Electrochemical DNA Biosensor Based on Nanoporous Gold

Anal. Chem. 2008, 80, 9124–9130

Electrochemical DNA Biosensor Based on Nanoporous Gold Electrode and Multifunctional Encoded DNA-Au Bio Bar Codes Kongcheng Hu, Dongxiao Lan, Xuemei Li, and Shusheng Zhang* Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, P. R. China A sensitive electrochemical DNA sensor based on nanoporous gold (NPG) electrode and multifunctional encoded Au nanoparticle (AuNP) was developed. The NPG electrode was prepared with a simple dealloying strategy, by which silver was dissoluted from silver/gold alloys in nitric acid, making the active surface area of NPG electrode 9.2 times higher than that of a bare flat one characterized by cyclic voltammetry. A DNA biosensor was fabricated by immobilizing capture probe DNA on the NPG electrode and hybridization with target DNA, which further hybridized with the reporter DNA loaded on the AuNPs. The AuNP contained two kinds of bio bar code DNA, one was complementary to the target DNA, while the other was not, reducing the cross reaction between the targets and reporter DNA on the same AuNP. Electrochemical signals of [Ru(NH3)6]3+ bound to the reporter DNA via electrostatic interactions were measured by chronocoulometry. Taking advantage of dual-amplification effects of the NPG electrode and multifunctional encoded AuNP, this DNA biosensor could detect the DNA target quantitatively, in the range of 8.0 × 10-17-1.6 × 10-12 M, with a limit of detection as low as 28 aM, and exhibited excellent selectivity even for single-mismatched DNA detection. In recent years, the development of highly sensitive and selective DNA sensors to bring down the limit of detection to picoand femtomolar levels is a field of ever increasing interest, so that the genoassays are suitable for various applications including clinical diagnosis, environmental control, and forensic analysis.1,2 Sensitive detection of specific nucleic acid sequences on the basis of the hybridization reaction can be improved by target or signal amplification strategies. Most of recent developments in ultrasensitive detection of DNA are based on nanomaterials and nanotechnologies.3,4 Particularly, use of nanomaterials, such as nanotubes, nanoparticles, and nanowires, as a medium of signal amplification supplied many opportunities to advance biomolecular and gene detection. Carbon nanotube coat, composite, or multi* To whom correspondence should be addressed. Phone: 86-532-84022750. Fax: 86-532-84022750. E-mail: [email protected]. (1) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192–1199. (2) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503–1506. (3) Hahm, J.; Lieber, C. M. Nano Lett. 2004, 4, 51–54. (4) Wang, J.; Liu, G.; Merkoci, A. J. Am. Chem. Soc. 2003, 125, 3214–3215.

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layer was used in nucleic acid detection.5-7 There are explorations on the PANI nanotube array modified electrode, which provides a high-efficiency route for ultrasensitive DNA hybridization detection.8 Mirkin reported on gold nanoparticle-based electrochemical DNA chips with catalytically deposited silver as an enhancing element.2 Wang et al. developed electrochemical assays based on quantum dot nanocrystals as tracers.9 The nanoporous gold (NPG) has attracted considerable attention in recent years due to its unique properties: high surface-tovolume reaction, stability, high in-plane conductivity, and biocompatibility. Several methods for the preparation of macroporous electrodes are by using colloidal crystal templating.10-12 Kuhn et al. demonstrated porous macroelectrodes by template synthesis using colloidal crystals and further developed macroporous ultramicroelectrodes for glucose detection.13,14 Choi’s group reported an electrochemical DNA biosensor based on a thin gold film sputtered on anodic porous niobium oxide.15 More recently, Zhu et al. reported a label-free immusensor for the detection of C-reactiveproteinbasedonathree-dimensionalorderedmacroporous (3DOM) gold film-modified electrode.16 Although the sensing enhancement was significantly higher than that of bare flat film, the preparation of 3DOM film is complicated and time-consuming. A simple dealloying strategy, by which silver was dissoluted from silver/gold alloys in nitric acid, to make free-standing noble metal membranes with controllable three-dimensional porosity, has been reported.17-19 These porous gold materials have been (5) Jung, D. H.; Kim, B. H.; Ko, Y. K.; Jung, M. S.; Jung, S.; Lee, S. Y.; Jung, H. T. Langmuir 2004, 20, 8886–8891. (6) Wang, J.; Dai, J.; Yarlagadda, T. Langmuir 2005, 21, 9–12. (7) Tang, X.; Bansaruntip, S.; Nakayama, N.; Yenilmez, E.; Chang, Y.; Wang, Q. Nano Lett. 2006, 6, 1632–1636. (8) Chang, H.; Yuan, Y.; Shi, N.; Guan, Y. Anal. Chem. 2007, 79, 5111–5115. (9) Liu, G.; Lee, T. M. H.; Wang, J. J. Am. Chem. Soc. 2005, 127, 38–39. (10) Ben-Ali, S.; Cook, D. A.; Evans, S. A. G.; Thienpont, A.; Bartlett, P. N.; Kuhn, A. Electrochem. Commun. 2003, 5, 747–751. (11) Wang, C.; Yang, C.; Song, Y.; Gao, W.; Xia, X. Adv. Funct. Mater. 2005, 15, 1267–1275. (12) Bartlett, P. N.; Baumberg, J. J.; Birkin, P. R.; Ghanem, M. A.; Netti, M. C. Chem. Mater. 2002, 14, 2199–2208. (13) Szamocki, R.; Reculusa, S.; Ravaine, S.; Bartlett, P. N.; Kuhn, A.; Hempelmann, R. Angew. Chem., Int. Ed. 2006, 45, 1317–1321. (14) Szamocki, R.; Velichko, A.; Holzapfel, C.; Mucklich, F.; Ravaine, S.; Garrigue, P.; Sojic, N.; Hempelmann, R.; Kuhn, A. Anal. Chem. 2007, 79, 533–599. (15) Rho, S.; Jahng, D.; Lim, J. H.; Choi, J.; Chang, J. H.; Lee, S. C.; Kim, K. J. Biosens. Bioelectron. 2008, 23, 852–856. (16) Chen, X.; Wang, Y.; Zhou, J.; Yan, W.; Li, X.; Zhu, J. Anal. Chem. 2008, 80, 2133–2140. (17) Erlebacher, J.; Aziz, M. J.; Karma, A.; Dimitrov, N.; Sieradzki, K. Nature 2001, 410, 450–453. 10.1021/ac8017197 CCC: $40.75  2008 American Chemical Society Published on Web 11/05/2008

Table 1. DNA Sequences Used in This Work name

sequence 5′-SH-(CH2)6-TCG TAC GAT CGA TCC-3′

target DNA (S2)

5′-GCC GCT CAC ACG ATA TTT TTT TTG GAT CGA TCG TAC GA-3′ 5′-TAT CGT GTG AGC GGC TTT TTT TT (CH2)6-SH-3′ 5′-GCT CAT ATG GAC CTC TTT TTT TT (CH2)6-SH-3′ 5′-GCC GCT CAC ACG ATA TTT TTT TTG GAT CGA TGG TAC GA-3′ 5′-ACA TGC TTG GAC TGC TTT TTT TTC AGG CTC ATC GTA CG-3′

reporter DNA (S3) signal DNA (S4) single-mismatched DNA (S5) noncDNA (S6)

widely used as catalyst for important reactions such as methanol20,21 or CO oxidation.22 In the present work, we described a sensitive DNA biosensor based on the use of the NPG electrode as a solid support for the immobilization of probe DNA, which is a critical step in the development of new biosensors and assays. DNA-Au bio bar code, which have become increasingly incorporated bioassays with its effective amplification based on AuNPs functionalized with a large number of oligonucleotide strands,23-26 was involved in this strategy. Different from the reported AuNPs with one kind of oligonucleotide, we used the AuNP label with two kinds of DNA bio bar code. One is complementary to the target, while the other is not, reducing the cross-reaction of targets with the cDNA loaded on the same AuNP. Taking advantage of dual-amplification effects of the NPG electrode and multifunctional encoded AuNP coupled with [Ru(NH3)6]3+ as indicator, our DNA biosensor has a limit of detection as low as 28 aM. EXPERIMENTAL SECTION Materials and Chemicals. All synthetic oligonucleotides designed according to the ref 27 were purchased from SBS Genetech Co. Ltd. (Beijing, China). Their sequences are presented in Table 1. The 9-carat white gold leaves (Ag/Au alloy, 50:50 wt %) 100 nm in thickness were obtained from Monarch. 6-Mercapto1-hexanol (MCH) was purchased from Fluka. Hexaammineruthenium(III) chloride ([Ru(NH3)6]3+) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were from Acros Organics, and HAuCl4 · 4H2O was obtained from Guoyao Chemical Co. All solutions were prepared with triply distilled water. The buffers involved in this work are as follows: DNA immobilization buffer, 10 mM Tris-HCl, 1.0 mM EDTA, 1.0 M NaCl, and 1.0 mM TCEP (pH 7.0); DNA hybridization buffer, 10 mM Tris-HCl, 1.0 mM EDTA, and 1.0 M NaCl (pH 7.0). Buffer for electrochemistry, 10 mM Tris-HCl buffer (pH 7.0), 10 mM Tris-acetate buffer (pH 8.2). (18) (19) (20) (21) (22) (23) (24) (25) (26)

description

capture DNA (S1)

Li, R.; Sieradzki, K. Phys. Rev. Lett. 1992, 68, 1168–1171. Ding, Y.; Erlebacher, J. J. Am. Chem. Soc. 2003, 125, 7772–7773. Ge, X.; Wang, R.; Liu, P.; Ding, Y. Chem. Mater. 2007, 19, 5827–5829. Yu, C.; Jia, F.; Ai, Z.; Zhang, L. Chem. Mater. 2007, 19, 6065–6067. Xu, C.; Su, J.; Xu, X.; Liu, P.; Zhao, H.; Tian, F.; Ding, Y. J. Am. Chem. Soc. 2007, 129, 42–43. Rosi, N.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547–1562. Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884–1886. He, P.; Shen, L.; Cao, Y.; Li, D. Anal. Chem. 2007, 79, 8024–8029. Shen, L.; Chen, Z.; Li, Y.; He, S.; Xie, S.; Xu, X.; Liang, Z.; Meng, X.; Li, Q.; Zhu, Z.; Li, M.; Le, X. C.; Shao, Y. Anal. Chem. 2008, 80, 6323–6328.

thiolated probe immobilized on NPG electrode complementary to S1 and S3 thiolated probe loaded on NPG electrode AuNP and complementary to S2 thiolated probe loaded on NPG electrode AuNP and noncomplementary to S2 italic was the mismatched base

Fabrication of NPG Electrode. NPG was prepared by selective dissolution of silver from silver/gold alloy.17,19,28 Briefly, a piece of commercially available 9-carat white gold leaf (Au/Ag alloy, 50:50, wt %, 100 nm thick) was floated onto 1:1 concentrated nitric acid for 1 h. Then the NPG foil was carefully coated onto a pretreated glassy carbon electrode (GCE) using a variation of electroless deposition in which the leaf adhered on the GCE surface via physical adsorption after being washed repeatedly with triply distilled water to remove the NO3- and Ag+, which would interfere with signal detection during electrochemical analysis. The electrode was then intentionally parched in infrared light for 1 h. Thus, NPG was modified onto the GCE. Preparation of Bio Bar Coded Gold Nanoparticles. Gold nanoparticles were prepared by citrate reduction of HAuCl4 according to the previous literature.29 Ten milliliters of 38.8 mM sodium citrate was immediately added to 100 mL of 1.0 mM HAuCl4 refluxing solution under stirring, and the mixture was kept boiling for another 15 min. The solution turned to a wine red, indicating the formation of gold nanoparticles. The solution was cooled to room temperature with continuous stirring. The sizes of the AuNPs were verified by scanning electron micrograph using a JEOL JSM-6700F microscope. The process of probe and reporter DNA labeling was performed as follows:30 The mixture of 5.0 × 10-10 mol of S3 and 2.0 × 10-9 mol of S4 was activated with acetate buffer (pH 5.2) and 1.5 µL of 10 mM TCEP for 1 h, then added to 1 mL of freshly prepared gold nanoparticles, and shaken gently overnight. Over the course of 16 h, the DNA-AuNP conjugates were aged in salts (0.1 M NaCl, 10 mM acetate buffer) for another 24 h. Excess reagents were removed by centrifuging at 16 000 rpm for 30 min. The red precipitate was washed and centrifuged repeatly for three times. The resulting nanoparticles were dispersed into a buffer solution (pH 8.2) and stored at 4 °C. Preparation of DNA Sensor Based on NPG Electrode and Bio Bar Code. NPG electrode freshly prepared was immediately used for the preparation of DNA biosensor by immersing the electrode into an immobilization buffer containing 1.0 × 10-8 M capture probe for 24 h. The DNA-modified electrode was further treated with 1.0 mM MCH for 2 h to obtain a well-aligned DNA monolayer, followed by washing with triple-distilled water to remove unspecific adsorbed DNA. For the hybridization reaction, ssDNA/NPG was immersed into stirred Tris-HCl solution contain(28) Newman, R. C.; Sieradzki, K. Science 1994, 263, 1708–1709. (29) Liu, J.; Lu, Y. Nat. Protoc. 2006, 1, 246–252. (30) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757–1760.


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Scheme 1. Chronocoulometry Determination of DNA Hybridization through Two Steps of Amplification

ing target DNA (S2) for a desired time at 37 °C. Temperature stability was accomplished by the temperature-controlled magnetic stirrer system. The DNA-modified NPG electrode was secondly hybridized with reporter probes loaded on AuNPs for 2 h at room temperature. After hybridization, the electrode was extensively rinsed with washing buffer (10 mM Tris-HCl, pH 7.0) and dried under a stream of nitrogen prior to electrochemical characterization. Electrochemical Measurements. All electrochemical measurements were carried out at room temperature in a singlecompartment, three-electrode glass cell using an electrochemical analyzer (CHI832B, CH Instruments). The three-electrode system used consisted of the working electrode of interest, a saturated calomel electrode reference electrode, and a platinum wire auxiliary electrode. Cyclic voltammetry (CV) and chronocoulometry (CC) was performed in 2 mL of 10 mM Tris-HCl solution (pH 7.0) containing 20 µM [Ru(NH3)6]3+, with a scan rate of 500 mV/s for CV and a pulse period of 250 ms for CC, respectively. Electrochemical impedance spectroscopy (EIS) was carried out in a degassed Tris-HCl buffer (pH 7.0) containing 0.1 M KCl and 5 mM Fe(CN)63-/Fe(CN)64-. RESULTS AND DISCUSSION Sensing System Based on NPG and Bio Bar Code. The S3 and S4 functionalized AuNP was prepared by thiolated DNA via gold-sulfur affinity, which significantly increased the stability of AuNP.30 S3 was complementary to target DNA S2, while S4 was noncomplementary. According to the method in our previous reports,31 it was found that there were 127 single strands per AuNP, consist of 27 S3 and 100 S4 on each AuNP (see Supporting Information (SI) for details). Comparing with the AuNP labeled with all complementary reporter DNA, the probe density was low, which facilitated one AuNP labeled with one target. (31) Zhang, S.; Zhong, H.; Ding, C. Anal. Chem. 2008, 80, 7206–7212.

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In the present study, an electrochemical DNA biosensor based on the NPG electrode and bio bar code was presented. The system contained a thiolated DNA sequence (S1) as capture probe, target DNA (S2), and AuNP-linked DNA sequence as reporter probe (S3), which is shown in Scheme 1. The 5′-thiol-modified single-strand DNA probe S1 was immobilized onto the NPG electrode surface via a thiol-Au interaction.32 Subsequently, the 15-base segment close to the 3′ end of the target DNA S2 was hybridized with S1, and the 5′ end was reacted with reporter DNA S3. When the electrode was immersed into a Tris-HCl solution containing [Ru(NH3)6]3+, the signal was measured, which was proportional to the amount of the reporter DNA and further corresponded to the concentration of target DNA. Since a single AuNP is loaded with ∼100 reporter DNA strands, this offers a significant amplification for DNA detection.33 Fabrication of NPG Electrode. The commercially available 9-carat white gold leaf (Ag/Au alloy, 50:50 wt %) in 100-nm thickness could be dealloyed to create free-standing NPG membranes that are inexpensive and crack-free over 80 cm2.19 A simple method to dealloy silver/gold alloys was carried out under corrosion-free conditions to float the leaf on a 1:1 concentrated nitric acid surface for 1 h. During etching, silver atoms were selectively dissolved, and the gold atoms left behind assembled into the 3D porous structure. One important characteristic of NPG is that its structural unit (pore/ligament size) can be tunable by varying the starting alloy composition or etching time or etching temperature.20 As shown in Figure S4 (SI), dealloying a bulk Ag/ Au foil at 25 °C resulted in a coarsened structure (Figure S4a, SI), which reduced the active surface area. Bulk nanoporous gold can be routinely fabricated with ligament sizes less than 30 nm (32) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916–8920. (33) Thaxton, C. S.; Hill, H. D.; Dimitra, G. G.; Stoeva, S. I.; Mirkin, C. A. Anal. Chem. 2005, 77, 8174–8178.

Figure 1. Cyclic voltammograms of different electrodes. (a) The NPG electrode; (b) the bare flat Au electrode. Supporting electrolyte, 0.5 M H2SO4; scan rate, 100 mV/s.

by annealing at 10 °C (Figure 4b).18 The resulting high surface area of the porous film would induce the film efficiency or sensing enhancement. Moreover, a closer look at the pores revealed small points, indicating that the complete inner surface should be accessible for electrochemical reactions. Electrochemical Characterization of the NPG Electrode. In comparison with other detection protocols, cyclic voltammetry of electroactive species in a conducting aqueous solution is a valuable means of probing the electrochemical characterization of the modified electrode.34 The NPG electrode was electrochemically characterized by CV in 0.5 M H2SO4. The bare flat gold electrode with the same geometric surface area was also characterized under the same conditions for comparison (Figure 1). The CV curves showed that the NPG electrode had a significant larger CV area compared with the bare electrode, indicating that the NPG electrode had a much larger effective surface area due to the porous morphology. Assuming that a specific charge of 386 µC/cm2 was required for gold oxide reduction,35 the NPG electrode had a total active surface of 109.7 mm2, while the corresponding bare flat gold electrode was 11.8 mm2. The significant enhancement of 9.2-fold was obtained, representing a much larger area of the three-dimensional NPG electrode. The number of pore layers could be calculated as 2.5, according to the equation of the relative surface enhancement between a flat and a porous electrode, on the basis of fundamental geometric considerations, supposing a close-packed structure,14 f ) nπ(4 ⁄ 3)1⁄2

(1)

where f is the enhancement factor and n is the number of pore layers. Characterization of the NPG-Based Biosensor. The assembly of oligonucleotides on electrodes and the formation of double-stranded DNA on the support can be followed by faradic (34) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559–3568. (35) Szamocki, R.; Velichko, A.; Holzapfel, C.; Mucklich, F.; Ravaine, S.; Garrigue, P.; Sojic, N.; Hempelmann, R.; Kuhn, A. Anal. Chem. 2007, 79, 533–599.

Figure 2. Nyquist plots corresponding to NPG electrode (A) and flat Au electrode (B). (a) The bare electrode, (b) after immobilization of probe 1, (c) hybridization with target DNA, and (d) hybridization with the reporter DNA loaded on the bio bar code AuNP. The data were recorded in the presence of [Fe(CN)6]3-/4-, 5.0 mM, as redox label, and upon application of the biasing potential 0.21 V, applying 5-mV alternative voltage in the frequency range of 50 mHz-10 kHz. Data were recorded in a PBS solution (10 mM) that included KCl (100 mM); pH 7.0.

impedance spectroscopy. 36 Figure 2 showed a Nyquist plot of impedance for the stepwise modification process with the NPG electrode (Figure 2A) and bare flat Au electrode (Figure 2B). For the bare flat Au electrode, the impedance spectra included a semicircle portion at higher frequencies relating to the electrontransfer-limited process and a linear part at lower frequencies corresponding to diffusion. The increase in the diameter of the semicircle reflects the increase in the interfacial charge-transfer resistance (Rct). For the bulk Au electrode, the value of Rct was 127.4 Ω, revealing a very small semicircle domain. After immobilization of probe 1, the value of Rct increased from 127.4 to 1675.0 Ω. The increase in Rct was due to the immobilization of negatively charged ODN probes on the electrode surface resulting in a negatively charged interface that electrostatically repels the negatively charged redox probe [Fe(CN)6]3-/4- and inhibits interfacial (36) Zayats, M.; Huang, Y.; Gill, R.; Ma, C. A.; Willner, I. J. Am. Chem. Soc. 2006, 128, 13666–13667.


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Figure 3. Chronocoulometry curves for the bare Au electrode (a-d) and the NPG electrodes (e-h) with capture probe hybridized with target DNA at a series of concentrations (40, 80, 160, and 640 fM). The electrolyte was 10.0 mM Tris buffer (pH 7.0) containing 20 µM [Ru(NH3)6]3+. Pulse period, 250 ms.

charge transfer.37 Subsequently, the target DNA was hybridized with probe 1, and the Rct increased again. After hybridization with probe 3 loaded on the AuNP, the value of Rct was increased greatly to 3327.0 Ω, due to the large amount of DNA linked on the AuNPs. The NPG electrode exhibited an almost straight line, which was just the characteristic of the diffusional limiting step of the electrochemical process. Even after probe immobilization and hybridization steps, the Rct values were also quite low, indicating that the NPG electrode represented high conductivity due to its nanoporous architecture.16 These results were consistent with the fact that the NPG electrode was fabricated as expected. Sensitive Detection of Target DNA. Since a large portion of [Ru(NH3)6]3+ molecules entrapped in the heterogeneous film are kinetically electroinactive during “dynamic” voltammetric scans, while nearly all [Ru(NH3)6]3+ molecules are electroactive in the “static” chronocoulometric measurements, it has been reported that the [Ru(NH3)6]3+/DNA/electrode system generated significantly more intense signal in CC than in CV. 38,39 We then carried out CC for the detection of target DNA. To examine the sensitivity of the protocol, the charges of the [Ru(NH3)6]3+ were measured after hybridization with the target DNA at different concentrations. Figure 3 showed an increase of the charges with the increase of target DNA concentration, implying that one could use this DNA sensor to perform quantitive target DNA detection. For comparison, CCs of a bare flat gold electrode-based biosensor with the same target concentrations were also investigated (Figure 3a-d). The Q observed on the NPG electrode was ∼30 times higher than that on the bare gold electrode with the same ssDNA target. We ascribed the enhancement of the signal of NPG electrode to a large effective surface area and high electrical conductivity. Additionally, it might be ascribed to the larger number of capture DNA probes at the NPG electrode than on the flat Au substrate. (37) Cho, M.; Lee, S.; Han, S. Y.; Park, J. Y.; Rahmen, M. A.; Shim, Y. B.; Ban, C. Nucleic Acids Res. 2006, 34, e75. (38) Lao, R.; Song, S.; Wu, H.; Wang, L.; Zhang, Z.; He, L.; Fan, C. Anal. Chem. 2005, 77, 6475–6480. (39) Katz, E.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2005, 127, 9191– 9200.

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Figure 4. (A) Chronocoulometry transients of [Ru(NH3)6]3+ for different hybrids. Concentrations of target DNA in the presence of AuNPs-reporter DNA: (a) 0, (b) 8.0 × 10-17, (c) 2.0 × 10-16, (d) 4.0 × 10-16, (e) 6.0 × 10-16, (f) 8.0 × 10-16, (g) 3.2 × 10-15, (h) 6.4 × 10-15, (i) 9.6 × 10-15, (j) 4.0 × 10-14, (k) 8.0 × 10-14, (l) 1.6 × 10-13, (m) 6.4 × 10-13, and (n) 1.6 × 10-12 M. (B) Calibration curve of the DNA sensor, where the definition of signal is the same as that in Figure 4A.

The influence of the concentration of [Ru(NH3)6]3+ on the CC charge was also investigated, and 20 µM [Ru(NH3)6]3+ was chosen in this study (see Figure S5, SI). The chronocoulometric response curves are converted to Anson plots by plotting charge versus t1/2. The linear part of the Anson plot is then extrapolated back to time zero to obtain the intercept for the plot in the presence and absence of target DNA. Figure 4A showed the chronocoulometric curves for the NPGbased sensor after hybridization with different concentrations of target DNA. Figure 4B represented the relationship between the charge changes and target DNA concentration. The results showed that chronocoulometry intensities increased with the increase of the concentration of target DNA ranging from 8.0 × 10-17 to 1.6 × 10-12 M. The logarithm function was y ) 3.8102 lnx - 2.8953 (shown in Figure 4B, where y was the charge (µC) and x was the concentration of target DNA, 10-16 M; n ) 12) with r ) 0.9729. The linear range was from 8.0 × 10-17 to 8.0 × 10-16 M, with the regression equation being y ) 1.0588 x + 0.4258 (shown in Figure 4B, y was the charge, µC; x was the concentration of target DNA, 10-16 M; n ) 6; r ) 0.9909). A detection limit

Table 2. Comparison between the Proposed Assay and Other Reported Techniques for the Determination of DNA Hybridization label or Indicator

analytical techniquesa

detection limit of ssDNA

ref

multiwalled carbon nanotubes conducting polyaniline nanotube array conducting polypyrrole poly(pyrrole-co-3-pyrrolylacrylic acid) AuNPs magnetic nanoparticles silver NPs ZnS, CdS, PbS NPs tin oxide nanoparticle AuNPs with Ag amplification AuNPs with Ag amplification AuNPs with Ag amplification liposome NPG electrode

ac impedance DPV CV EIS CC CC and EIS ASV stripping voltammetry photoelectrochemical detection electrical detection scanometric detection bio bar code amplified scanometric detection liposome-amplified electrochemical detection bio bar code amplified CC

10 nM 1.0 fM 0.16 or3.5fmol 0.98nM 10 fM 1.7 nm 0.5 pM 270 pM 0.18nM 500 fM 50 fM 500 aM 50 fM 28 aM

40 8d 41 42 27 43 44 4 45 2 31 46 47 thiswork

a Abbreviations: ASV, anodic stripping voltammetry; CC, chronocoulometry; CV, cyclic voltammetry; DPV, differential pulse voltammetry; EIS, electrochemical impedance spectroscopy.

of 2.8 × 10-17 M target DNA can be estimated using 3σ. The use of our NPG electrode indicated that the detection limit had a 300fold improvement on the bare flat Au electrode.27 As shown in Table 2, our strategy was more sensitive than those of nanoparticle, nanotube, conductive polymer, and other nanostructuremodified assay as reported previously. Moreover, the electrochemical signal was reproducible, with coefficient of 95% for target DNA, when samples containing 4.0 × 10-16 M target DNA were measured 4 times. Selectivity of the DNA Biosensor. The selectivity of the present biosensor in discriminating perfect cDNA from singlebase mismatched and noncDNA sequences was investigated under three concentrations as shown in Figure 5. Referring to the complementary hybridization at each concentration as 100%, the ratio of hybridization efficiencies were as follows: target:Ms: Nc ) 100:20:4, 100:21:4, and 100:28:6 for 6.0 × 10-13, 6.0 × 10-14, and 6.0 × 10-15 M targets, respectively. We found that only the perfectly matched DNA produced prominent signals, while signals corresponding to GG mismatched DNA were not less than 15% even at the femtomolar range. In comparison with the polyaniline nanotube array-based DNA biosensor, which had hybridization efficiencies of 22 and 54% for TC and TG mismatch, respectively,8 our NPG-based DNA biosensor represented a much better specificity, showing that the presented NPG-based sensor could be satisfactory to single-nucleotide polymorphisim assays. Regeneration of the NPG-Based DNA Biosensor. Besides sensitivity and selectivity, reusability is also an extremely important feature for biosensors in the practical applications such as (27) Zhang, J.; Song, S. P.; Wang, L. H.; Wu, H. P.; Pan, D.; Fan, C. H. J. Am. Chem. Soc. 2006, 128, 8575–8580. (40) Cai, H.; Xu, Y.; He, P.; Fang, Y. Electroanalysis 2003, 15, 1864–1870. (41) Riccardi, C.; Yamanaka, H.; Josowicz, M.; Kowalik, J.; Mizaikoff, B.; Kranz, C. Anal. Chem. 2006, 78, 1139–1145. (42) Peng, H.; Soeller, C.; Vigar, N.; Caprio, V.; Travas-Sejdic, J. Biosens. Bioelectron. 2007, 22, 1868–1873. (43) Pumera, M.; Castaneda, M. T.; Pividori, M. I.; Eritja, R.; Merkocui, A.; Alegret, S. Langmuir 2005, 21, 9625–9629. (44) Cai, H.; Xu, Y.; Zhu, N.; He, P.; Fang, Y. Analyst 2002, 127, 803–808. (45) Liu, S.; Li, C.; Cheng, J.; Zhou, Y. Anal. Chem. 2006, 78, 4722–4726. (46) Nam, J. M.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 5932– 5933. (47) Patolsky, F.; Lichtenstein, A.; Willner, I. Angew. Chem., Int. Ed. 2000, 39, 940–943.

Figure 5. Comparison of chronocoulometry signal for NPG electrodes hybridized with a series of target DNA (6.0 × 10-13, 6.0 × 10-14, and 6.0 × 10-15 M) the cDNA (A), one-base mismatched DNA (B), and completely mismatched target DNA (C). The definition of signal was the same as that in Figure 2. Error bars showed the standard deviations of measurements.

clinical diagnoses. In our test, the NPG-based biosensor could be regenerated by incubation of the modified electrode in hot water (90 °C) for 1 min, by which hybridized DNA was removed via thermal denaturation. After the regeneration procedure was performed three times, the NPG-based biosensor almost retained its original hybridization efficiency, with the relative standard deviation of these measurements of 5.6% (see Figure S6 in Supporting Information). The DNA-modified NPG electrode could be stored in the refrigerator for one week with negligible loss of the immobilized probe DNA. CONCLUSIONS The electrochemical DNA biosensor based on NPG electrode and multifunctional encoded AuNP exhibited excellent sensitivity and selectivity, with the limit of detection for target DNA as low as 28 aM, which could be comparable with that of PCR. The main advantages of the present biosensor contributed to two aspects. First, the fabrication of NPG electrode with high active surface area, through which the immobilization of capture DNA was enhanced, was quite simple and economical, avoiding the use of template. Second, compared with the reported single bio bar code Analytical Chemistry, Vol. 80, No. 23, December 1, 2008

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assay, AuNPs containing two DNA bio bar codes were used to avoid cross-reaction. Moreover, this method was stable and could extend to the application in the ultramicroassay technique. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (20775038), the Scientific and Technical Development Project of Qingdao (06-3-1-4-yx), and the National High-tech R&D Program (863 Program, 2007AA09Z113).

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SUPPORTING INFORMATION AVAILABLE Calculation of the ratios of S3 and S4 loaded on per AuNP as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review August 16, 2008. Accepted October 11, 2008. AC8017197

Electrochemical DNA Biosensor Based on Nanoporous Gold

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