Ultrasensitive Electrochemical Detection For DNA Arrays Based on

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Anal. Chem. 2010, 82, 5477–5483

Ultrasensitive Electrochemical Detection For DNA Arrays Based on Silver Nanoparticle Aggregates Hui Li,† Ziyin Sun,† Wenying Zhong,‡ Nan Hao,† Danke Xu,*,† and Hong-Yuan Chen† Key Lab of Analytical Chemistry for Life Science, Ministry of Education, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, and Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210009, China Multiplexed DNA target detection is of great significance in many fields including clinical diagnostics, environmental monitoring, biothreat detection and forensics. Although the emergence of DNA chip technology has accelerated this process, it is still a challenge to perform ultrasensitive DNA assay at low attomol concentrations so that DNA detection can be directly achieved without a PCR protocol. In this work, an oligonucleotide-functionalized silver nanoparticle tag has been successfully developed for multiplexed DNA electrochemical detection with ultrahigh sensitivity. The multiprobes containing oligo(d)A and the reporting probes were anchored onto the silver nanoparticles, followed by hybridizing with the silver nanoparticle conjugate modified with oligo(d)T. The hybridizationinduced tag was found to show an aggregated nanostructure 10 times larger than the individual nanoparticle, as revealed by TEM. For sandwich-based assays, the tag was specifically coupled to a gold electrode surface via target DNA. Compared to a single nanoparticle label, this novel tag has shown excellent electroactive property and produces 103-fold amplification in the differential pulse voltammetric (DPV) method. Hepatitis B virus (HBV) sequence was employed as a sample model, and we have achieved a detection limit of 5 aM (∼120 molecules in 40 µL volume), demonstrating ultrasensitive measurement for DNA. The property of the electrochemical process involving silver aggregates was further investigated and the integrative oxidation of the silver tag was observed. We further demonstrated the multiplexed DNA target detection using array chips functionalized with Herpes simplex virus (HSV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) sequences, which shows effective recognition of the relative sequences individually or simultaneously. The method offers a uniquely new approach for DNA detection with ultrahigh sensitivity as well as advantages of rapidity, throughput, and miniaturization. Multiplexed DNA detection is important to clinical diagnostics, environmental monitoring, bioterrorism, and forensics since more genomic information has become known with the acceleration of * To whom correspondence should be addressed. Phone: +86 25 83595835. Fax: +86 25 83595835. E-mail: [email protected]. † Nanjing University. ‡ China Pharmaceutical University. 10.1021/ac101193e  2010 American Chemical Society Published on Web 06/15/2010

genomics discoveries.1,2 Although DNA chips with fluorescence detection are a powerful tool that provides complex and informative data from nucleic acid sequences, they need fluorescence scanners that are inherently costly and not transportable.3 In addition, PCR amplification is still required due to insufficient detection sensitivity of the optical methods, especially for ultralow concentrations.4 These issues have limited the widespread use of DNA chips for point-ofcare testing or as a routine diagnostics tool. Electric transducers coupled with electrode arrays have been used for detection of multiple species and DNA owing to high sensitivity, small size, low cost, and compatibility with micromanufacturing technology.5,6 Many electrochemical detection methods have been developed to test DNA targets via label7-11 or label free approaches.12,13 Generally, the detection sensitivity with label systems is higher due to their signal amplification procedure. Although enzyme-labeling methods are considered to have the larger amplification through generation of a large number of electroactive molecules from the enzymes, their detection limits are currently only in the fmol/L concentration range.5 This method is difficult to adopt for the detection of arrayed electrodes because diffusion of electroactive molecules on the arrayed electrodes causes a severe crosstalking effect.14 (1) Mikhailovich, V.; Gryadunov, D.; Kolchinsky, A.; Makarov, A. A.; Zasedatelev, A. BioEssays 2008, 30, 673–682. (2) Barken, K. B.; Haagensen, J. A. J.; Tolker-Nielsen, T. Clin. Chim. Acta 2007, 384, 1–11. (3) Lassiter, S. J.; Stryjewski, W. J.; Wang, Y.; Soper, S. A. Spectroscopy 2002, 17, 14–23. (4) Albelda, S. M.; Shepard, J. R. E. Am. J. Respir. Cell Mol. Biol. 2000, 23, 265–269. (5) Sassolas, A.; Leca-Bouvier, B. D.; Blum, L. J. Chem. Rev. 2008, 108, 109– 139. (6) Park, S.-J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503–1506. (7) Nebling, E.; Grunwald, T.; Albers, J.; Scha1 fer, P.; Hintsche, R. Anal. Chem. 2004, 76, 689–696. (8) Elsholz, B.; Worl, R.; Blohm, L.; Albers, J.; Feucht, H.; Grunwald, T.; Jurgen, B.; Schweder, T.; Hintsche, R. Anal. Chem. 2006, 78, 4794–4802. (9) Farabullini, F.; Lucarelli, F.; Palchetti, I.; Marrazza, G.; Mascini, M. Biosens. Bioelectron. 2007, 22, 1544–1549. (10) Diercks, S.; Metfies, K.; Medlin, L. K. Biosens. Bioelectron. 2008, 23, 1527– 1533. (11) Neugebauer, S.; Zimdars, A.; Liepold, P.; Gebala, M.; Schuhmann, W.; Hartwich, G. Chem. Bio. Chem. 2009, 10, 1193–1199. (12) Takahashi, M.; Okada, J.; Ito, K.; Hashimoto, M.; Hashimoto, K.; Yoshida, Y.; Furuichi, Y.; Ohta, Y.; Mishiro, S.; Gemma, N. Analyst 2005, 130, 687– 693. (13) Nakamura, N.; Ito, K.; Takahashi, M.; Hashimoto, K.; Kawamoto, M.; Yamanaka, M.; Taniguchi, A.; Kamatani, N.; Gemma, N. Anal. Chem. 2007, 79, 9484–9493. (14) Marchand, G.; Delattre, C.; Campagnolo, R.; Pouteau, P.; Gin, F. Anal. Chem. 2005, 77, 5189–5195.

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Nanoparticle probes are alternative signal amplification materials and have shown great promise for electrochemical detection of DNA.15-17 Gold or silver nanoparticle labels have attracted the most interest, based on their unique physical and chemical properties. Although various nanoparticle tags have been suggested as detection agents, these labels in most cases are used to promote reactions such as catalysis,18-20 dissolution by acids,21-24 or electrostatic binding.25,26 However, there are few reports to show their advantages in multiplexed detection. In this paper, we present an ultrasensitive and direct electrochemical DNA detection method based on Ag aggregate tag and differential pulse voltammtery. The scheme of detection is shown in Scheme 1a. The silver tags consist of Conjugate 1 (functionalized with capture probes and oligo A) and Conjugate 2 (modified with oligo T). Hybridization between complementary oligo(d) A and oligo(d) T anchored on the silver nanoparticles produced aggregate tags. The novel hybridization-induced tags are successfully applied to bind with the DNA target via sandwich hybridization format and offer direct and amplified readout by differential pulse voltammetric method. We have found that the detection sensitivity by use of the aggregate tags can be improved by 3 orders of magnitude as compared to the single silver nanoparticle labels and a detection limit of 5 amol/L could be obtained. In addition, this strategy is applicable to multiplexed target measurement on DNA arrays (Scheme 1b). EXPERIMENTAL SECTION Materials and Instrumentation. Silver nitrate (Shanghai Shengbo Chemical. Co. Ltd.) and sodium borohydride (Institute of chemical reagents, Tianjing, China) were used to synthesize colloidal silver nanoparticles. Phosphate buffered saline (PBS), tween-20 (Nanjing Bokkman Biotechnology Ltd.), NaCl (Nanjing Chemical Reagent. Co. Ltd.), NaNO3 (Nanjing Zhenxin Reagent Company) were used for preparation of the following phosphate buffers: 0.1 M PBS (0.1 M NaCl+10 mM sodium phosphate buffer pH 7.0), PBST (0.1 M PBS+0.05% tween-20), 0.2 M PBS (0.2 M NaCl+10 mM sodium phosphate buffer pH 7.0), and 0.1 M PBN (0.1 M NaNO3+10 mM sodium phosphate buffer pH 7.0). Doubly distilled deionized water was used in all experiments. All the synthetic oligonucleotides used in this study were purchased from Shanghai Sengon Biotechnology Co. UV-vis spectra was performed on UV-3600 spectrophotometer (Shimadzu, Japan). A transmission electron microscope (JEM(15) Wang, J. Anal. Chim. Acta 2003, 500, 247–257. (16) Katz, E.; Willner, I.; Wang, J. Electroanal. 2004, 16, 19–44. (17) Castaneda, M. T.; Alegret, S.; Merkoci, A. Electroanal. 2007, 19, 743– 753. (18) Rochelet-Dequaire, M.; Limoges, B.; Brossiera, P. Analyst 2006, 131, 923– 929. (19) Konga, J. M.; Zhang, H.; Chen, X. T.; Balasubramanian, N.; Kwong, D. L. Biosens. Bioelectron. 2008, 24, 793–797. (20) Fanjul-Bolado, P.; Hernandez-Santos, D.; Gonzalez-Garcı´a, M. B.; CostaGarca´, A. Anal. Chem. 2007, 79, 5272–5277. (21) Wang, J.; Xu, D.; Kawde, A.-N.; Polsky, R. Anal. Chem. 2001, 73, 5576– 5581. (22) Wang, J.; Polsky, R; Xu, D. Langmuir 2001, 17, 5739–5741. (23) Cai, H.; Xu, Y.; Zhu, N.; He, P.; Fang, Y. Analyst 2002, 127, 803–808. (24) Rijiravanich, P.; Somasundrum, M.; Surareungchai, W. Anal. Chem. 2008, 80, 3904–3909. (25) Zhang, J.; Ting, B. P.; Jana, N. R.; Gao, Z.; Ying, J. Y. Small 2009, 5, 1414– 1417. (26) Hu, K.; Lan, D.; Li, X.; Zhang, S. Anal. Chem. 2008, 80, 9124–9130.

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Scheme 1. Schematic Illustration of the Electrochemical Assay (a) and Multiplexed Assay (b) with Silver Nanoparticle Conjugates;Preparation of the Aggregates Is Shown As Well

200CX, Japan) was used for collecting TEM images. Atomic force microscopy (Agilent Technologies, Inc. U.S.) was used for AFM experiments. The aggregate tag solution were measured by dynamic light scattering technique using a commercial particle sizer (BI-200SM, Brookhaven Instruments, Corporation, U.S.). The oligonucleotides for electrode modification were derived with alkyl thiol group at the 3′ terminus and their sequences are as following. C-HBV: 5′-GGGTG CTCCC CCTAG-3′-SH; C-HSV: 5′GGCCC TCAGG GAGAG3′-SH; C-EBV: 5′TAGCC TGTGC TCTTC3′-SH; C-CMV: 5′TCGCC ACCCG GCACC3′-SH. The capture probes for functionalized silver nanoparticles were modified with an alkyl thiol group at the 5′ terminus and their sequences are shown as the following. D-CMV: SH-5′GGGTC CACAG GGTAC3′; D-HSV: SH-5′GTGCG CCACT GCGTC3′ DEBV: SH-5′CGGGC GCAGG CCGGC3′; D-HBV: SH-5′-TTGGC CAGGA CACGT-3′. An oligo(d)A (SH-A15: SH-5′AAAAA AAAAA AAAAA-3′) and oligo(d)T (SH-T15: SH-5′-TTTTT TTTTT TTTTT3′) were also used for modification of silver nanoparticles. The oligonucleotide fragments were employed for sandwich hybridization and their sequences are as follows: T-CMV: 5′GGTGC CGGGT GGCGA GTACC CTGTG GACCC3′; T-HSV: 5′CTCTC CCTGA GGGCC GACGC AGTGG CGCAC3′; T-EBV: 5′GAAGA GCACA GGCTA GCCGG CCTGC GCCCG3′; T-HBV: 5′-CTAGG GGGAG CACCC ACGTG TCCTG GCCAA3′.

Preparation and Modification of Silver Nanoparticles. Silver nanoparticles were prepared using the Munro’s method36 with some modification. Briefly, ice cold AgNO3 (2 × 10-3 M) and two times volume of NaBH4(3 × 10-3 M) were mixed dropwise with vigorously stirring in an ice bath, and the mixture was continuously stirred until the temperature naturally increased to the room temperature. Oligo(d)T-derived silver nanoparticle were prepared according to the previously published method.37 Briefly, 1 mL of silver nanoparticle solution with SH-T15(100 µL, 10 µM) was incubated for at least 18 h. To adjust the pH value and increase ionic strength of the resulting solution, 122 µL of 1 × PBS was added to the solution and allowed to react for 6 h. Then 21 µL of 2 M NaCl was added to the solution and this procedure was repeated two times at the interval of 3 h such that the total NaCl concentration could be increased gradually. After an additional standing for at least 48 h, the nanoparticles were isolated by centrifugation for 15 min,14 °C, 3 times at 15 000 rpm (Beckman Centrifuge, Allergra 64R, U.S.). The resulting silver-oligonucleotide conjugate precipitate was washed, recentrifuged and redispersed in 0.1 M PBS. The final silver nanoparticles (Conjugates 2) have an average diameter of 17 nm as measured by TEM. To prepare bistranded functionalized silver nanoparticles (Conjugate 1), 1 mL of silver nanoparticle solution was mixed with D-HBV, SH-A15(50 µL, 10 µM) and the next procedure is the same as described above. One mL of silver nanoparticle solution was mixed with D-CMV, D-HSV, D-EBV, and SH-A15 (25 µL, 10 µM) to prepare multiprobe functionalized silver nanoparticles (Conjugate 3). Preparation of oligonucleotidefunctionalized silver nanoparticle aggregates was carried out by mixing 200 µL of Conjugate 1 and 200 µL of Conjugate 2 at room temperature for 2 h for aggregation Tag 1. In addition, 200 µL of Conjugate 2 and 200 µL of Conjugate 3 were mixed at room temperature for 2 h for preparation of aggregation Tag 2. The resulting aggregate tag solution was diluted 40 times with PBS solution and then used for hybridization procedures. Dynamic Light Scattering. Samples were prepared by adding 800 µL of the aggregate tag solution to 2.4 mL 0.2 M PBS (pH 7.0). Measurements were performed at 90° scattering angle using a 532 nm laser, and the data gives distribution characterized by a mean diameter and variance. Electrode Arrays. To perform the electrochemical assay with the nanoparticle tags, a 200 nm thick gold arrayed electrode and the setup were reported previously by our lab.38 In brief, the electrodes were fabricated by photolithography and the array contains four electrodes with 1.5 mm diameter. Au electrode array chips were cleaned with freshly prepared Piranha solution (H2SO4: H2O2 ) 3:1, handled with extreme caution) for 30 min, then sonicated in water for 30 min, followed by repeating this procedure three times. The Au chip was then rinsed thoroughly by deionized water and sonicated in ethanol for 10 min and then in a detergent solution for 10 min. The chip was then washed with deionized water and dried by nitrogen. Hybridization Procedures. To improve hybridization efficiency, mercaptopropanol was selected to mix with the thiolmodified DNA capture probes. The capture probe solutions containing 1 µM capture oligonucleotides and 10 µM mercapto-

propanol in 0.1 M PBS solution were spotted onto individual gold electrodes and allowed for immobilization based on thiol chemisorption by incubating overnight at 4 °C. It was then washed by 0.1 M PBST twice (8 min/time) at 30 rpm performed on BioMixer II (Beijing CapitalBio Co.Ltd.,China). In a typical assay, 40 µL target oligonucleotides were added onto the Au chip at 37 °C for 45 min with gentle shaking at 10 rpm. After washing twice with 0.1 M PBST at 30 rpm, 40 µL detection tag solution was added at 37 °C for 15 min with gentle shaking at 10 rpm. Finally, the Au chip was thoroughly washed three times with 0.1 M PBST and one time with 0.1 M PBN to remove residual Cl-. Hybridization procedures for multiplexed target. Four kinds of capture oligonucleotides C-CMV(10 µL 1 µM), C-HBV(10 µL 1 µM), C-EBV(10 µL 1 µM), C-HSV(10 µL 1 µM) were mixed separately with 10 µM mercaptopropanol and then the resulting mixture solution was added to the electrodes of the Au chip. The immobilization was carried out by incubating overnight at 4 °C. It was then washed by 0.1 M PBST twice (8 min/time) at 30 rpm. The 40 µL mixture sample containing all three sequences (each containing 100 amol/L oligos) were added onto the Au chip at 37 °C for 45 min with gentle shaking at 10 rpm. After washing twice with 0.1 M PBST at 30 rpm, 40 µL of detection tag solution was added at 37 °C for 15 min with gentle shaking at 10 rpm. Finally, the Au chip was thoroughly washed three times with 0.1 M PBST and one time with 0.1 M PBN to remove residual Cl-. Measurement. The silver aggregate tags were then measured by using differential pulse voltammetric detection. All DPV data were collected by model 660 Electrochemical Workstation. (CH Instruments Inc., Austin, TX). The DNA hybrids were assessed with DPV method based on oxidation current of silver nanoparticles in 0.1 M PBS (pH 7.0). The concentration of target oligonucleotide was quantified by the peak area of oxidation current of the silver nanoparticles coupled on the electrodes. A two-electrode system consisted of the gold electrode on the arrays as the working electrode and a saturated calomel electrode (SCE) as a reference electrode were employed to carry out DPV measurements. Potential window spans from -100 to 600 mV; pulse amplitude, 25 mV; pulse width, 60 ms; scan rate, 20 mV/s. The reference electrode was separated from the working electrode by a double electrolytic salt bridge filled with saturated 0.1 M NaNO3 in order to avoid the interference of chloride. RESULTS AND DISCUSSION Preparation of Oligonucleotide-Functionalized Silver Aggregate Tags. Silver based DNA tags have a number of advantages for electrochemical detection such as lower oxidation potential and facile dissolution condition over a gold nanoparticle.19,20,22-25,27-29 However, most electrochemical analytical methods use metallic gold nanoparticles due to the difficulty in the modification of silver nanoparticles. Main problems of chemical degradation of the silver nanoparticles and the susceptibility of silver surface to oxidation limit their widespread use.30,31 (27) Wang, J.; Xu, D.; Polsky, R. J. Am. Chem. Soc. 2002, 124, 4208–4209. (28) Karadeniz, H.; Erdem, A.; Caliskan, A.; Pereira, C. M.; Pereira, E. M.; Ribeiro, J. A. Electrochem. Commun. 2007, 9, 2167–2173. (29) Gao, M.; Qi, H.; Gao, Q.; Zhang, C. Electroanalysis 2008, 20, 123–130. (30) Vidal, B. C., Jr.; Deivaraj, T. C.; Yang, J.; Too, H.-P.; Chow, G.-M.; Gan, L. M.; Lee, J. Y. New J. Chem. 2005, 29, 812–816.

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Figure 1. UV-visible spectrum of the synthesized silver colloid (black line), silver Conjugate 1 solution before (red line) and after centrifugation (yellow line), hybridization-induced silver aggregate 1 solution (blue line).

Recently, various stabilizers including dendrimers and hyperbranched polymers as well as derived anchoring groups have been developed to modify silver nanoparticles and enhance their stability.32-35 It has also been reported that specific DNA sequences such as poly adenine (A) could be successfully coupled with AgNPs and enable particle stabilization.30,31 The preparation of silver aggregate tags is schematically drawn in Scheme 1a. Silver nanoparticles were first synthesized in a NaBH4 solution according to Munro’s method.36 The resulting silver nanoparticles were further functionalized by a mixture containing 5′-SH-oligo(d)A15 and 5′-SH TTGGC CAGGA CACGT3′, where the sequence is from the virus hepatitis B. The obtained bistranded -modified nanoparticles (Conjugate 1) have the same wavelength absorbance maximum as the bare silver nanoparticles (400 nm, Figure 1), which suggests that the size of the nanoparticles unchanged after the modification. The loss of silver nanoparticles in the modification reaction leads to decrease in absorbance of UV-vis after centrifugation since unmodified silver nanoparticles are unstable. The oilgo(d)Tderived silver nanoparticles (Conjugate 2) were prepared by introducing SH-oligo(d)T15 into the silver colloidal dispersion. The yield of Conjugate 2 was lower than that of Conjugate 1. The preparation of silver nanoparticle aggregates was accomplished by mixing an equal number of Conjugates 1 and 2 in NaCl. After hybridization, the color of the solution changed to pale red from bright yellow, with the absorption spectrum showing a red shift from 400 to 525 nm. The absorbance maximum decreased slightly and band broadening was observed after hybridization-induced aggregation, suggesting the increase in the (31) Tokareva, I.; Hutter, E. J. Am. Chem. Soc. 2004, 126, 15784–15789. (32) Lee, J.-S.; Lytton-Jean, A.K. R.; Hurst, S. J.; Mirkin, C. A. Nano Lett. 2007, 7, 2112–2115. (33) Dougan, J. A.; Karlsson, C.; Smith, W. E.; Graham, D. Nucleic Acids Res. 2007, 35, 3668–3675. (34) Liu, S.; Zhang, Z.; Han, M. Anal. Chem. 2005, 77, 2595–2600. (35) Sun, Y.; Wang, D.; Gao, J.; Zheng, Z.; Zhang, Q. J. Appl. Polym. Sci. 2007, 103, 3701–3705. (36) Munro, C. H.; Smith, W, E.; Garner, M.; Clarkson, J; White, P. C. Langmuir 1995, 11, 3712–3720. (37) Liu, C.-H.; Li, Z.-P.; Du, B.-A.; Duan, X.-R.; Wang, Y.-C. Anal. Chem. 2006, 78, 3738–3744. (38) Wang, J.; Xu, D.; Chen, H.-Y. Electrochem. Commun. 2009, 11, 1627–1630.

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Figure 2. TEM images of silver nanoparticle Conjugate 2 (a); silver nanoparticle Conjugate 3 (b); and silver nanoparticle agggregate Tag 2 (c-e).

size of nanoparticles produced a corresponding decrease in the particle numbers. For the preparation of Conjugate 3, the silver nanoparticles were modified by using capture probe mixture containing oligonucleotides for EBV, HSV, CMV, and oligo(d) A (see the inset of Scheme 1b). No significant change in color between Conjugate 1 and Conjugate 3 was observed. The size and morphology of quadruple functionalized nanoparticles and the aggregate tag were characterized by TEM (Figure 2). Most silver nanoparticles are spheres and have average diameters of 16.9 ± 3.3 nm and 15.5 ± 2.2 nm for Conjugates 2 and 3, respectively. To describe an average diameter of the aggregated nanoparticles, dynamic light scattering measurement has been carried out. The results show that an average diameter of the aggregated nanoparticles is 410.4 nm and relatively variation is 11.6%. It was estimated that an aggregate tag contained about 1.7 × 104 16-nm-diameter nucleotide-derived Ag nanoparticles. Since the coverage of oligo(d)A15 on the surface of Conjugate 1 was larger than that of Conjugate 3, a higher concentration of salt was necessary to increase the chance of proximity while irreversible conglomeration should be avoided. In our experiment, we found that 0.1 M PBS was sufficient to make hybridization of Conjugate 1 and Conjugate 2 (AgNPs-T15) possible, but a higher concentration (0.2 M PBS) was found to be needed when Conjugate 3 was intended to hybridize with AgNPs-T15. It has been reported that thiolated-oligo(d)A can be more readily attached to silver nanoparticles due to the greater affinity of adenine than thiolated-oligo(d)T.30 Thus in our modification strategy oligo(d)A was employed as a stabilizer to mix with the probes and facilitate functionalization of silver nanoparticles. Oligo(d)A can induce aggregation of silver nanoparticles as a programmable linker. The hybridization-induced aggregation was a reversible process and the color of the aggregates would be reversed to the original bright yellow when heated. Although it was reported previously that the success of DNA functionalization strongly depends on the sequence of the oligonucleotides, no detailed information has been reported on how to select and design sequences for functionalizing the silver nanoparticles. Our results show that oligo(d)A can play an important role in the multioligonucleotide functionalization of the nanoparticles. Hy-

Figure 3. Detection of HBV target at different concentrations. (a) Different pulse voltammograms recorded for a series concentration of HBV target (1) blank, (2) 10 aM, (3) 100 aM, (4) 1 fM, (5) 10 fM, (6) 100 fM; (b) calibration curves of HBV target based on silver nanoparticle aggregates and (c) single silver nanoparticle conjugates; (d) different pulse voltammograms recorded for 0.1 M PBS (1), a control (2), and 5 aM HBV target (3).

bridization-induced aggregates shown here are more stable and thus readily controllable. To our best knowledge, the multiprobe hybridization-induced DNA aggregate tag has not been reported yet. Development of Electrochemical Detection Strategy. Most previous methods of electrochemical measurement involving silver labels first oxidize metallic silver into silver ions, which are then reduced and assayed electrochemically.21-24 The differential pulse voltammetric detecting metallic silver label was first reported to assay biotinylated DNA target with metallic silver deposited on the gold nanoparticles by the addition of a silver enhancer. It showed advantages of in situ testing, simplicity and rapidity over the detection of silver ions.27 Recently, amine-functionalized Ag nanoparticles have been used to assay DNA targets via interaction between positively charged Ag nanoparticles and negatively charged single-strand DNA target.25 We develop here oligonucleotide-functionalized Ag nanoparticles, which have not yet been reported for electrochemical assay of DNA targets. Figure 3a shows a serial of voltammgrams, corresponding to different target concentrations. The peak current is found at 0.15 V vs Hg/Hg2Cl2, which corresponds to the oxidation of metallic silver, and it increases with the concentrations of the DNA targets. The dynamic concentration range was determined by varying the concentration of the DNA target in the sample solution while keeping the concentration of silver aggregate tags constant, and the experiment was repeated three times. As shown in Figure 3b, a calibration curve is linear in the range from 10 aM to 100 fM, in which the difference of peak areas corresponds to the increased peak area values (subtracting the peak area values of the blank sample).

Figure 4. AFM images of the gold electrode surface modified with C-HBV oligomer and mercaptoethanol before (a), after hybridization with 100 aM HBV target (b), chronoamperometric treatment at the initial stage (c), and at the last stage (d). Chronoamperometric parameters: initial potential, 0 V; high potential, 1 V; pulse width, 600 s; sample interval, 0.01 s.

To determine the limit of detection, 5 aM of the DNA target, a noncomplementary target and a blank solution were measured at the same analytical conditions (Figure 3d). As a result, the signal of 5 aM target DNA significantly larger than the signal sum (signal plus 3 times of standard deviation) of the blank or the control. This corresponds to the detection of 20 zmol (∼120 molecules) of the target in the 40 µL sample solution. For comparison, single silver nanoparticles labeled with D-HBV and oligo(d)A were also tested. The results showed that the peak area of the current increased with the concentration of the DNA targets (Figure 3b inset) and a detection limit of 10 fmol/L was obtained. This detection limit is in the same range with a previous report25 but is substantially higher than the aggregate tag developed here. Based on this result, silver aggregate tags show a significant increase in the detection sensitivity and produced a 1000-fold signal amplification. Surface Characterization. To explore the possible mechanism of amplification with silver aggregate tags, the morphological changes on the electrode surface was examined by using atomic force microscopy. Figure 4a-d shows the changes in the surface morphology before and after electrochemical oxidation. Compared to the electrode surface modified with capture probes (Figure 4a), the conglomeration with 400-500 nm of diameter appear after hybridization with the aggregate tags (Figure 4b). This agrees well with the size of aggregate tags obtained by TEM (Figure 2c). To study the effect of electrochemical oxidation on the silver aggregates, a constant potential was applied to initiate oxidation of silver tags. To our surprise, a large aggregate was formed whereas the gaps between the individual tags gradually disapAnalytical Chemistry, Vol. 82, No. 13, July 1, 2010

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Figure 5. Signal intensity of the arrayed electrodes obtained by DPV for human virus oligonucleotides. (a) 100 aM T-CMV, (b) 100 aM T-EBV, (c) 100 aM T-HSV, (d) mixture sample containing 100 aM T-CMV, EBV, and HSV. Insert are the voltammograms for the mixture sample, (e) different pulse voltammogram of mixture sample containing T-CMV, EBV, and HSV, each concentration is 100 aM. Curve 1, 2, 3, and 4 represent C-CMV, C-EBV, and C-HSV and C-HBV modified electrodes, respectively.

peared (Figure 4c). In addition, the surface density of the aggregate decreased considerably. This could be attributed to simultaneously oxidation due to their conductive property of the individual aggregates composed of metallic silver and oligonucleotides, and the aggregates fuse together in response to potential applied on the electrodes. With further oxidation, the formed conglomeration diffused into the margin and the height of the structure became lower (Figure 4d). This is likely due to loss of the materials to the bulk upon the pass of a large current. Given these, we propose that the amplified signal stems from cooperative oxidation of whole silver nanoparticles in the aggregates, which is different from that with deposited silver on gold nanoparticles.27 Multiplexed DNA Measurements with Multiprobe Tag and Arrayed Electrode. The advantage of silver aggregate tags for detection of DNA arrays is further demonstrated with parallel detection of DNA sequences of human viruses. Three oligonucleotide targets were selected as a concept-of-principle and their sequences from EBV, HSV, CMV have been reported previously on DNA arrayed electrodes.7 The detection scheme, shown in Scheme 1b, involves the use of multiprobe-derived silver aggregate tags, which consist of Conjugates 2 and 3 and can simultaneously recognize three target sequences due to the complementary probes on Conjugate 3. An array chip comprised of 10 subarray units was fabricated by thermal deposition and each subarray contained four gold electrodes.38 The capture probes corresponding to EBV, HSV, CMV, and HBV (as a negative control) 5482

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sequences were immobilized on the gold electrodes by thiol chemisorption. Figure 5a-c show the measurement of DNA target fragments (HSV, EBV and CMV, each containing 100 amol/L) by use of silver aggregate tags in combination with DPV. HBV sequence (C-HBV) was used as a control and its peak area was used as a reference to normalize the current signals for all other DNA samples and maximum analytical signals were observed only at positions modified with the corresponding capture probe. The sample containing all three sequences (each containing 100 amol/L oligos) was assayed (Figure 5d). The comparable signal indicates that the multiprobe tag can simultaneously detect multiplexed targets in one shot with satisfactory sensitivity. Tag 2 produces a positive shift of the peak potential (Figure 5e). This might be attributed to different sizes between Tags 1 and 2 since there is dependence of potential on silver colloid size.39 When the ratio of oligo A decreased in the modification of DNA conjugated silver particles, the degree of aggregation reduced. However, this shift does not affect the accuracy of the electrochemical measurements. A low degree of nonspecific signal was observed on the negative control electrode, which could be attributed to the weak adsorption of silver nanoparticle aggregates on the electrode surface. This observation suggests that arrayed electrodes are advantageous for assessing specificity as compared (39) Jones, S. E. W.; Campbell, F. W.; Baron, R.; Xiao, L.; Compton, R. G. J. Phys. Chem. C 2008, 112, 17820–17827.

to single biosensing electrodes such that this background influence could be deducted by normalization. A high degree of homology could be the source of cross reactivity since the signals from irrelevant electrodes remained existence. For example, T-CMV/T-EBV and T-CMV/T-HSV have 36.7% (11 in 30 bases) and 33.3% same nucleotide bases in both sequences. This interference could decrease by further optimization of target sequences. Compared to the classic electrochemical stripping method, differential pulse voltammetric method could directly and rapidly assay metallic nanoparticles. Based on this biosensing method, electrochemical aggregate tag could open a new approach to gain high detection sensitivity and high throughput.

aggregates. We have proved that oligonucleotide-silver nanoparticle aggregates are attractive probes for ultrasensitive detection and enable multiplexed electrochemical measurement of low concentrations of DNA sequences via a cooperative oxidation mechanism. In addition, the differential pulse voltammteric method shows advantages of in situ testing and simplicity over traditional stripping voltammetry, especially for detection of metallic nanoparticle tags. The presented method is thus applicable to multiplexed target assay on the arrayed electrodes by skipping dissolution process of the labeled nanoparticls. This work may open new avenues for ultra sensitive, PCR-less detection of low concentrations of genomics materials.

CONCLUSIONS Nanoparticle-based analytical materials have played an important role in the development of detection methodologies. Compared to gold nanoparticles in optical detection, excellent property of the silver nanoparticles for electrochemical assays has not been extensively exploited. This is possibly due to the difficulty in synthesis and proper modification of silver nanoparticles. In this work, we have successfully developed multiprobe-modified silver nanoparticles and hybridization-induced silver nanoparticle aggregates. The use of oligo(d)A not only facilitates stability of silver nanoparticles, but also acts as a programmable linker for the

ACKNOWLEDGMENT We acknowledge financial support of National Natural Foundation of China (Grant Nos. 20975050, 20575079, 20890020, and 20775033), The National Science Funds for Creative Research Groups (No. 20821063) and National Basic Research Program of China (973 Program, Nos. 2006CB910803 and 2007CB936404).

Received for review January 22, 2010. Accepted June 2, 2010. AC101193E

Analytical Chemistry, Vol. 82, No. 13, July 1, 2010

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