J. Phys. Chem. C 2008, 112, 9891–9895
9891
Label-Free Electronic Detection of DNA Using Simple Double-Walled Carbon Nanotube Resistors Xiaochen Dong,*,†,‡ Dongliang Fu,† Yanping Xu,† Jinquan Wei,| Yumeng Shi,† Peng Chen,§ and Lain-Jong Li*,† School of Materials Science and Engineering, Nanyang Technological UniVersity, 50 Nanyang AVenue, Singapore, 637819, Institute of AdVanced Materials (IAM), Nanjing UniVersity of Posts and Telecommunications, 9 Wenyuan Road, Nanjing 210046, China, School of Chemical and Biomedical Engineering, Nanyang Technological UniVersity, 50 Nanyang AVenue, Singapore, 637819, and Department of Mechanical Engineering, Key Laboratory for AMMPT of Education Ministry, Tsinghua UniVersity, Beijing 100084, China ReceiVed: December 31, 2007; ReVised Manuscript ReceiVed: March 27, 2008
Label-free electrical detection of DNA hybridization using resistive sensing devices based on drop-casted doubled-walled carbon nanotube (DWNT) networks and as-grown films is presented. Sensitive, selective, and reliable detection of DNA hybridization is achieved by simple two-terminal measurements, which is suitable for the hand-held type of device applications. Our approach is able to detect DNA concentrations ranging from 50 to 500 nM. Utilizing location-selective capping by photoresists, we designed experiments to reveal the sensing mechanism and found that the carbon nanotube (CNT)-metal junction may not play a dominant role in sensing as it does in single-walled carbon nanotube-based transistor devices. A chargetransfer model is proposed to explain the detection mechanism. Introduction The development of label-free and sequence-selective detection of DNA based on electrochemical1–3 or electroluminescence methods4,5 and direct electrical detection using field-effect transistors (FETs) has become a subject of intense research.6–11 Carbon nanotubes (CNTs) have attracted much attention in this endeavor because their conductance is highly sensitive to the molecular adsorption on tube walls12 or within tubes13 as a result of their unique electronic structures. In addition, CNTs are biocompatible and chemically stable in ambient environment. Therefore, there is a growing body of research on the development of CNT-based devices for DNA sensing. Star et al. reported the detection of DNA immobilization and hybridization using FETs made from single-walled carbon nanotube (SWNT) networks.14 Additionally, a charge transfer mechanism underlying the detection was proposed by the authors, in which the drain currents were thought to be altered by the adsorption of electron-donating groups (for example, amino groups in DNA molecules) and subsequent changes in the charge carrier density in SWNTs. Similar transduction mechanisms have also been reported in the experiments utilizing semiconducting CNTs as chemical sensors.12,15,16 But alternatively, several studies on the electronic sensing mechanism of SWNT devices suggested that the DNA-molecule-induced modulations at metal-CNT junctions rather than to the channel conductance actually play dominant roles in FETs based on either individual SWNTs17,18or SWNT networks.19 This theory is consistent with the Schottky
barrier model,20 highlighting that responses from semiconducting CNTs originates from the change of the Schottky barrier at metal-CNT contact.18 So far, most of CNT sensors are based on semiconducting CNT-transistors. We hereby, for the first time, report a simple resistive sensing approach for label-free electrical detection of DNA based on metallic CNT networks. This type of detector provides advantages over conventional CNT transistors, such as low-cost, solution-based, room temperature and ink-jet compatible process, and feasibility on flexible substrates. The catalytic chemical vapor deposition methods for high throughput production (∼0.5 g/h) of double-walled carbon nanotubes (DWNTs) or as-grown film-type DWNTs have been documented.21–23 Previous theoretical calculations show that the band structure of the DWNT outer tube is not significantly affected by the inner tube,24 and the electronic properties of an individual DWNT is essentially determined by its outer tube. In the approach presented here, simple two-terminal resistive devices based on drop-cast DWNT networks and separately asgrown DWNT films are used for electrical detection of DNA immobilization and hybridization. DWNTs are used because they can be easily fabricated as good resistors. Our method is highly sensitive to discriminate one-base-pair mismatch DNA, and is suitable for the development of low-power and low-cost hand-held DNA sensor devices. The possible sensing mechanism is explored using location-selective capping. The results can be explained by a charge-transfer model. Experimental Section
* Correspondingauthor.E-mail:
[email protected](X.D.);
[email protected] (L.-J.L.). † School of Materials Science and Engineering, Nanyang Technological University. ‡ Nanjing University of Posts and Telecommunications. § School of Chemical and Biomedical Engineering, Nanyang Technological University. | Tsinghua University.
Materials and Solutions. The raw DWNT samples, containing several to tens of thin layers, were prepared by a chemical vapor deposition method.23 The sequences of the DNA strands used in the experiments are as follows: probe DNA: 5′-AGGTCG-CCG-CCC-(CH2)3-NH2; target DNA: 5′-GGG-CGG-CGACCT-TTT-TTT-TTT-TT; single base mismatched DNA: 5′-
10.1021/jp7121714 CCC: $40.75 2008 American Chemical Society Published on Web 06/10/2008
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Figure 1. (a) Schematic illustration of the structure of DWNT network devices. (b, c) TEM image of purified DWNTs and AFM image of DWNTs network in the device channel area, (d) Id-Vd curves of the DWNT resistors immobilized with probe-DNA, blocked with PEG, and hybridized with target DNA. (e) Id-Vd curves of the DWNT resistors immobilized with probe DNA, blocked with PEG, and hybridized with single-base mismatched DNA (device channel length ) 40 µm, DNA concentration was 500 nM).
Figure 2. Comparison of electrical sensing characteristics for hybridization with target and one-base mismatched DNA for DWNT resistor devices. The solid squares and circles represent the statistical percentage decreases of Id versus the concentration of target DNA and one-base mismatched DNA, respectively. The dash line represents the result (4.1%) of noncDNA (each point was obtained by averaging the results from six separate devices).
GTG-CGG-CGA-CCT-TTT-TTT-TTT-TT; noncDNA: 5′-AGGTCG-CCG-CCC. Tris-ethylenediaminetetraacetic acid (TrisEDTA) buffer solution for DNA immobilization contains 10 mM tris-HCl, 1.0 mM EDTA, and 0.10 M NaCl. Phosphate buffered saline (PBS, pH 7.4) solution for DNA hybridization contains 0.25 M NaCl and 10 mM phosphate buffer. All solutions were prepared with Milli-Q water (18 MΩ · cm) from a Millipore system. Fabrication of Carbon Nanotube Resistors. The as-grown DWNT films were treated in H2O2 and HCl solution and subsequently rinsed with distilled water to remove the impurities of catalytic particles and amorphous carbon. After drying, the DWNTs were suspended in 1 wt % of sodium dodecyl benzene sulfonate (SDBS) aqueous solution using tip-sonication followed by ultracentrifugation at 140 000 g-force. The supernatant was
then drop-casted on SiO2/Si substrate between two thermalevaporated electrodes (illustrated in Figure 1), followed by water rinsing. The metallic electrodes were composed of 1 nm of Ti as a glue layer and 10 nm of Au on top of the Ti. The channel lengths of the devices were 20, 30, and 40 µm, while the channel width was fixed at 400 µm. For the resistors based on DWNT films, the purified DWNT film was suspended in water/ethanol solution to be fully expanded into an ultrathin and floated film,25 and then transferred to a SiO2/Si substrate with prepatterned gold electrodes. DNA Immobilization and Hybridization. It has been reported that DNA can be physically absorbed onto the surface of CNTs.14 We use the same strategy to decorate our CNT resistors with probe DNA. It is noted that some of the probe DNA can be securely attached to the DWNT surface and those loosely bound to the surface will be removed by rinsing. To immobilize probe DNAs, the CNT resistors were immersed in Tris-EDTA buffer solution containing 1 µM probe DNA for a period of 16-24hrs. The samples were then rinsed with TrisEDTA buffer to remove the weakly bound DNA, and were blow-dried before electrical characterization. To avoid nonspecific adsorption of target DNAs, the CNT devices were immersed in an 80% poly(ethylene glycol) (PEG, molecular weight 380-420) aqueous solution for 12 h after DNA immobilization to block the uncovered CNT surface. The devices were then rinsed with water and followed by blow drying. Ten microliter target DNA solutions were pipetted onto the DNA decorated resistors for 1 h to allow hybridization. Finally, the device was washed and dried for analyses. Electrical Characterization and Electrical Measurements. Transmission electronic microscopy (TEM) and atomic force microscopy (AFM) were used to characterize the DWNTs and DWNT films. A confocal Raman microscope (WITec CRM200) equipped with a 488 nm laser was used in this study for locationselective exposure of positive photoresists (EPG510). All
Detection of DNA Using DWNT Resistors
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Figure 3. (a) Id-Vd curves of as-grown DWNT film resistors immobilized with probe DNA, blocked with PEG, and hybridized with target DNA (channel length ) 40 µm, DNA concentration was 200 nM). (b) Optical and (c) AFM images of the DWNT film in the device channel area.
Figure 4. Electrical sensing characteristics of resistor devices made from arc-discharge-produced SWNTs. The solid squares and empty circles represent the statistical percentage decreases of Id versus the concentration of target DNA and single-base mismatched DNA, respectively. (Each point was obtained by averaging the results from six separate devices.)
electrical measurements were carried out in ambient environment using a Kiethley semiconductor parameter analyzer, model 4200SCS. Results and Discussion Figure 1a schematically illustrates the structure of a DWNT network device. A representative TEM image of purified DWNTs and an AFM image for the DWNTs networks in the device channel area are depicted in Figure 1b,c, respectively. It is evident from the drain current (Id) versus drain voltage (Vd) relation shown in Figure 1d that the electrical characteristics of the DWNT network are essentially ohmic. The corresponding changes of Id after immobilization of probe DNAs, PEGblocking, and hybridization with target DNAs are also shown in Figure 1d. The results indicate that Id is decreased after immobilization with probe-DNA. And such reduction usually is slightly recovered by the subsequent PEG-blocking. PEG has been previously used as a suppressant for protein nonspecific binding (NSB) on CNT surfaces.26 These molecules are expected to modify the CNT bare surfaces (places not occupied by the probe-DNA) to hydrophilic and charge-neutral states, thereby eliminating hydrophobic interactions and electrostatic binding
with DNA molecules.27 The increase in Id after PEG-blocking could be attributed, at least partly, to competitive binding of PEG to CNTs with nonstably immobilized probe-DNAs. The DNA hybridization was electrically reported as the percentage decrease in Id from “PEG-blocked” to “target DNA-hybridized′ states. In Figure 1d, the hybridization with 500 nM of target DNA results in about 44.4 % of Id decrease in DWNT network resistors. In contrast, in a parallel experiment demonstrated in Figure 1e, the introduction of single-base mismatched DNA strands only caused about 14.2 % decrease in Id. The relatively large reduction of Id produced by the target DNA is believed to be due to more successful capturing of target DNA as the result of sequence-specific binding between probe- and target-DNA molecules. To further verify that the DWNT resistive sensor is able to differentiate perfectly complementary and one-base mismatched DNA molecules, we investigated the dose responses of drain current (Id) to various DNA concentrations. Figure 2 shows the statistics of the percentage Id decrease versus the concentrations of target and single-base mismatched DNA, respectively, where each point was obtained by averaging the results from six devices (with various channel lengths and thus resistances that range from 104 to 106 Ω). It is noteworthy that the electrical differentiation was achieved without any particular controls in the device channel length or the resistance. As expected, higher DNA concentrations lead to higher percentage decrease of Id. When the target DNA concentration is 500 nM, the Id reduction is as large as about 44%. At a low concentration (50 nM), perfectly matched target DNA is still able to make an appreciable change (∼10.5%) in Id. By contrast, when single-base mismatched DNA was tested, the same DNA concentration range (50-500 nM) was only able to produce small changes in Id from 6.1 to 14.9%. On the contrary, the noncomplementary strand DNA at 50 nM only results in about 4.1% decrease in Id. The detection limit (for reliable differentiation) is around 50 nM. We also have explored the possibility of using purified asgrown DWNT films for similar electrical detection of DNA hybridization. Figure 3a demonstrates the sensing performance of DWNT films (resistance ∼140 Ω), while the optical and AFM images for the as-grown films are shown in Figure 3b,c. It is observed that the decrease in Id is also measurable (∼13.9%)
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Figure 5. Id-Vd curves for photoresist-capped DWNT resistors with (a) one junction exposed and (b) a channel exposed before immobilization, after immobilization, and upon hybridization with its cDNA (device channel length ) 40 µm, DNA concentration was 200nM). The inset for each graph shows the exposed photoresist pattern under an optical microscope.
after the hybridization of target DNA at a concentration of 200 nM, indicating good DNA sensing capabilities. The electrical detection capability of metallic networks is not limited to DWNTs. We found that resistive network devices formed by arc-discharge-produced SWNTs29 can also serve as bioelectric sensors. As shown in Figure 4, SWNT resistors are also able to discriminate the perfectly complementary and single-base mismatched DNA sequences. The detection concentration limit for this type of resistors is around 100 nM, comparable to that of DWNT resistors (50 nM). As discussed in our previous work, the mechanism for electrical detection of DNA hybridization in semiconducting SWNT network devices is likely due to the modification of junction barrier energy,18,28 whereas the conductance change from a nanotube-nanotube junction or the nanotubes themselves could be less significant than that from a nanotube-metal junction. However, the transduction mechanism in the case of metallic networks is still not clear. Selective capping using photoresists has been used to study the detection mechanism of CNT based sensors.18,30,31 Here we perform the area-selective photoresist capping on the Au-contacted DWNT resistors to selectively examine the contributions from the junction or the channel in device sensing performance. The DWNT resistors were covered with a positive photoresist, and the desired patterns (channel exposed or junction exposed) were realized by optical lithography using a confocal microscope equipped with a 488 nm laser as the light source (focused to a spot size around 1 µm) for photoresist exposure, followed by standard photoresist stripping and washing. The width of the exposed area was kept at ∼20 µm. Because of the photoresist dissolve in PEG solution, these experiments are measured without PEG blocking. The experiments results are show in Figure 5. The percentage of Id decrease for the channel-exposed pattern is larger than that of the junction-exposed pattern for DWNT resistor devices, which is significantly different from the reported results for SWNT transistors, where the Id decrease in junction-exposed SWNT transistor devices was significantly larger than that in channelexposed ones. These results imply that the modulation of the CNT-metal junction in DWNT resistor devices is not as significant as that in transistor-type SWNT devices, probably because the DWNTs we prepared are very metallic and therefore the CNT-metal contact here is ohmic (i.e., the energy barrier between CNTs and metallic electrodes is low). It is likely that the interface between Au and DWNTs may not be sensitive to the DNA absorption, and therefore the channel conductance
change becomes dominant in the DWNT resistors. Recently, Lee et al. reported that the charge transfer from gas molecule to SWNT is the dominant mechanism in the detection of nerve agents when metallic SWNTs are used as sensors.30 The electrical detection of DNA hybridization observed here could be explained by the similar charge transfer mechanism, where the electron-rich group (NH2) present in DNA may donate part of the electron density to CNTs14 and reduce the number of majority carriers in CNTs and hence the conductivity. Conclusions In summary, the label-free electrical detection of DNA hybridization using the resistor devices based on drop-cast DWNTs and as-grown DWNT films has been realized. Easy electrical discrimination between perfectly cDNAs and one-basepair mismatched DNAs is achieved, with the detection limit of 50 nM. Similar performance has also been demonstrated with SWNT metallic networks. Although the detection concentration from this approach is not as competitive as transistor sensors made of semiconduting SWNT networks (whose detection limit is several order of magnitudes lower), the metallic network devices based on simple fabrication processes and detection schemes are advantageous in cost-efficiency, reproducibility, and scalability. It has great potentials in onsite low-cost diagnostics. This study also suggests a previously ignored sensing mechanism resulting from charge-transfer between target biomolecules and CNTs. Acknowledgment. We acknowledge with thanks the support from Nanyang Technological University (Singapore) RG32/06, MINDEF fund, and A*Star grant (#072 101 0020). References and Notes (1) Zhang, J.; Song, S. P.; Zhang, L. Y.; Wang, L. H.; Wu, H. P.; Pan, D.; Fan, C. H. J. Am. Chem. Soc. 2006, 128, 8575. (2) Ma, Y. F.; Ali, S. R.; Dodoo, A. S.; He, H. X. J. Phys. Chem. 2006, 110, 16359. (3) Drummond, T. G.; Hill, M. G.; Barton, G. K. Nat. Biotechnol. 2003, 21, 1192. (4) Ho, Y. P.; Kung, M. C.; Yang, S.; Wang, T.H. Nano Lett. 2005, 9, 1693. (5) Zhao, X. J.; Dytioco, R. T.; Tan, W. H. J. Am. Chem. Soc. 2003, 125, 11474. (6) Stern, E.; Klemic, J. F.; Routenberg, D. A.; Wyrembak, P. N.; Turner-Evans, D. B.; Hamilton, A. D.; LaVan, D. A.; Fahmy, T. M.; Reed, M. A. Nature 2007, 445, 519.
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