. J. Ma, X. Y. Shan, L. X. Shao, J. Zhou, J. R. Chen, H. Feng, RSC Adv. 2013, 3, 14571 – 14579; b) Z. S. Qian, J. J. Ma, X. Y. Shan, H. Feng, L. X. Shao, J. R. Chen, Chem. Eur. J. 2014, 20, 2254 – 2263; c) J. Zhou, X. Y. Shan, J. J. Ma, Y. M. Gu, Z. S. Qian, J. R. Chen, H. Feng, RSC Adv. 2014, 4, 5465 – 5468; d) Z. S. Qian, X. Y. Shan, L. J. Chai, J. J. Ma, J. R. Chen, H. Feng, ACS Appl. Mater. Interfaces 2014, 6, 6797 – 6805; e) X. Y. Shan, L. J. Chai, J. J. Ma, Z. S. Qian, J. R. Chen, H. Feng, Analyst 2014, 139, 2322 – 2325. [18] Z. S. Qian, X. Y. Shan, L. J. Chai, J. J. Ma, J. R. Chen, H. Feng, Nanoscale 2014, 6, 5671 – 5674. [19] P. Yu, X. M. Wen, Y. Toh, Yu. Lee, K. Huang, S. J. Huang, S. Shrestha, G. Conibeer, J. Tang, J. Mater. Chem. C. 2014, 2, 2894 – 2901.
Keywords: carbon nanotubes · graphene quantum dots · multiple DNA targets · sensors · simultaneous detection [1] D. A. Giljohann, C. A. Mirkin, Nature 2009, 462, 461 – 464.
Chem. Eur. J. 2014, 20, 16065 – 16069
www.chemeurj.org
Received: August 4, 2014 Published online on October 21, 2014
16069
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
& Nanotechnology | Hot Paper|
Simultaneous Detection of Multiple DNA Targets by Integrating Dual-Color Graphene Quantum Dot Nanoprobes and Carbon Nanotubes Zhaosheng Qian, Xiaoyue Shan, Lujing Chai, Jianrong Chen, and Hui Feng*[a] Abstract: Simultaneous detection of multiple DNA targets was achieved based on a biocompatible graphene quantum dots (GQDs) and carbon nanotubes (CNTs) platform through spontaneous assembly between dual-color GQDbased probes and CNTs and subsequently self-recognition between DNA probes and targets.
Developments in biomarker sensing methodologies are of great importance owing to their versatile potential applications in gene expression profiling for early diagnosis and treatment.[1] Notably, many diseases are associated with multiple biomarkers and the detection of a single biomarker may cause false diagnosis.[2] Simultaneous detection of multiple targets brings new opportunities for improving the accuracy of early disease detection over the single-marker assay. Moreover, simultaneous determination of multiple biomarkers within a single sample has many advantages over monoplex assays such as less sample requirement, lower cost per test, shorter analysis time, and fewer repetitions of tedious procedures. However, up to date, convenient and simultaneous detection of multiple biomarkers such as DNA and proteins with biocompatible materials and good analytical performance still remains a challenge. Several nanosensors have been reported to achieve separate detection of different targets. Most proceeding research focused on respective detection of multiplex targets or species with dye-labeled probes.[3–7] However, organic dyes as fluorophores in the probes have non-negligible disadvantages such as poor photostability, easy photobleaching, and small Stokes shifts. To overcome these problems, novel fluorescence platforms based on semiconductor QDs and AgNPs were recently established.[8–11] Nevertheless, the drawbacks of semiconductor QDs[12] and AgNPs[13] including high toxicity, relatively high cost and hard manipulation still do not enable them as excellent fluorophores of probes for biomolecules, which urges scientists to discover and exploit alternative fluorescent materials with good biocompatibility because the safety and biocompatibility [a] Dr. Z. Qian, X. Shan, L. Chai, Prof. J. Chen, Dr. H. Feng Department of Chemistry, Zhejiang Normal University 688 Yingbin Road, Jinhua, Zhejiang Province (P.R. China) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201404730. Chem. Eur. J. 2014, 20, 16065 – 16069
of used materials are also of great significance for the further application of constructed biosensors in cellular tissue or in vivo.[14] Graphene quantum dots (GQDs) have been used in photovoltaic devices, photocatalysis and biological imaging because of their distinctive optical and electronic properties.[15] With respect to conventional dye molecules and semiconductor QDs, GQDs have many superiorities such as stable light emitting, high quantum yield, good photostability, easy modulation, and excellent biocompatibility. To date, the synthesis and optical properties of GQDs have been extensively investigated and great progress was made in the development of facile preparations of GQDs with high and controllable fluorescence.[16] Our previous work showed that modified-GQDs with small organic compounds and heteroatom-doped GQDs possess strong fluorescence, low toxicity, and excellent biolabeling to human Hela cells.[17] Based on these doped GQDs, a multifunctional fluorescence sensing platform for selective detection of AgI, FeIII and hydrogen peroxide, glucose, and melamine were constructed. Recently, we employed GQDs as the fluorophore in the probe and graphene oxide as the platform to achieve highly sensitive and selective detection of a single DNA target for the first time.[18] In this communication, we report simultaneous detection of multiplex DNA targets with high sensitivity and selectivity based on a biocompatible GQDs and CNTs platform by taking advantage of intense and dual-color fluorescence of GQDs, efficient quenching capability of CNTs, specific recognition between probes and targets, and unique self-assembly between GQDs and CNTs. Dual-color GQDs were employed to synthesize blue probe (P1) and green probe (P2) for recognition of two DNA targets respectively. To realize simultaneous determination, two GQD-labeled probes were assembled on the CNTs surface at the same time. By the integration of their specific interactions, the dual-functional nanosensor assembled with CNTs and two probes succeeded for convenient and simultaneous detection of multiplex DNA through two cycles of fluorescence on-off-on for the first time. Scheme 1 displays the schematic illustration of simultaneous detection strategy for multiplex DNA targets through the simultaneous assembly of two probes on the CNTs surface and synchronous recognition between probes and targets. Simultaneous detections of two DNA targets are accomplished in the following procedure: In the first step, two probes are mixed with CNTs so that the probes can adsorb on the surface of CNTs through electrostatic attraction and p–p stacking interactions, where assembly of (P1 and P2)/CNTs occurs. The formation of the corresponding
16065
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication
Figure 1. TEM (A) and HRTEM (B) images of ssDNA-eGQDs (P1) and ssDNApGQDs (P2) adsorbed on CNTs.
Scheme 1. Schematic illustration of the proposed detection strategy for simultaneous detection of multiplex DNA targets. P1: ssDNA-eGQDs (DNA probe 1), P2: ssDNA-pGQDs (DNA probe 2), T1: DNA target 1, T2: DNA target 2, P1/T1: dsDNA-eGQDs, P2/T2: dsDNA-pGQDs.
assembly leads to substantial fluorescence quenching of the original probe through static quenching between probes and CNTs. It has been proven that efficient static quenching between GQDs and CNTs occurs and subsequently leads to effective fluorescence quenching of GQDs.[19] In the second step, upon the addition of DNA target 1 (T1) and DNA target 2 (T2), T1 and T2 can hybridize with P1 and P2 in the assembly (P1 + P2)/CNTs to produce double-stranded DNA assembly P1/T1 and P2/T2 through their corresponding base pairing. The formation of double-stranded DNA assembly breaks up the electrostatic attraction and p–p stacking interaction between the probes and CNTs, and thus liberates double-stranded DNA assemblies from CNTs. The free double-stranded DNA assemblies P1/T1 and P2/T2 re-emit blue and green fluorescence, respectively, and lead to gradual recovery of the two fluorescences upon addition of two DNA targets. Highly fluorescent 1,2-ethylenediamine-functionalized GQDs (eGQDs) and pristine GQDs (pGQDs) were prepared and well characterized in our previous paper.[17] eGQDs and pGQDs exhibit blue and green fluorescence, respectively, and thus they are used as fluorophores for P1 and P2 probes, respectively. eGQDs possess a great deal of NH2 groups, while pGQDs are rich in the COOH group.[17] Through the condensation reaction with the same reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), eGQDs react with COOH-functionalized DNA (ssDNA) to form a ssDNA-eGQDs probe (P1) bonded with a CO NH group, whereas pGQDs bind with NH2-functionalized DNA (ssDNA) to produce ssDNA-pGQDs probe (P2) through CO NH group. TEM images in Figure 1 clearly show that the two probes are adsorbed on the surface of multiwalled carbon nanotubes (MWCNTs), and their size distribution falls in the range of 3–5 nm. P1 and P2 present blue and green fluorescence under UV lamp as shown in Figure 2, inset. Normalized fluorescence spectra illustrate that P1 shows intense fluorescence emission centered at l = 414 nm, whereas the P2 probe Chem. Eur. J. 2014, 20, 16065 – 16069
www.chemeurj.org
Figure 2. Fluorescence spectra of eGQDs, ssDNA-eGQDs (P1), dsDNA-eGQDs (P1/T1), pGQDs, ssDNA-pGQDs (P2), and dsDNA-pGQDs (P2/T2). Inset: the fluorescence images of ssDNA-eGQDs (P1) and ssDNA-pGQDs probes (P2).
strongly emits yellowlish–green light at around l = 514 nm when they are excited by their own optimal excitation light. The large gaps in fluorescence emission between the probes and their corresponding GQDs provide solid evidence for successful binding link between GQDs and DNA sequence, that is, successful preparation of the probes. It is worth noting that eGQD-labeled probe P1 separates well in fluorescence emission from the pGQD-labeled probe P2. This good separation in fluorescence emission enables the subsequent accomplishment of simultaneous detection of two DNA targets. After addition of DNA target 1 (T1), the fluorescence emission is shifted to l = 438 nm, which is from the assembly P1/T1 due to their self-recognition through base paring. However, no apparent shift in fluorescence emission can be observed for the P2/T2 assembly, which is consistent with the observation in our previous report. The large difference in the time-resolved decay curve between P2 and P2/T2 in Figure S1 (Supporting Information) offers the proof for the formation of P2/T2 assembly with addition of T2 into P2 solution. Moreover, it should be noted that the high quantum yields of probes shown in Table S1 (Supporting Information) has been attained due to high emission efficiency from eGQDs or pGQDs, which endows the probes with a high sensitivity of the assembled sensors.
16066
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication A dual-functional biosensor based on two-colored probes and CNTs was further constructed to explore simultaneous detection of two DNA targets. Here we used synchronous fluorescence spectroscopy to differentiate the respective emission of the mixed probes because of the broad emission band of the probes. The two probes show an apparently distinct fluorescence emission peaks at l = 364 and 440 nm, respectively, with a Dl of 60 nm in the synchronous spectra, which enables simultaneous detection of two DNA targets. After the addition of 3.48 mg mL 1 of CNTs into the mixed probes system, the two probes were assembled onto the surface of CNTs at the same time. The formation of the assembly (P1 and P2)/CNTs resulted in substantial fluorescence quenching due to static quenching between probes and CNTs. Figure 3 shows a similarly strong quenching effect of CNTs to both probes, and the presence of 3.48 mg mL 1 of CNTs leads to a reduction of more than 60 % in intensity. The formation of the assembly (P1 + P2)/CNTs was also verified by the TEM images shown in Figure 1, which demonstrates that a great deal of GQDs-labeled probes are adsorbed on the surface of CNTs as the assembly. Condition optimization including amounts of added CNTs, the incubation time for fluorescence quenching and recovery were carried out. According to Figure 3A and Figures S3 and S4 (Supporting Information), 3.48 mg mL 1 of CNTs and 2 and 40 min mixing for quenching and recovery were chosen to assess the performance of the dual-functional biosensor in quantitative analysis of T1 and T2 at the same time. Figure 3B
shows a gradual increase in fluorescence intensity of two types of dsDNA-GQDs with the concentration of both T1 and T2 up to 140.0 nm. The fluorescence enhancement can be attributed to the increase of fluorescent P1/T1 and P2/T2 in amount due to hybridization between probe DNA and target DNA. Figure 3C displays the linear relationship of fluorescence intensity to concentration of T1 from 6.9 to 80.0 nm, and this equation can be expressed as y = 1.23x + 215.2, in which R2 = 0.994. Its detection limit is estimated as 4.2 nm from derived calibration curve ( 3 standard deviations). The method for T2 in Figure 3D has a broad detection range of 8.2–100 nm with a detection limit of 3.6 nm, and this equation can be expressed as y = 0.80x + 191.2, in which R2 = 0.999. The detection limits for T1 and T2 are comparable to those of DNA sensors for single detection (0.1–1.0 nm),[5,6,9,18] which indicates that this dualfunctional biosensor does not lose its high sensitivity while largely expanding its detection scope. Furthermore, the selectivity of this nanosensor was also assessed with target DNA and mismatched DNA sequences as shown in Figure 4. The presence of T1 and T2 leads to respective fluorescence recovery of P1 and P2, but does not have an influence on each other. The mismatched DNA with one, two, and three bases with respect to T1 shows nearly no fluorescence response to this sensor, whereas the fluorescence intensity doubles relative to the blank with the presence of T1. The high selectivity is consistent with our previous results.[18] To demonstrate the feasibility of the practical application of the proposed method, we detected the target DNA in real samples (Bovine serum). Before analysis, the 100-fold diluted serum sample was spiked with 2.0 mL of target DNA (T1) and target DNA (T2), and then added into the ssDNA-eGQDs and ssDNA-pGQDs probe solution which has been quenched by CNTs, respectively. After incubation for 60 min at room temperature, the fluorescence intensity of the resulting solutions was measured. As shown in Figure S5 (Supporting Information), comparable responses were obtained for the detection of target DNA in real samples with those results in buffer, which indicated the potentiality of the proposed method for real biological sample analysis. In summary, the present study has introduced biocompatible carbon-based materials to assemble multifunctional biosenFigure 3. A) Fluorescence quenching spectra of P1 and P2 with various concentrations of CNTs (0, 0.29, 0.58, 0.87, sors for the simultaneous detec1 1.16, 1.45, 1.74, 2.32, 2.90, 3.48, 4.06, 5.22 mg mL ). B) Fluorescence recovery of the (P1 + P2)/CNTs system after intion of multiplex DNA. Benign cubation with various concentrations of DNA targets (T1 and T2) (0, 20.0, 40.0, 60.0, 80.0, 100.0, 120.0, 140.0 nm). GQDs and CNTs were used as C,D) Linear response in fluorescence to DNA target 1 and 2. Chem. Eur. J. 2014, 20, 16065 – 16069
www.chemeurj.org
16067
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication Synthesis of 1,2-ethylenediamine functionalized graphene quantum dots (eGQDs)
Figure 4. Fluorescence intensity changes (F/F0) of the aptasensor in the presence of T1, T2, mT1, mT2, and mT3 (100.0 nm) in buffer.
fluorophores in two-colored probes and platforms respectively. High emission efficiency of GQDs guarantees high sensitivity of the constructed biosensors, and good biocompatibility provides promising opportunities for further utilization of the biosensors in vivo. This dual-functional biosensor assembled with two GQDs-labeled probes and CNTs platform possesses the capability to simultaneous and quantitative determination of two DNA targets with a broad linear range and low detection limit, which can serve as a general detection model for synchronous detection of multiple species in complex systems.
Experimental Section Materials and reagents Triplex distilled water was used in the whole experimental process and to prepare PBS buffers. Graphite powder, sodium borohydride, tetrahydrofuran, 1,2-ethylenediamine, thionyl dichloride, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and N-hydroxylsuccinimide sodium salt (NHS) were purchased from Aladdin Ltd. (Shanghai, China). Phosphate buffer solution (PBS) was prepared by mixing stock solutions of NaH2PO4 and Na2HPO4. All reagents were of analytical grade and without any further purification. DNA oligonucleotides were purchased from Shanghai Sangon Biotechnology (Shanghai, China). Their sequences are listed as follows: Capturing ssDNA sequence in probes: P1 = 5’COOH-CTG ATT ACT ATT GCA TGA GGC CTT-3’, P2 = 5’-NH2-TTG GTG AAG CTA ACG TTG AGG-3’; target ssDNA sequence (tDNA: perfectly matched with capturing ssDNA) T1 = 5’-AAG GCC TCA TGC AAT AGT AAT CAG-3’, T2 = 5’-CCT CAA CGT TAG CTT CAC CAA3’. Single-base mismatched ssDNA sequence (mT1): mT1 = 5’-CCT CAA CGT TCG CTT CAC CAA-3’; two-base mismatched ssDNA sequence (mT2): mT2 = 5’-CCT CAT CGT TCG CTT CAC CAA-3’; threebase mismatched ssDNA sequence (mT3): mT3: 5’-CCT CAT CGT TCG CTT CTC CAA-3’.
Synthesis of oxidized carbon nanotubes (CNTs) Briefly, MWNTs (1.0 g) were oxidized with HNO3/H2SO4 (100 mL, 1:3, v/v) in an ultrasonic bath for 24 h. After repetitive washing with water to neutral pH, the resulted CNTs were dried in the vacuum drying oven at 60 8C for 24 h. Chem. Eur. J. 2014, 20, 16065 – 16069
www.chemeurj.org
The preparation of eGQDs was described in our previous paper in detail.[17] Graphite powder (0.3 g) was added into a mixture of concentrated sulfuric acid (180 mL) and nitric acid (60 mL). The solution was sonicated for 2 h and heated at 80 8C for 24 h. The mixture was cooled and diluted with deionized water (800 mL). The dark-brown GQDs solution was neutralized with sodium carbonate. The final product solution was further dialyzed in a dialysis bag (1000 Da) for 3 days. A mixture of as-prepared GQDs (0.1 g) and SOCl2 (20 mL) reacted for 2 h at 80 8C, and then vacuum-distillation was carried out to remove excessive SOCl2. 1,2-Ethylenediamine (20 mL) was added into the chlorinated GQDs and the mixture was heated at 100 8C for 4 h. The excessive 1,2-ethylenediamine of the resulting mixture was removed by vacuum-distillation, and the residue was washed with ethanol several times.
Synthesis of pristine graphene quantum dots (pGQDs) Carbon fiber (1.0 g) was added into a mixture of concentrated sulfuric acid (180 mL) and nitric acid (60 mL). The solution was sonicated for 2 h and heated at 120 8C for 24 h. The mixture was cooled and diluted with deionized water (800 mL). The dark-brown GQDs solution was neutralized with sodium carbonate. The final product solution was further dialyzed in a dialysis bag (1000 Da) for 3 days.
Preparation of the ssDNA-eGQDs probe (P1) and ssDNA– pGQDs probe (P2) The obtained eGQDs were first dissolved in PBS solution (10.0 mL, 10 mm, pH 7.4), and then EDC (80.0 mg) and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) (80.0 mg) were added to the proceeding solution at room temperature with continuous stirring. After 30 min activation, capturing ssDNA (DNA 1 in P1) (15 mL, 100.0 mm) was added into the proceeding solution. The condensation reaction was allowed for 24 h at room temperature with stirring. The DNA probes were obtained by the following centrifugation and washing with PBS buffer. For the preparation of ssDNApGQDs probe (P2), as-prepared pGQDs were first dissolved in phosphate buffer (5.0 mL, 10 mm, pH 7.0), and then the solution was adjusted to pH 5 for protonation of carboxyl groups of pGQDs with dilute hydrochloric acid. After that, EDC (80.0 mg) and sulfoNHS (80.0 mg) were added into the proceeding solution to activate the carboxylic group of pGQDs for 30 min at room temperature with stirring. Capturing ssDNA (DNA 2 in P2) (20.0 mL, 100.0 mm) was subsequently added into the above solution, and the reaction was allowed to process for 24 h. Finally, the ssDNA-pGQDs probe was attained by centrifugation and washing with PBS buffer.
Simultaneous detection of two DNA targets ssDNA-eGQDs probe (1.5 mL) and the ssDNA-pGQDs probe (400.0 mL) were mixed, and then the mixture was diluted with PBS to a volume of 500.0 mL. CNTs (6.0 mL, 0.29 mg mL 1) was introduced into the resultant mixture at room temperature to quench fluorescence with 2 min of incubation. Then, different concentrations of DNA target 1 (T1) (0, 20, 40, 60, 80, 100, 120, 140 nm) and DNA target 2 (T2) (0, 20, 40, 70, 100, 130, 160, 190 nm) were added into the above solution to initiate the fluorescence recovery. After incubation for 40 min at room temperature, the fluorescence intensity of resulting solutions was measured by using synchronous
16068
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Communication fluorescence ranging from 260 to 600 nm with the constant value of the Dl = 60 nm.
Target DNA detection in bovine serum sample Bovine serum sample was diluted 100-fold with PBS solution (10 mm, pH 7.4) before analysis. ssDNA-eGQDs probe (30.0 mL) or the ssDNA-pGQDs probe (200.0 mL) was diluted with the 100-fold diluted serum sample to a volume of 1.0 mL. CNTs (2.0 mL, 0.29 mg mL 1) was introduced into the resultant mixture at room temperature to quench fluorescence with 2 min of incubation. Then, target DNA (T1) (2.0 mL) was added into the above ssDNAeGQDs probe solution and target DNA (T2) (2.0 mL) was added into the above ssDNA–pGQDs solution to initiate the fluorescence recovery, respectively. After incubation for 60 min at room temperature, the fluorescence intensity of resulting solutions was measured using fluorescence spectrometer.
Characterization methods The morphologies of all samples were characterized by TEM, which was performed on a JEOL-2100F instrument with an accelerating voltage of 200 kV. Samples were prepared by dropping aqueous suspensions of the separated fractions of samples onto Cu TEM grids coated with a holey amorphous carbon film and following solvent evaporation in a dust protected atmosphere. The UV/Vis spectra were recorded on a Perkin–Elmer Lambda 950 spectrometer, in which the sample was dispersed in water after ultrasonication for 30 min. The photoluminescence spectra were conducted on a Perkin–Elmer LS-55 fluorescence spectrometer, and lifetimes were determined by using a FLS920 fluorescence spectrophotometer.
Acknowledgements We are thankful for the support by the National Natural Science Foundation of China (No. 21405142, 21005073 and 21275131) and Zhejiang Province (No. LY13B050001, LQ13B050002 and ZJHX201403).
[2] D. Sidransky, Science 1997, 278, 1054 – 1058. [3] R. H. Yang, Z. W. Tang, J. L. Yan, H. Z. Kang, Y. M. Kim, Z. Zhu, W. H. Tan, Anal. Chem. 2008, 80, 7408 – 7413. [4] J. Chen, Y. Huang, M. Shi, S. L. Zhao, Y. C. Zhao, Talanta 2013, 109, 160 – 166. [5] S. J. He, B. Song, D. Li, C. F. Zhu, W. P. Qi, Y. Q. Wen, L. H. Wang, S. P. Song, H. P. Fang, C. H. Fan, Adv. Funct. Mater. 2010, 20, 453 – 459. [6] M. Zhang, B. C. Yin, W. H. Tan, B. C. Ye, Biosens. Bioelectron. 2011, 26, 3260 – 3265. [7] N. Li, C. Y. Chang, W. Pan, B. Tang, Angew. Chem. Int. Ed. 2012, 51, 7426 – 7430; Angew. Chem. 2012, 124, 7544 – 7548. [8] Q. S. Mei, Z. P. Zhang, Angew. Chem. Int. Ed. 2012, 51, 5602 – 5606; Angew. Chem. 2012, 124, 5700 – 5704. [9] X. Q. Liu, F. Wang, R. Aizen, O. Yehezkeli, I. Willner, J. Am. Chem. Soc. 2013, 135, 11832 – 11839. [10] L. Chen, X. W. Zhang, G. H. Zhou, X. Xiang, X. H. Ji, Z. H. Zheng, Z. K. He, H. Z. Wang, Anal. Chem. 2012, 84, 3200 – 3207. [11] H. Zhang, L. Zhang, R. P. Liang, J. Huang, J. D. Qiu, Anal. Chem. 2013, 85, 10969 – 10976. [12] A. Nagy, A. Steinbrck, J. Gao, N. Doggett, J. A. Hollingsworth, R. Lyer, ACS Nano 2012, 6, 4748 – 4762. [13] S. Choi, R. M. Dickson, J. H. Yu, Chem. Soc. Rev. 2012, 41, 1867 – 1891. [14] H. Xu, Q. Li, L. H. Wang, Y. He, J. Y. Shi, B. Tang, C. H. Fan, Chem. Soc. Rev. 2014, 43, 2650 – 2661. [15] a) S. N. Baker, G. A. Baker, Angew. Chem. Int. Ed. 2010, 49, 6726 – 6744; Angew. Chem. 2010, 122, 6876 – 6896; b) J. H. Shen, Y. H. Zhu, X. L. Yang, C. Z. Li, Chem. Commun. 2012, 48, 3686 – 3699; c) H. T. Li, Z. K. Kang, Y. Liu, S.-T. Lee, J. Mater. Chem. 2012, 22, 24230 – 24253. [16] a) L. L. Li, J. Ji, R. Fei, C. Z. Wang, Q. Lu, J. R. Zhang, L. P. Jiang, J. J. Zhu, Adv. Funct. Mater. 2012, 22, 2971 – 2979; b) J. Peng, W. Gao, B. K. Gupta, Z. Liu, R. Romero-Aburto, L. Ge, L. Song, L. B. Alemany, X. Zhan, G. Gao, S. A. Vithayathil, B. A. Kaipparettu, A. A. Marti, T. Hayashi, J. J. Zhu, P. M. Ajayan, Nano Lett. 2012, 12, 844 – 849. [17] a) Z. S. Qian, J. J. Ma, X. Y. Shan, L. X. Shao, J. Zhou, J. R. Chen, H. Feng, RSC Adv. 2013, 3, 14571 – 14579; b) Z. S. Qian, J. J. Ma, X. Y. Shan, H. Feng, L. X. Shao, J. R. Chen, Chem. Eur. J. 2014, 20, 2254 – 2263; c) J. Zhou, X. Y. Shan, J. J. Ma, Y. M. Gu, Z. S. Qian, J. R. Chen, H. Feng, RSC Adv. 2014, 4, 5465 – 5468; d) Z. S. Qian, X. Y. Shan, L. J. Chai, J. J. Ma, J. R. Chen, H. Feng, ACS Appl. Mater. Interfaces 2014, 6, 6797 – 6805; e) X. Y. Shan, L. J. Chai, J. J. Ma, Z. S. Qian, J. R. Chen, H. Feng, Analyst 2014, 139, 2322 – 2325. [18] Z. S. Qian, X. Y. Shan, L. J. Chai, J. J. Ma, J. R. Chen, H. Feng, Nanoscale 2014, 6, 5671 – 5674. [19] P. Yu, X. M. Wen, Y. Toh, Yu. Lee, K. Huang, S. J. Huang, S. Shrestha, G. Conibeer, J. Tang, J. Mater. Chem. C. 2014, 2, 2894 – 2901.
Keywords: carbon nanotubes · graphene quantum dots · multiple DNA targets · sensors · simultaneous detection [1] D. A. Giljohann, C. A. Mirkin, Nature 2009, 462, 461 – 464.
Chem. Eur. J. 2014, 20, 16065 – 16069
www.chemeurj.org
Received: August 4, 2014 Published online on October 21, 2014
16069
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
