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A glass nanopore electrode for single molecule detection
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Chinese Chemical Letters 21 (2010) 1115–1118 www.elsevier.com/locate/cclet
A glass nanopore electrode for single molecule detection Guo Xia Li, Xiang Qin Lin * Department of Chemistry, University of Science and Technology of China, Hefei 230026, China Received 28 December 2009
Abstract We have developed a simple method for fabricating robust and low noise glass nanopore electrodes with pore size 10 5 nm to detect single molecules. b-Cyclodextrin was used as model compound for characterization. In 1.0 mol/L NaCl solution, the molecules generated current pulses of 2–5 pA with noise level less than 0.8 pA. A slide mode and a plug mode were suggested for the way of b-cyclodextrin single molecule moving into the glass nanopores. # 2010 Xiang Qin Lin. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Glass nanopores; Single molecule detecting; b-Cyclodextrin
Solid-state nanopores have recently been developed for sequencing and analysis [1,2] with single molecule resolution, which are comparable to biological phospholipids embedded protein channels [3] and have significant advantages of being functional and offering possibilities for device integration and adjustment in the pore dimensions [4]. The nanopore applications entail mounting a membrane containing a single nanopore between two halves of an electrochemical cell filled with electrolyte solutions. A transmembrane potential is applied and the ion current flowing through the nanopore is recorded versus time. As an analyte, with dimensions comparable to the nanopore diameter, is driven through the channel. When the analyte particle enters the channel, the electrical impedance of the channel increases and the momentary block in the ion current is observed in the form of current pulse. There have been several means for fabricating solid nanopores with Si or Si3N4 substrate, in which ion beam sculpting [5] and latent track etching [6] are most widely used. However, these methods require expensive instruments, well-trained operators and special materials. Glass capillaries can be easily manipulated with a programmable puller to form nanopipets [2]. This technique is inexpensive and Gu and co-workers [7] have created a glass nanopore electrode by etching the capillaries’ tip in 40% NH4F/49% HF solution. However, the problem is that HF solution is poisonous, and the etching process may also damage the nanochannels. Here we reported our achievement in the development of a much more efficient process for creating low-noise nanopores at the molecular scale using regular glass pipettes and the preliminary study of these nanopores for single-molecule detection. We were able to control the pore size by monitoring the level of pore conductance.
* Corresponding author. E-mail address: [email protected] (X.Q. Lin). 1001-8417/$ – see front matter # 2010 Xiang Qin Lin. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2010.04.026
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G.X. Li, X.Q. Lin / Chinese Chemical Letters 21 (2010) 1115–1118
1. Experimental A Model CHI 660A electrochemical workstation equipped with CHI 200 Picoamp Booster (Chenhua Instruments Co., Ltd., Shanghai, China) with Faraday cage was used for electrochemical measurements. An electrochemical cell with two electrode system was used, in which two Ag/AgCl (sat. KCl) electrodes were used to apply the potential difference on the both side of the nanopore, as shown in Scheme 1. The Ag/AgCl electrode inside the micropipette was connected to WE (working electrode), and the other one was connected to RE + CE (reference and counter electrode). In this way, the electrode potentials reported in this article were the voltages from WE to RE + CE. The 1.0 mol/L NaCl solution was used as the electrolyte. For measurements, certain amount of b-cyclodextrin (b-CD, diluted in 1 mol/L NaCl, Alfa Aesar) were added into the external solution of the nanopore electrode. Borosilicate capillaries (1.5 mm o.d. and 0.86 mm i.d., Spring Teaching Experiment Luhe County Equipment Factory, China) were used for making the nanopore electrodes. Before use, the capillary was washed with ethanol and double-deionized water (ddH2O), and dried in oven. The clean capillary was first pulled to a cone with cone-angle about 608 by PN-30 electrode puller (Narishige, Japan). Then the tip was melt on an alcohol burner until it was completely closed forming a spherical terminal with a radius of about 0.2 mm. The terminal enclosed an asymmetrical gradually narrowed cavity with cone-angle about 38 and cone-length 20–30 mm, which were monitored under an optical microscope (Olympus, Japan). After cooling, the spherical terminal was polished to expose the nanopore on metallographic sandpapers with grain size of 800# to 4000#, successively. A plastic pipet (1 mL, Axygen biotechnology (Taizhou) Co., Ltd.) was fixed on the capillary with the nanopore by heat melting. After washed by ddH2O, the electrode was filled with the electrolyte, and checked under microscope to ensure that no air bubbles were isolated inside. The resistance of the nanopore was characterized by the slope of cyclic voltammetry in the 1.0 mol/L NaCl solution. Repeated polishing was needed if the resistance was larger than the value expected. By this way we could effectively control the final pore size, because the resistance of the system is condensed in the nanochannel. It was so difficulty to visually find a nano-hole on the end surface of the capillary that no satisfied SEM images were obtained. However, the size of the nanopore could be estimated from the resistance measured according to the equation [8]: 1 1 1 Rp ¼ þ (1) kap p tan u 4 where Rp is resistance of the nanopore, k is conductivity of the electrolyte solution (k 9 (V m)1 for 1.0 mol/L NaCl), ap is the radius of the nanopore, and u the half-cone-angle of the nanopore. b-CD has been interested as a probe of single molecule detections since its special size and complexation properties. As shown in Fig. 1, the translocation of each b-CD molecules can be clearly identified by the individual
[(Schem_1)TD$FIG]
Scheme 1. (a) Experimental setup for single molecule detection using the glass nanopore electrode: (1) Ag/AgCl (sat. KCl), (2) plastic pipet connection; (3) nanopore capillary; (4) external electrolyte solution. (b) Optical micrograph of the spherical terminal enclosed an asymmetrical gradually narrowed cavity. (c) Local enlargement of the circle region in (b).
[(Fig._1)TD$IG]
G.X. Li, X.Q. Lin / Chinese Chemical Letters 21 (2010) 1115–1118
1117
Fig. 1. Chronoamperometric response of the glass nanopore electrode in 1.0 mol/L NaCl. The outside solution contains 1.2 mmol/L b-CD. The applied electrode potential was 100 mV. The pore diameter was 10 5 nm.
current pulses with different amplitude and time duration. The current pulses were almost 10 times larger than the noise level. This meant that the nanopore electrode was perfect to realize single molecule detecting. Although there have been some studies on the interaction of b-CD with protein pores [9], we found some characteristic phenomena when b-CD molecules went through the glass nanopores. Interestingly, there were four different current levels (1–4) appearing on the response curve, which generated three characteristic pulse amplitudes calculated in the light of level 1 as the baseline. These phenomena could be explained by different orientation of b-CD to the nanopore. The current of level 1 should be generated with no b-CD in the pore and the current reached to the maximal. The level 2 with pulse amplitude of about 1.0 pA should be the case that a couple of b-CD were moving close to the nanopore orifice and striking at the side wall of the pore in the form of molecular thermal motion, which lead to the minor reduce of the current. So, the location of b-CD outside the pore like Scheme 2(A) may cause both of current levels 1 and 2. Level 3 with pulse amplitude of 3.4 pA corresponds to the b-CD having come into the pore with a slide mode just as shown in Scheme 2(B). However, level 4 with pulse amplitude of 6.0 pA corresponds to the b-CD having come into the pore with a plug mode just as shown in Scheme 2(C). Also, if level 4 generated by following up molecules, much longer pulse time should have been generated. The fact was that the pulse width of level 4 was little shorter than that of level 3. And the phenomenon of the pulse ‘‘*’’ clearly showed a switch between the different mode when b-CD passed through the nanochannel. Although different pulse amplitudes of current signals generated with nanopores have been used to distinguish the chiral enantiomers of ibuprofen [7], two different orientations of b-CD in the nanopore were first observed with these distinct pulse amplitudes. Because b-CD molecule looks like a hollow cylinder with the outer diameter is 1.53 nm and the height is 0.79 nm [10], the blocking area of the two modes for b-CD coming into the pore can be calculated as 1.21 nm2 for the slide mode and 1.84 nm2 for the plug mode. Because the cavity of cyclodextrin is hydrophobic, the blocking area was estimated as disc-shaped for the plug mode. The ratio of the blocking area for the slide mode to the plug mode was 1: 1.52, which meant the same ratio of resistance and was in well agreement with the pulse amplitude ratio of 1:1.76. The theoretical calculation also confirms the two modes suggestion. As we have known, this was the first report about that b-CD could generated two current pulse signals in a nanopore electrochemical sensor. Besides, we also noted that the pulse duration time was much longer in our case than what has been seen in the silicon nanochannels reported by Rant and co-workers [11]. The amplitude of the current change and the time duration for the translocation could depend on a number of experimental factors (e.g., electrolyte concentration, electrical field), properties of the nanochannels (e.g., the length and surface charges) and properties of nanoparticles (e.g., the size and surface charges). So there were a number of possible reasons leading to this large time duration. First, the channel length in our case was estimated as about 25 mm, which was about 5-fold larger than the length of the [(Schem_2)TD$FIG]
Scheme 2. Orientation of b-CD molecules going through the nanopore. (A) b-CD outside the pore (B) the slide mode (C) the plug mode.
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G.X. Li, X.Q. Lin / Chinese Chemical Letters 21 (2010) 1115–1118
membrane channel (6 mm) [11]. Second, there may be electroosmosis which was in the convert direction with b-CD diffusion in our experimental arrangement. Bayley and co-workers [9] have pointed out that the electroosmosis could be a driving force to push the molecules in or out the pore. Third, due to the nature of the nanochannel, there could be a stronger interaction between the nanoparticles and the wall, which is worthy for further investigation. 2. Conclusion We developed a simple conventional way for preparation of glass capillary nanopore electrode with the pore size of 10 5 nm for single molecule detection. The electrode was mechanically strong, easy to handle, can generate higher current pulse amplitude with lower noise level. The model molecule b-CD was used to characterize the basic function of the nanopore. Three distinct amplitudes of pulse signals have been identified from the chronoamperometric response, based on which a slide mode and a plug mode for b-CD single molecule moving into the pore orifice have been proposed. These results were significant both for fabrication of nanopore electrodes and for better understanding of the nano-confinement. Acknowledgments We gratefully acknowledge the financial support from the National Natural Science Foundation of China (No. 20575062) and The Graduate Innovation Fund of USTC. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
S. Howorka, Z. Siwy, Chem. Soc. Rev. 38 (2009) 2360. B. Zhang, M. Wood, H. Lee, Anal. Chem. 81 (2009) 5541. D.Q. Wang, Q.T. Zhao, R.S.S. de Zoysa, et al. Sens. Actuators B: Chem. 139 (2009) 440. B.M. Venkatesan, B. Dorvel, S. Yemenicioglu, et al. Adv. Mater. 21 (2009) 2771. M.Y. Wu, R.M.M. Smeets, M. Zandbergen, et al. Nano Lett. 9 (2009) 479. Y.B. Xie, J.M. Xue, L. Wang, et al. Langmuir 25 (2009) 8870. C.L. Gao, S. Ding, Q.L. Tan, et al. Anal. Chem. 81 (2009) 80. B. Zhang, J. Galusha, P.G. Shiozawa, et al. Anal. Chem. 79 (2007) 4778. L.Q. Gu, S. Cheley, H. Bayley, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 15498. J. Szejtli, Chem. Rev. 98 (1998) 1743. D. Pedone, M. Firnkes, U. Rant, Anal. Chem. 81 (2009) 9689.
Chinese Chemical Letters 21 (2010) 1115–1118 www.elsevier.com/locate/cclet
A glass nanopore electrode for single molecule detection Guo Xia Li, Xiang Qin Lin * Department of Chemistry, University of Science and Technology of China, Hefei 230026, China Received 28 December 2009
Abstract We have developed a simple method for fabricating robust and low noise glass nanopore electrodes with pore size 10 5 nm to detect single molecules. b-Cyclodextrin was used as model compound for characterization. In 1.0 mol/L NaCl solution, the molecules generated current pulses of 2–5 pA with noise level less than 0.8 pA. A slide mode and a plug mode were suggested for the way of b-cyclodextrin single molecule moving into the glass nanopores. # 2010 Xiang Qin Lin. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Glass nanopores; Single molecule detecting; b-Cyclodextrin
Solid-state nanopores have recently been developed for sequencing and analysis [1,2] with single molecule resolution, which are comparable to biological phospholipids embedded protein channels [3] and have significant advantages of being functional and offering possibilities for device integration and adjustment in the pore dimensions [4]. The nanopore applications entail mounting a membrane containing a single nanopore between two halves of an electrochemical cell filled with electrolyte solutions. A transmembrane potential is applied and the ion current flowing through the nanopore is recorded versus time. As an analyte, with dimensions comparable to the nanopore diameter, is driven through the channel. When the analyte particle enters the channel, the electrical impedance of the channel increases and the momentary block in the ion current is observed in the form of current pulse. There have been several means for fabricating solid nanopores with Si or Si3N4 substrate, in which ion beam sculpting [5] and latent track etching [6] are most widely used. However, these methods require expensive instruments, well-trained operators and special materials. Glass capillaries can be easily manipulated with a programmable puller to form nanopipets [2]. This technique is inexpensive and Gu and co-workers [7] have created a glass nanopore electrode by etching the capillaries’ tip in 40% NH4F/49% HF solution. However, the problem is that HF solution is poisonous, and the etching process may also damage the nanochannels. Here we reported our achievement in the development of a much more efficient process for creating low-noise nanopores at the molecular scale using regular glass pipettes and the preliminary study of these nanopores for single-molecule detection. We were able to control the pore size by monitoring the level of pore conductance.
* Corresponding author. E-mail address: [email protected] (X.Q. Lin). 1001-8417/$ – see front matter # 2010 Xiang Qin Lin. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2010.04.026
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G.X. Li, X.Q. Lin / Chinese Chemical Letters 21 (2010) 1115–1118
1. Experimental A Model CHI 660A electrochemical workstation equipped with CHI 200 Picoamp Booster (Chenhua Instruments Co., Ltd., Shanghai, China) with Faraday cage was used for electrochemical measurements. An electrochemical cell with two electrode system was used, in which two Ag/AgCl (sat. KCl) electrodes were used to apply the potential difference on the both side of the nanopore, as shown in Scheme 1. The Ag/AgCl electrode inside the micropipette was connected to WE (working electrode), and the other one was connected to RE + CE (reference and counter electrode). In this way, the electrode potentials reported in this article were the voltages from WE to RE + CE. The 1.0 mol/L NaCl solution was used as the electrolyte. For measurements, certain amount of b-cyclodextrin (b-CD, diluted in 1 mol/L NaCl, Alfa Aesar) were added into the external solution of the nanopore electrode. Borosilicate capillaries (1.5 mm o.d. and 0.86 mm i.d., Spring Teaching Experiment Luhe County Equipment Factory, China) were used for making the nanopore electrodes. Before use, the capillary was washed with ethanol and double-deionized water (ddH2O), and dried in oven. The clean capillary was first pulled to a cone with cone-angle about 608 by PN-30 electrode puller (Narishige, Japan). Then the tip was melt on an alcohol burner until it was completely closed forming a spherical terminal with a radius of about 0.2 mm. The terminal enclosed an asymmetrical gradually narrowed cavity with cone-angle about 38 and cone-length 20–30 mm, which were monitored under an optical microscope (Olympus, Japan). After cooling, the spherical terminal was polished to expose the nanopore on metallographic sandpapers with grain size of 800# to 4000#, successively. A plastic pipet (1 mL, Axygen biotechnology (Taizhou) Co., Ltd.) was fixed on the capillary with the nanopore by heat melting. After washed by ddH2O, the electrode was filled with the electrolyte, and checked under microscope to ensure that no air bubbles were isolated inside. The resistance of the nanopore was characterized by the slope of cyclic voltammetry in the 1.0 mol/L NaCl solution. Repeated polishing was needed if the resistance was larger than the value expected. By this way we could effectively control the final pore size, because the resistance of the system is condensed in the nanochannel. It was so difficulty to visually find a nano-hole on the end surface of the capillary that no satisfied SEM images were obtained. However, the size of the nanopore could be estimated from the resistance measured according to the equation [8]: 1 1 1 Rp ¼ þ (1) kap p tan u 4 where Rp is resistance of the nanopore, k is conductivity of the electrolyte solution (k 9 (V m)1 for 1.0 mol/L NaCl), ap is the radius of the nanopore, and u the half-cone-angle of the nanopore. b-CD has been interested as a probe of single molecule detections since its special size and complexation properties. As shown in Fig. 1, the translocation of each b-CD molecules can be clearly identified by the individual
[(Schem_1)TD$FIG]
Scheme 1. (a) Experimental setup for single molecule detection using the glass nanopore electrode: (1) Ag/AgCl (sat. KCl), (2) plastic pipet connection; (3) nanopore capillary; (4) external electrolyte solution. (b) Optical micrograph of the spherical terminal enclosed an asymmetrical gradually narrowed cavity. (c) Local enlargement of the circle region in (b).
[(Fig._1)TD$IG]
G.X. Li, X.Q. Lin / Chinese Chemical Letters 21 (2010) 1115–1118
1117
Fig. 1. Chronoamperometric response of the glass nanopore electrode in 1.0 mol/L NaCl. The outside solution contains 1.2 mmol/L b-CD. The applied electrode potential was 100 mV. The pore diameter was 10 5 nm.
current pulses with different amplitude and time duration. The current pulses were almost 10 times larger than the noise level. This meant that the nanopore electrode was perfect to realize single molecule detecting. Although there have been some studies on the interaction of b-CD with protein pores [9], we found some characteristic phenomena when b-CD molecules went through the glass nanopores. Interestingly, there were four different current levels (1–4) appearing on the response curve, which generated three characteristic pulse amplitudes calculated in the light of level 1 as the baseline. These phenomena could be explained by different orientation of b-CD to the nanopore. The current of level 1 should be generated with no b-CD in the pore and the current reached to the maximal. The level 2 with pulse amplitude of about 1.0 pA should be the case that a couple of b-CD were moving close to the nanopore orifice and striking at the side wall of the pore in the form of molecular thermal motion, which lead to the minor reduce of the current. So, the location of b-CD outside the pore like Scheme 2(A) may cause both of current levels 1 and 2. Level 3 with pulse amplitude of 3.4 pA corresponds to the b-CD having come into the pore with a slide mode just as shown in Scheme 2(B). However, level 4 with pulse amplitude of 6.0 pA corresponds to the b-CD having come into the pore with a plug mode just as shown in Scheme 2(C). Also, if level 4 generated by following up molecules, much longer pulse time should have been generated. The fact was that the pulse width of level 4 was little shorter than that of level 3. And the phenomenon of the pulse ‘‘*’’ clearly showed a switch between the different mode when b-CD passed through the nanochannel. Although different pulse amplitudes of current signals generated with nanopores have been used to distinguish the chiral enantiomers of ibuprofen [7], two different orientations of b-CD in the nanopore were first observed with these distinct pulse amplitudes. Because b-CD molecule looks like a hollow cylinder with the outer diameter is 1.53 nm and the height is 0.79 nm [10], the blocking area of the two modes for b-CD coming into the pore can be calculated as 1.21 nm2 for the slide mode and 1.84 nm2 for the plug mode. Because the cavity of cyclodextrin is hydrophobic, the blocking area was estimated as disc-shaped for the plug mode. The ratio of the blocking area for the slide mode to the plug mode was 1: 1.52, which meant the same ratio of resistance and was in well agreement with the pulse amplitude ratio of 1:1.76. The theoretical calculation also confirms the two modes suggestion. As we have known, this was the first report about that b-CD could generated two current pulse signals in a nanopore electrochemical sensor. Besides, we also noted that the pulse duration time was much longer in our case than what has been seen in the silicon nanochannels reported by Rant and co-workers [11]. The amplitude of the current change and the time duration for the translocation could depend on a number of experimental factors (e.g., electrolyte concentration, electrical field), properties of the nanochannels (e.g., the length and surface charges) and properties of nanoparticles (e.g., the size and surface charges). So there were a number of possible reasons leading to this large time duration. First, the channel length in our case was estimated as about 25 mm, which was about 5-fold larger than the length of the [(Schem_2)TD$FIG]
Scheme 2. Orientation of b-CD molecules going through the nanopore. (A) b-CD outside the pore (B) the slide mode (C) the plug mode.
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G.X. Li, X.Q. Lin / Chinese Chemical Letters 21 (2010) 1115–1118
membrane channel (6 mm) [11]. Second, there may be electroosmosis which was in the convert direction with b-CD diffusion in our experimental arrangement. Bayley and co-workers [9] have pointed out that the electroosmosis could be a driving force to push the molecules in or out the pore. Third, due to the nature of the nanochannel, there could be a stronger interaction between the nanoparticles and the wall, which is worthy for further investigation. 2. Conclusion We developed a simple conventional way for preparation of glass capillary nanopore electrode with the pore size of 10 5 nm for single molecule detection. The electrode was mechanically strong, easy to handle, can generate higher current pulse amplitude with lower noise level. The model molecule b-CD was used to characterize the basic function of the nanopore. Three distinct amplitudes of pulse signals have been identified from the chronoamperometric response, based on which a slide mode and a plug mode for b-CD single molecule moving into the pore orifice have been proposed. These results were significant both for fabrication of nanopore electrodes and for better understanding of the nano-confinement. Acknowledgments We gratefully acknowledge the financial support from the National Natural Science Foundation of China (No. 20575062) and The Graduate Innovation Fund of USTC. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
S. Howorka, Z. Siwy, Chem. Soc. Rev. 38 (2009) 2360. B. Zhang, M. Wood, H. Lee, Anal. Chem. 81 (2009) 5541. D.Q. Wang, Q.T. Zhao, R.S.S. de Zoysa, et al. Sens. Actuators B: Chem. 139 (2009) 440. B.M. Venkatesan, B. Dorvel, S. Yemenicioglu, et al. Adv. Mater. 21 (2009) 2771. M.Y. Wu, R.M.M. Smeets, M. Zandbergen, et al. Nano Lett. 9 (2009) 479. Y.B. Xie, J.M. Xue, L. Wang, et al. Langmuir 25 (2009) 8870. C.L. Gao, S. Ding, Q.L. Tan, et al. Anal. Chem. 81 (2009) 80. B. Zhang, J. Galusha, P.G. Shiozawa, et al. Anal. Chem. 79 (2007) 4778. L.Q. Gu, S. Cheley, H. Bayley, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 15498. J. Szejtli, Chem. Rev. 98 (1998) 1743. D. Pedone, M. Firnkes, U. Rant, Anal. Chem. 81 (2009) 9689.