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Fabrication of a highly sensitive sensor electrode using a nano-gapped gold particle film
Solid State Ionics 177 (2006) 2317 – 2320 www.elsevier.com/locate/ssi
Fabrication of a highly sensitive sensor electrode using a nano-gapped gold particle film Shiho Tokonami, Masashi Iwamoto, Ken Hashiba, Hiroshi Shiigi ⁎, Tsutomu Nagaoka Frontier Science Innovation Center, Osaka Prefecture University, 1-2 Gakuen-cho, Sakai 599-8570, Japan Received 6 October 2005; received in revised form 7 July 2006; accepted 31 July 2006
Abstract Controlling a gap between electrodes arranged in nanometer size has a great potential for a highly sensitive detection. We have successfully fabricated a sensor based on a nano-gapped gold particle film consisting of gold nanoparticle–alkylchain–gold nanoparticle repeated sequences in a straightforward manner. Here we report on its application to direct DNA analysis, enabling us to read out a specific complementary DNA through electronic protocols. © 2006 Elsevier B.V. All rights reserved. Keywords: Nano-gap; Electrical detection; Gold nanoparticle; DNA sensing
1. Introduction DNA detection technology plays an important role in the field of modern life science including drug discovery, diagnosis of single nucleotide polymorphism (SNP) and tailor-made cure. Fluorescence-based DNA chips have already been leveraged in researches on gene expression analysis. However, there are still many problematic aspects, such as the requirement of sophisticated optical set-up, high cost and instability in the fluorescence intensity. Therefore, the development of a simple, rapid and user friendly detection method for specific DNA sequences has become increasingly important. In order to take over the above problems, some attempts to use metal nanoparticles for chemical and biological sensing, such as colorimetric [1,2], surface plasmon resonance [3] and microgravimetric [4] DNA detections have been reported. In particular, electronic detection has been recognized as a promising technique because of its straightforwardness in signal interfacing and processing as well as in the integration of a DNA microarray system. Although a native double-stranded (ds)DNA had been regarded as an insulator, some recent studies have suggested that π overlapping between adjacent base pairs makes dsDNA superconductive [5],
conductive [6] or semiconductive [7,8] through electron transfer mechanisms, such as electron and hole hopping. Mirkin et al. have reported that Au particles were captured in between a micrometer-gapped electrode by the hybridization of DNA strands [9]. However, the distance between the particles was too large to monitor the current between the particles without a post procedure, such as silver staining. In this connection, precise control of the electrode gap in nanometer range is a crucial factor for direct DNA detection. Therefore, we have focused on a selfassembly property of Au particle and alkylthiol, and attempted to fabricate a sensor based on a nano-gapped Au particle film consisting of Au nanoparticle–alkylchain–Au nanoparticle repeated sequences in which each gap between abutting particles can be precisely controlled by the length of alkylchain [10–14]. In this paper, the electrical property and the surface condition change of the film in the film formation process were discussed and the film was applied to direct DNA detection, reading out the very little resistance change of DNA itself caused by hybridization and decomposition purely with the electrical protocols. 2. Experimental 2.1. Preparation of Au nanoparticle
⁎ Corresponding author. E-mail address: [email protected] (H. Shiigi). 0167-2738/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ssi.2006.07.048
Au nanoparticles with 12, 30 and 80 nm diameter were prepared in the literature procedure.
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80 nm: 10 mL of 3.0% citric acid as a reducer was added into 200 mL of 0.03% aqueous chloroauric acid, and then the mixture was stirred at 80 °C for 20 min [15]. 30 nm: 4.5 mL of 2.0% sodium citrate as a reducer was added into 200 mL of 0.03% aqueous chloroauric acid, and then the mixture was stirred at 80 °C for 20 min [16]. 12 nm: 5.2 mL of 2.0% L(+)-ascorbic acid sodium salt as a reducer and 6 mL of potassium carbonate was added into 200 mL of 0.03% aqueous chloroauric acid, and then the mixture was stirred at 5 °C. The color of the solution at this point turns to a purple-red. The mixture was stirred at 80 °C for 20 min until the color of the suspension turned from purple-red to red [16].
2.2. Preparation of Au particle film A comb-shaped microelectrode having two rows of 65 teeth of Pt electrodes with 5 μm space fabricated on a quartz glass plate (3.6 mm2, NTT-AT, Japan) was utilized for the preparation of Au–alkylchain hybrid monolayer. After preliminary electrochemical cleaning by repeated potential sweeps between − 0.25 and + 1.3 V vs. Ag/AgCl in an aqueous solution of 0.1 M H2SO4, the electrode was immersed into a binder solution, 5 mM ethanolic 1,10-decanedithiol, for 30 min and then immersed in the Au dispersion for 30 min at room temperature. These procedures were repeated up to 6 times to modify the glass surface with the Au particle film (Fig. 1A).
Fig. 1. (A) Illustration of the procedure for the fabrication of the Au particle film. (B) FE–SEM images of the comb-shaped microelectrode for bare (a), after 1 (b), 6 (c) and 9 (d) dipping cycles to the binder solution and Au dispersion. The inset is the close-up AFM image of the Au particle film.
S. Tokonami et al. / Solid State Ionics 177 (2006) 2317–2320
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The surface of the Au particle film was characterized by using a scanning electron microscope (S-4700, Hitachi, Japan) with an applied voltage of 10 kV and atomic force microscopy (SPM-9500, Shimadzu, Japan) equipped with a dynamic modemicrocantilever (AC160TS, Olympus, Japan) [17]. 2.3. DNA detection using nano-gapped electrode Artificially synthesized DNA used in this study was purchased from Nisshinbo Co. Japan. We used a 12-mer single-stranded (ss) deoxyoligonucleotide probe modified with thiol at 5′-phosphate end (HS-5′-TCT CAA CTC GTA-3′). The Au particle film was modified by 5 pmol of the probe in a 5 μL of pH 7.5 TE buffer solution (10 mM Tris–HCl, 1 mM EDTA and 1 M NaCl). The modified electrode was then left at rest for 30 min. Finally, the surface of the film was rinsed with the buffer. The modification of the probe DNA on the Au surface was confirmed by XPS analysis through an intense N1s peak at 399.0 eV arising from purine and pyrimidine bases in DNA. After the probe DNA modification, 1 μL TE buffer solution was spread over the Au particle film to measure a base resistance. Afterwards, 5 μL TE buffer involving a target ssDNA (5′-TAC GAG TTG AGA-3′) which was complemented to the ss-probe DNA strand was seeped onto the probe modified Au particle film. The film resistance changes due to the hybridization between the probe and sample DNA strands were measured with a standard digital multimeter (HewlettPackard Model 34401A) operating at a constant current mode of 1 mA. All the measurements were made in a Faraday cage regulated at 298 K. Followed by resistance measurement due to DNA hybridization, 10 μL of DeoxyribonucleaseI (DNaseI: 10 mg/mL) derived from bovine pancreas (Wako, Japan) and 5 mM MgCl2 as an enzyme activator, both dissolved in 5 mM Tris–HCl solution, was added over the Au particle film. 3. Results and discussion 3.1. Electrical property and surface analysis of Au particle film The film formation process is illustrated in Fig. 1A; decanedithiol was adsorbed on the Pt-comb electrode by
Fig. 2. Dependence of the Au particle film resistance on the repeated immersing sequence time to the binder and Au particle in a respective Au particle diameter of 80 (a), 30 (b) and 12 nm (c).
Fig. 3. Time course of the film resistance by applying a complementary DNA (a) and DNaseI (b) at the respective arrow marks on the Au particle film.
dipping it into the binder solution (1), and then, the Au particle was self-assembled through the sulfur group of the binder molecule (2). The electrode was again dipped into the binder solution (3). These repeated dipping cycles enabled us to produce a film with an equally spaced nano-gap created by the binder molecule. The gap interval between each particle can be adjusted by the length of alkylchain of dithiol (ca. 1.3 nm) [13,17]. We have observed the surface of the comb-shaped microelectrode in the course of film formation using 80 nm particle (Fig. 1B). The surface of the bare electrode was flat and smooth (a) whereas particles adsorbed not only on the Pt electrode but also on the glass substrate even in the first dipping cycle (b), which corresponds to the state of the second dipping process (2) in Fig. 1A. This indicates the existence of the unspecific adsorption of the binder molecule to the glass since particles hardly adsorbed to the electrode without binder, which led to the adsorption of the Au nanoparticle on the glass. While it was observed that the particles adsorbed thickly and uniformly both on the Pt electrode and glass substrate, and the film grew monolayer mostly after 6 dipping cycles (c). Therefore, it was figured out that the optimal number of dipping cycles for fabricating the monolayer film was approximately 6 cycles, while the larger dipping cycles, approximately over 9 dipping cycles, makes the film multilayer about 3 layers (d). The electrical resistance of the Au particle film decreased dramatically with an increase in the number of dipping cycles, as shown in Fig. 2. In the case of using 80 nm particle, the resistance decreased precipitously by 6 orders of magnitude at the second immersion and became constant ∼ 100 Ω for further dipping. The film resistance prepared from 30 nm particle dropped in a less drastic way and then diminished gradually at third dipping. Such reduction of resistance occurred at the fourth immersion in the case of 12 nm diameter particle. These indicated that the Pt electrode gap was filled with a conducting metal particle with the repetitive procedures. Larger particle size, such as 80 nm, can form a conductive path at a much earlier stage of the immersion than smaller particles (30 and 12 nm). Moreover, the resistivity of the film prepared here (30 Ω cm) gave a close agreement with the value (20 Ω cm) reported by L. Han et al. for nonanedithiol (ca. 1.1 nm) [10].
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It was expected geometrically that the formation of the crosslinked structure with Au particles of the gap interval (5 μm) between the Pt electrodes requires over 30, 80 and 200 dipping sequences for 80, 30 and 12 nm particles, respectively for overall coverage. However, 2, 3 and 4 times repeated sequences were enough to build up such a Au particle film. It can be attributed to the unspecific adsorption of Au nanoparticles on the glass substrate observed in the first dipping cycle in Fig. 1B (b). Such particles on the glass work as a nucleus for the next adsorption, accelerating the film accomplishment much earlier than geometric anticipation. The gap interval between abutting particles is regulated by the used binder molecule. It was explained by our previous report about the fabrication of a Au particle film on a polymer substrate using Au nanoparticle and alkylthiol as a binder [13]. The resistance of the film depended on the length of the alkylchain of the binder; the shorter binder led to lower film resistance, while the larger binder led to higher film resistance. It suggests that the space between particles is well-controlled by the length of the alkylchain of the binder. Further, even if Au particles are adsorbed excessively by excessive dipping sequences up to the formation of a multilayer as shown in Fig. 1B(d), the resistance of the film never go down under 70 Ω, suggesting that each abutting particle never come into contact and keep distance from each other. It is therefore, the space formed between abutting particles that must be kept by the length of the alkylchain of the binder. Moreover, the film had a mechanical strength enough to carry out following electrical measurements since it did not change in morphology and resistance after repeated potential sweeps in the range − 0.25 to + 1.3 V vs. Ag/AgCl in an 0.1 M aqueous H2SO4. 3.2. Response to the complementary DNA To measure a resistance change caused by DNA hybridization, the electrode was preconditioned by adding a 1 μL of TE buffer solution on the electrode for 2 min. As seen in Fig. 3, the resistance was decreased rapidly on the addition of 500 pmol ss-complementary DNA (a). Even with this simple experimental set-up, high S/N ratios above 20 were obtained. Furthermore, no detectable change in film morphology by hybridization, observed by AFM, explains stability in resistance. This resistance reduction, ΔR, would be attributed to the formation of dense π overlapping of the base pairs in the DNA double helix, promoting the electron transfer between Au particles. To make sure that the DNA hybridization caused the resistance change, DNaseI enzyme that specifically breaks dsDNA down at its phosphodiester positions was applied to the film after hybridization [7]. After the addition of DNaseI, the resistance was restored in 40 min to the original value. The resistance recovery can be attributed to the decomposition of dsDNA located between the Au particles with DNaseI, which
breaks the conducting path having been created by hybridization. On the contrary, the resistance remained constant over 1 h after hybridization unless the enzyme was added. These indicated that the resistance reduction was surely caused by the DNA hybridization [6–8]. 4. Conclusions We have successfully fabricated a highly sensitive sensor based on a nano-gapped gold particle film, utilizing alkyldithiol as a binder molecule. This technique allows us not only to control the resistance of the film but also to adjust the electrode gap through self-assembly procedures. Precisely controlled electrode gap enabled us to detect a small change caused by 12 base pair DNA both by hybridization and degradation directly with the electronic protocols. Acknowledgements This study was supported by the Industrial Technology Research Grant Program in '05 from the New Energy and Industrial Technology Development Organization (NEDO). One of authors, S.T., is thankful for the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (JSPS). References [1] C.A. Mirkin, R.L. Letsinger, R.C. Mucic, J.J. Storhoff, Nature 382 (1996) 607. [2] K. Sato, K. Hosokawa, M. Maeda, J. Am. Chem. Soc. 125 (2003) 8102. [3] K. Nakatani, S. Sando, I. Saito, Nat. Biotechnol. 19 (2001) 51. [4] T. Liu, J. Tang, H. Zhao, Y. Deng, L. Jiang, Langmuir 18 (2002) 5624. [5] A.Yu. Kasumov, M. Kociak, S. Guéron, B. Reulet, V.T. Volkov, D.V. Klinov, H. Bouchiat, Science 291 (2001) 280. [6] H.-W. Fink, C. Schönenberger, Nature 398 (1999) 407. [7] D. Porath, A. Bezryadin, S. de Vries, C. Dekker, Nature 403 (2000) 635. [8] Y. Okahata, T. Kobayashi, K. Tanaka, M. Shimomura, J. Am. Chem. Soc. 120 (1998) 6165. [9] S.-J. Park, T.A. Taton, C.A. Mirkin, Science 295 (2002) 1503. [10] L. Han, D.R. Daniel, M.M. Maye, C.J. Zhong, Anal. Chem. 73 (2001) 4441. [11] P. Zamborini, M.C. Leopold, J.F. Hicks, P.J. Kulesza, M.A. Malik, R.W. Murray, J. Am. Chem. Soc. 124 (2002) 8958. [12] T. Sato, H. Ahmed, D. Brown, B.F.G. Johnson, J. Appl. Phys. 82 (1997) 696. [13] H. Shiigi, Y. Yamamoto, H. Yakabe, S. Tokonami, T. Nagaoka, Chem. Commun. (2003) 1038. [14] T. Ogawa, K. Kobayashi, G. Masuda, T. Takase, S. Maeda, Thin Solid Films 393 (2001) 374. [15] J. Turkevich, P.C. Stevenson, J. Hillier, Discuss. Faraday Soc. 11 (1951) 55. [16] G.T. Hermanson, Bioconjugate Techniques, Academic Press, 1996, p. 598. [17] H. Shiigi, S. Tokonami, H. Yakabe, T. Nagaoka, J. Am. Chem. Soc. 127 (2005) 3280.
Fabrication of a highly sensitive sensor electrode using a nano-gapped gold particle film Shiho Tokonami, Masashi Iwamoto, Ken Hashiba, Hiroshi Shiigi ⁎, Tsutomu Nagaoka Frontier Science Innovation Center, Osaka Prefecture University, 1-2 Gakuen-cho, Sakai 599-8570, Japan Received 6 October 2005; received in revised form 7 July 2006; accepted 31 July 2006
Abstract Controlling a gap between electrodes arranged in nanometer size has a great potential for a highly sensitive detection. We have successfully fabricated a sensor based on a nano-gapped gold particle film consisting of gold nanoparticle–alkylchain–gold nanoparticle repeated sequences in a straightforward manner. Here we report on its application to direct DNA analysis, enabling us to read out a specific complementary DNA through electronic protocols. © 2006 Elsevier B.V. All rights reserved. Keywords: Nano-gap; Electrical detection; Gold nanoparticle; DNA sensing
1. Introduction DNA detection technology plays an important role in the field of modern life science including drug discovery, diagnosis of single nucleotide polymorphism (SNP) and tailor-made cure. Fluorescence-based DNA chips have already been leveraged in researches on gene expression analysis. However, there are still many problematic aspects, such as the requirement of sophisticated optical set-up, high cost and instability in the fluorescence intensity. Therefore, the development of a simple, rapid and user friendly detection method for specific DNA sequences has become increasingly important. In order to take over the above problems, some attempts to use metal nanoparticles for chemical and biological sensing, such as colorimetric [1,2], surface plasmon resonance [3] and microgravimetric [4] DNA detections have been reported. In particular, electronic detection has been recognized as a promising technique because of its straightforwardness in signal interfacing and processing as well as in the integration of a DNA microarray system. Although a native double-stranded (ds)DNA had been regarded as an insulator, some recent studies have suggested that π overlapping between adjacent base pairs makes dsDNA superconductive [5],
conductive [6] or semiconductive [7,8] through electron transfer mechanisms, such as electron and hole hopping. Mirkin et al. have reported that Au particles were captured in between a micrometer-gapped electrode by the hybridization of DNA strands [9]. However, the distance between the particles was too large to monitor the current between the particles without a post procedure, such as silver staining. In this connection, precise control of the electrode gap in nanometer range is a crucial factor for direct DNA detection. Therefore, we have focused on a selfassembly property of Au particle and alkylthiol, and attempted to fabricate a sensor based on a nano-gapped Au particle film consisting of Au nanoparticle–alkylchain–Au nanoparticle repeated sequences in which each gap between abutting particles can be precisely controlled by the length of alkylchain [10–14]. In this paper, the electrical property and the surface condition change of the film in the film formation process were discussed and the film was applied to direct DNA detection, reading out the very little resistance change of DNA itself caused by hybridization and decomposition purely with the electrical protocols. 2. Experimental 2.1. Preparation of Au nanoparticle
⁎ Corresponding author. E-mail address: [email protected] (H. Shiigi). 0167-2738/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ssi.2006.07.048
Au nanoparticles with 12, 30 and 80 nm diameter were prepared in the literature procedure.
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S. Tokonami et al. / Solid State Ionics 177 (2006) 2317–2320
80 nm: 10 mL of 3.0% citric acid as a reducer was added into 200 mL of 0.03% aqueous chloroauric acid, and then the mixture was stirred at 80 °C for 20 min [15]. 30 nm: 4.5 mL of 2.0% sodium citrate as a reducer was added into 200 mL of 0.03% aqueous chloroauric acid, and then the mixture was stirred at 80 °C for 20 min [16]. 12 nm: 5.2 mL of 2.0% L(+)-ascorbic acid sodium salt as a reducer and 6 mL of potassium carbonate was added into 200 mL of 0.03% aqueous chloroauric acid, and then the mixture was stirred at 5 °C. The color of the solution at this point turns to a purple-red. The mixture was stirred at 80 °C for 20 min until the color of the suspension turned from purple-red to red [16].
2.2. Preparation of Au particle film A comb-shaped microelectrode having two rows of 65 teeth of Pt electrodes with 5 μm space fabricated on a quartz glass plate (3.6 mm2, NTT-AT, Japan) was utilized for the preparation of Au–alkylchain hybrid monolayer. After preliminary electrochemical cleaning by repeated potential sweeps between − 0.25 and + 1.3 V vs. Ag/AgCl in an aqueous solution of 0.1 M H2SO4, the electrode was immersed into a binder solution, 5 mM ethanolic 1,10-decanedithiol, for 30 min and then immersed in the Au dispersion for 30 min at room temperature. These procedures were repeated up to 6 times to modify the glass surface with the Au particle film (Fig. 1A).
Fig. 1. (A) Illustration of the procedure for the fabrication of the Au particle film. (B) FE–SEM images of the comb-shaped microelectrode for bare (a), after 1 (b), 6 (c) and 9 (d) dipping cycles to the binder solution and Au dispersion. The inset is the close-up AFM image of the Au particle film.
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The surface of the Au particle film was characterized by using a scanning electron microscope (S-4700, Hitachi, Japan) with an applied voltage of 10 kV and atomic force microscopy (SPM-9500, Shimadzu, Japan) equipped with a dynamic modemicrocantilever (AC160TS, Olympus, Japan) [17]. 2.3. DNA detection using nano-gapped electrode Artificially synthesized DNA used in this study was purchased from Nisshinbo Co. Japan. We used a 12-mer single-stranded (ss) deoxyoligonucleotide probe modified with thiol at 5′-phosphate end (HS-5′-TCT CAA CTC GTA-3′). The Au particle film was modified by 5 pmol of the probe in a 5 μL of pH 7.5 TE buffer solution (10 mM Tris–HCl, 1 mM EDTA and 1 M NaCl). The modified electrode was then left at rest for 30 min. Finally, the surface of the film was rinsed with the buffer. The modification of the probe DNA on the Au surface was confirmed by XPS analysis through an intense N1s peak at 399.0 eV arising from purine and pyrimidine bases in DNA. After the probe DNA modification, 1 μL TE buffer solution was spread over the Au particle film to measure a base resistance. Afterwards, 5 μL TE buffer involving a target ssDNA (5′-TAC GAG TTG AGA-3′) which was complemented to the ss-probe DNA strand was seeped onto the probe modified Au particle film. The film resistance changes due to the hybridization between the probe and sample DNA strands were measured with a standard digital multimeter (HewlettPackard Model 34401A) operating at a constant current mode of 1 mA. All the measurements were made in a Faraday cage regulated at 298 K. Followed by resistance measurement due to DNA hybridization, 10 μL of DeoxyribonucleaseI (DNaseI: 10 mg/mL) derived from bovine pancreas (Wako, Japan) and 5 mM MgCl2 as an enzyme activator, both dissolved in 5 mM Tris–HCl solution, was added over the Au particle film. 3. Results and discussion 3.1. Electrical property and surface analysis of Au particle film The film formation process is illustrated in Fig. 1A; decanedithiol was adsorbed on the Pt-comb electrode by
Fig. 2. Dependence of the Au particle film resistance on the repeated immersing sequence time to the binder and Au particle in a respective Au particle diameter of 80 (a), 30 (b) and 12 nm (c).
Fig. 3. Time course of the film resistance by applying a complementary DNA (a) and DNaseI (b) at the respective arrow marks on the Au particle film.
dipping it into the binder solution (1), and then, the Au particle was self-assembled through the sulfur group of the binder molecule (2). The electrode was again dipped into the binder solution (3). These repeated dipping cycles enabled us to produce a film with an equally spaced nano-gap created by the binder molecule. The gap interval between each particle can be adjusted by the length of alkylchain of dithiol (ca. 1.3 nm) [13,17]. We have observed the surface of the comb-shaped microelectrode in the course of film formation using 80 nm particle (Fig. 1B). The surface of the bare electrode was flat and smooth (a) whereas particles adsorbed not only on the Pt electrode but also on the glass substrate even in the first dipping cycle (b), which corresponds to the state of the second dipping process (2) in Fig. 1A. This indicates the existence of the unspecific adsorption of the binder molecule to the glass since particles hardly adsorbed to the electrode without binder, which led to the adsorption of the Au nanoparticle on the glass. While it was observed that the particles adsorbed thickly and uniformly both on the Pt electrode and glass substrate, and the film grew monolayer mostly after 6 dipping cycles (c). Therefore, it was figured out that the optimal number of dipping cycles for fabricating the monolayer film was approximately 6 cycles, while the larger dipping cycles, approximately over 9 dipping cycles, makes the film multilayer about 3 layers (d). The electrical resistance of the Au particle film decreased dramatically with an increase in the number of dipping cycles, as shown in Fig. 2. In the case of using 80 nm particle, the resistance decreased precipitously by 6 orders of magnitude at the second immersion and became constant ∼ 100 Ω for further dipping. The film resistance prepared from 30 nm particle dropped in a less drastic way and then diminished gradually at third dipping. Such reduction of resistance occurred at the fourth immersion in the case of 12 nm diameter particle. These indicated that the Pt electrode gap was filled with a conducting metal particle with the repetitive procedures. Larger particle size, such as 80 nm, can form a conductive path at a much earlier stage of the immersion than smaller particles (30 and 12 nm). Moreover, the resistivity of the film prepared here (30 Ω cm) gave a close agreement with the value (20 Ω cm) reported by L. Han et al. for nonanedithiol (ca. 1.1 nm) [10].
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It was expected geometrically that the formation of the crosslinked structure with Au particles of the gap interval (5 μm) between the Pt electrodes requires over 30, 80 and 200 dipping sequences for 80, 30 and 12 nm particles, respectively for overall coverage. However, 2, 3 and 4 times repeated sequences were enough to build up such a Au particle film. It can be attributed to the unspecific adsorption of Au nanoparticles on the glass substrate observed in the first dipping cycle in Fig. 1B (b). Such particles on the glass work as a nucleus for the next adsorption, accelerating the film accomplishment much earlier than geometric anticipation. The gap interval between abutting particles is regulated by the used binder molecule. It was explained by our previous report about the fabrication of a Au particle film on a polymer substrate using Au nanoparticle and alkylthiol as a binder [13]. The resistance of the film depended on the length of the alkylchain of the binder; the shorter binder led to lower film resistance, while the larger binder led to higher film resistance. It suggests that the space between particles is well-controlled by the length of the alkylchain of the binder. Further, even if Au particles are adsorbed excessively by excessive dipping sequences up to the formation of a multilayer as shown in Fig. 1B(d), the resistance of the film never go down under 70 Ω, suggesting that each abutting particle never come into contact and keep distance from each other. It is therefore, the space formed between abutting particles that must be kept by the length of the alkylchain of the binder. Moreover, the film had a mechanical strength enough to carry out following electrical measurements since it did not change in morphology and resistance after repeated potential sweeps in the range − 0.25 to + 1.3 V vs. Ag/AgCl in an 0.1 M aqueous H2SO4. 3.2. Response to the complementary DNA To measure a resistance change caused by DNA hybridization, the electrode was preconditioned by adding a 1 μL of TE buffer solution on the electrode for 2 min. As seen in Fig. 3, the resistance was decreased rapidly on the addition of 500 pmol ss-complementary DNA (a). Even with this simple experimental set-up, high S/N ratios above 20 were obtained. Furthermore, no detectable change in film morphology by hybridization, observed by AFM, explains stability in resistance. This resistance reduction, ΔR, would be attributed to the formation of dense π overlapping of the base pairs in the DNA double helix, promoting the electron transfer between Au particles. To make sure that the DNA hybridization caused the resistance change, DNaseI enzyme that specifically breaks dsDNA down at its phosphodiester positions was applied to the film after hybridization [7]. After the addition of DNaseI, the resistance was restored in 40 min to the original value. The resistance recovery can be attributed to the decomposition of dsDNA located between the Au particles with DNaseI, which
breaks the conducting path having been created by hybridization. On the contrary, the resistance remained constant over 1 h after hybridization unless the enzyme was added. These indicated that the resistance reduction was surely caused by the DNA hybridization [6–8]. 4. Conclusions We have successfully fabricated a highly sensitive sensor based on a nano-gapped gold particle film, utilizing alkyldithiol as a binder molecule. This technique allows us not only to control the resistance of the film but also to adjust the electrode gap through self-assembly procedures. Precisely controlled electrode gap enabled us to detect a small change caused by 12 base pair DNA both by hybridization and degradation directly with the electronic protocols. Acknowledgements This study was supported by the Industrial Technology Research Grant Program in '05 from the New Energy and Industrial Technology Development Organization (NEDO). One of authors, S.T., is thankful for the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (JSPS). References [1] C.A. Mirkin, R.L. Letsinger, R.C. Mucic, J.J. Storhoff, Nature 382 (1996) 607. [2] K. Sato, K. Hosokawa, M. Maeda, J. Am. Chem. Soc. 125 (2003) 8102. [3] K. Nakatani, S. Sando, I. Saito, Nat. Biotechnol. 19 (2001) 51. [4] T. Liu, J. Tang, H. Zhao, Y. Deng, L. Jiang, Langmuir 18 (2002) 5624. [5] A.Yu. Kasumov, M. Kociak, S. Guéron, B. Reulet, V.T. Volkov, D.V. Klinov, H. Bouchiat, Science 291 (2001) 280. [6] H.-W. Fink, C. Schönenberger, Nature 398 (1999) 407. [7] D. Porath, A. Bezryadin, S. de Vries, C. Dekker, Nature 403 (2000) 635. [8] Y. Okahata, T. Kobayashi, K. Tanaka, M. Shimomura, J. Am. Chem. Soc. 120 (1998) 6165. [9] S.-J. Park, T.A. Taton, C.A. Mirkin, Science 295 (2002) 1503. [10] L. Han, D.R. Daniel, M.M. Maye, C.J. Zhong, Anal. Chem. 73 (2001) 4441. [11] P. Zamborini, M.C. Leopold, J.F. Hicks, P.J. Kulesza, M.A. Malik, R.W. Murray, J. Am. Chem. Soc. 124 (2002) 8958. [12] T. Sato, H. Ahmed, D. Brown, B.F.G. Johnson, J. Appl. Phys. 82 (1997) 696. [13] H. Shiigi, Y. Yamamoto, H. Yakabe, S. Tokonami, T. Nagaoka, Chem. Commun. (2003) 1038. [14] T. Ogawa, K. Kobayashi, G. Masuda, T. Takase, S. Maeda, Thin Solid Films 393 (2001) 374. [15] J. Turkevich, P.C. Stevenson, J. Hillier, Discuss. Faraday Soc. 11 (1951) 55. [16] G.T. Hermanson, Bioconjugate Techniques, Academic Press, 1996, p. 598. [17] H. Shiigi, S. Tokonami, H. Yakabe, T. Nagaoka, J. Am. Chem. Soc. 127 (2005) 3280.