- Email: [email protected]
A Low Noise Amplifier System for Nanopore-based Single Molecule Analysis
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 43, Issue 7, July 2015 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2015, 43(7), 971–976.
RESEARCH PAPER
A Low Noise Amplifier System for Nanopore-based Single Molecule Analysis YAN Bing-Yong, GU Zhen, GAO Rui, CAO Chan, YING Yi-Lun, MA Wei, LONG Yi-Tao* Department of Automation& Key Laboratory for Advanced Materials, East China University of Science and Technology, Shanghai 200237, China
Abstract: A novel amplifier system was designed and prepared for low-noise recording of pico-ampere current in nanopore experiment (< 100 pA). As an example, the amplifier system was applied in α-hemolysin based nanopore detection of DNAPEG-DNA conjugate to record the signals of translocation and bumping events in buffer solution (1 M KCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The amplified current signal was filtered by a 3 kHz Bessel filter and sampled by a 100 kHz analog-digital convertor. As a result, the presented amplifier system could lower the noise in recording the current. Current blockages (< 10 pA) of single molecules with low amplitude were recovered due to the high signal-to-noise ratio. Key Words: Nanopore; Single molecule detection; Current amplifier; Denoising
1
Introduction
In the last two decades, studies based on monitoring the current fluctuation across nanopore received a great deal of attention, due to their promising applications for rapid, sensitive and label-free detection of individual molecules[1‒4]. Most of the studies were carried out by using the nanopores for resistive-pulse sensing experiments. Transient current change induced by a single molecule could inform their unique properties which were hardly obtained in ensemble measurements. For example, the sequence of DNA was directly read out as the bases passed through the nanopore one after another, inducing different degrees of current blockages[5,6]. In addition, the versatility of nanopore was demonstrated in the studies such as sensing the structures of RNA[7‒9], peptides[10‒12] and proteins[13‒15], detecting the interactions between supramolecules[16,17], measuring the size of nanoparticles[18,19] and concentrations of metal ions[20]. However, the ionic current across nanopore was extremely low (picoampere to nanoampere) owing to their high resistance. For example, the open pore current of α-hemolysin
nanopore was as low as 100 pA at a voltage bias of 100 mV (1 M KCl), and the blockage of current was less than 20 pA at some cases[21]. Therefore, those small current signals could be submerged by the noise in the recording process[22,23]. Though the development in data analysis enables extracting the blockage information from current trace at condition of high noise, the missing of short-lived events is still inevitable[24,25]. Physically suppressing the noise in signal remains a feasible and direct way to improve the performance of nanopore measurement. Conventionally, the current was measured and converted into a voltage signal by using a resistor-feedback amplifier[26]. The resistance of the feedback resistor on the order of 0.1‒10 GΩ controls the gain of current, whereas it also contributes to the majority noise in the signal. Though using a large resistance can lower the noise, the dynamic range for current measurement will also be narrowed due to the saturation of amplifier[27]. Other methods for lowering noise have been developed. For example, Rosenstein et al[28] fabricated an integrated nanopore sensing platform to improve the signal-to-noise ratio at high bandwidth. Balan et al[29] suppressed the current noise by reducing the nanopore chip
________________________ Received 2 March 2015; accepted 4 May 2015 * Corresponding author. Email: [email protected] This work was supported by by the National Natural Science Foundation of China (Nos. 21125522, 21327807, 51407078) and the Fundamental Research Funds for the Central Universities of China (Nos. 222201313004, WH1514049). Copyright © 2015, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(15)60843-X
YAN Bing-Yong et al. / Chinese Journal of Analytical Chemistry, 2015, 43(7): 971–976
capacitance. In this study, a novel design of the circuit of amplifier system was presented for low-noise current measurement (Fig.1a). The amplifier system features in a capacitorfeedback amplifier (A1) and an inverting differential integrator (A3). The advantages of capacitor-feedback amplifier were demonstrated by Goldstein et al[30], and the thermal noise on the absence of feedback resistor was effectively reduced. The differential integrator could highly suppress the commonmode noise between the current signal and reference voltage. Furthermore, a drift compensation was introduced for the reference voltage.
2 2.1
Experimental Materials
α-Hemolysin and decane (≥ 99%) were purchased from Sigma-Aldrich (St.Louis, MO, USA). 1,2-diphytanoly-snglycero-3-phosphocholine (chloroform, ≥ 99%) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Ultrapure water (18.2 MΩ·cm at 25°C) was obtained by the Milli-Q System (EMD Millipore, Billerica, MA, USA.). Buffer solution (1 M KCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was prepared before use. All other chemicals were of analytical grade. The detected DNA-PEG-DNA conjugate
Fig.1
(5’-GTCACGATGGCCCAGTAGTT-HPO4-(CH2-CH2-O)9-T TGATGACCCGGTAGCACTG-3’) was synthesized and HPLC-purified by Sangon Biotech (Shanghai) Co. Ltd. (China). 2.2 Nanopore experiments As described previously, 1,2-diphytanoly-sn-glycero-3phosphocholine in decane (30 mg mL‒1) was applied to form a bilayer across a 150 μm orifice in a lipid bilayer chamber (Warner Instruments, Hamden, CT, USA) which was filled with buffer solution. The solution of α-hemolysin was injected to the cis chamber proximal to the bilayer. Then, seven monomers of α-hemolysin would assemble to form a hydrophilic channel in the bilayer. The two compartments of the bilayer cell were termed cis and trans (shown in Fig.1). A pair of Ag/AgCl electrode was inserted into the two compartments, respectively. After a single nanopore was formed on the bilayer, analyte was injected into the cis chamber. The voltage was set to +120 mV during the experiments. The presented amplifier and a commercial resistor-feedback amplifier system (ChemClamp, Dagan Corporation, Minneapolis, MN, USA) were used to amplify and measure the ionic current flowing through the nanopore, respectively. The amplified currents were filtered at 3 kHz and converted to digital signals by DigiData 1440A hardware (Axon Instruments,
Schematic (a) and photograph (b) of the setup of the nanopore experiment and the amplifier circuit
YAN Bing-Yong et al. / Chinese Journal of Analytical Chemistry, 2015, 43(7): 971–976
Forest City, CA, USA) at 100 kHz sampling rate. Nanopore data were recorded by PClamp software (Axon Instruments, Forest City, CA, USA). A homemade program called NANOPORE ANALYSIS was used to recognize the current blockages, evaluate and distribute the current amplitudes of blockages[24]. As the current traces had different signal-noise rate, the thresholds were adapted as 4σ, where σ was the standard deviation of the whole current trace. For pulse-like events, the current amplitude was evaluated by using integration method as described previously. For multi-level events, the minimum point was chosen for measuring the current amplitude.
3 3.1
Results and discussion Circuit analysis of the amplifier system
During detection, the amplifier system worked as a voltage clamp to measure the ionic current across the nanopore while holding the reference voltage at a certain level. The first stage integrated the current, and its output voltage (V1) was related to the quantity of electric charge poured into the feedback capacitor (Cf). The V1was evaluated by using the Equation (1). dt (1) where, a 0.1 pF feedback capacitor is selected to achieve a high bandwidth for the amplifier. Then, the signal of V1 is differentiated by differential amplifier (A2). The output voltage (V2) can be calculated as Equation (2). (2) Since reference voltage was not changed during recording, it was applied at the positive lead prior to a RC circuit for drift compensation. The differential integrator (A3) integrates the difference between V2 and reference voltage, whose output follows Equation (3). (3) It is notable that the common-mode noise from the reference voltage is eliminated owing to the advantage of differential integrator[31]. To avoid the saturation of Cf and C2, CMOS switchs (S1, S2 and S3, ADG452, Analog Devices) are used to discharge the capacitors, which have low leakage current and introduce limited noise. V1 and Vout are sampled by the analog-to-digital chip (AD7626, Analog Devices). The final differentiated output is linear to the input current i(t) as in Equation (4). (4) According to the value of the elements as shown in Table 1, the current was evaluated based on the final output voltage as i(t)/Vout = 1 pA mV‒1. The dynamic range of the amplifier was as large as ±10 nA with this configuration.
3.2
Noise of the amplifier system
To testify the presented amplifier system, α-hemolysin nanopore was fabricated and detected at 120 mV. As shown in Fig.2a, the open pore current measured (gray) by a resistor-feedback amplifier system (Fig.2b) had higher noise than that detected by the presented amplifier system (blue). The noise spectral density of the two current traces indicated that the high frequency noise was suppressed by the presented amplifier. 3.3
Application for detecting single molecule
Subsequently, the presented amplifier was applied for recording the current trace in real experiment. The detected molecule was a DNA-PEG-DNA conjugate, which could translocate through the α-HL nanopore, and induce obvious current blockage. Besides the translocation events, bumping of the molecules with the nanopore was common in the nanopore studies due to the nature of single molecule behavior, which appeared to be a pulse with low blockage current[32]. However, this part of events was often missed or not concerned, when locating the blockages through a strong threshold (20 pA) due to the high noise level[24]. In comparison with conventional amplifier, the presented amplifier had lower noise in recording the current trace for single molecular sensing than the conventional amplifier. Blockages with low current amplitude could be easily distinguished from the baseline by applying a low threshold. Examples of individual blockages with different current amplitude were depicted in Fig.3b. Table 1
Typical value of capacitors and resistors for amplifier systema
Capacitor (PF)
C0 105
Cf 0.1
C1 10
C2 10
Resistor (kΩ)
R0 100
R1 100
R2 100
R3 100
C3 10
a
This configuration of capacitors and resistors were used in the following nanopore measurements.
Fig.2
(a) Current trace measured by resistor-feedback amplifier (gray) and the proposed amplifier (blue); (b) Noise spectral density of the two current trace in (a), respectively
YAN Bing-Yong et al. / Chinese Journal of Analytical Chemistry, 2015, 43(7): 971–976
Fig.3
(a) Current trace measured in the real detection experiment by resistor-feedback amplifier (gray) and the proposed amplifier (blue); (b) Examples of the blockage events detected in the experiment with different current amplitude. The current amplitudes measured by resistor-feedback amplifier system (c) and the presented amplifier system (d) were distributed into histogram, respectively. The peaks were fitted by Gaussian function
Obviously, with lower threshold (10 pA) used in the data analysis, blockages lower than 20 pA were recognized. The detailed structures in the blockage such as multi-level blockage could be distinguished by the amplifier system. Furthermore, we distributed the current amplitude of the blockages into histograms. The distribution of current amplitudes measured by the conventional amplifier system was a single Gaussian peak (P0) centered at 0.58 (I/I0), which suggested that there was only translocation events existing during the detection (Fig.3c). However, two Gaussian peaks emerge in the histogram of current amplitude measured by the presented amplifier system (Fig.3d). The first peak (PI) with low current amplitude (0.21, I/I0) was related to the bumping blockages, while the second peak (PII) centered at 0.55 (I/I0) was consistent with the P0 in Fig.3c, therefore this part of events was also corresponding to the translocation event. It was clear that the recovery of PI contributed to the less noise in the current trace as measured by the presented amplifier system.
monitoring the momentary fluctuation of ionic current across nanopore. Electrical noise was suppressed by the following strategies: (1) using capacitor-feedback amplifier; (2) employing a differential integrator to eliminate the common-mode noise in current trace; (3) introducing drift compensation for the reference voltage. We characterized the performance of the amplifier system in real nanopore measurement. As a result, thermal noise and noise at high frequency were highly reduced. The example study for detecting DNA-PEG-DNA conjugate in real nanopore experiment demonstrated the significant improvement of the presented amplifier system for recovering the blockages with low current amplitude.
References [1]
Ying Y L, Cao C, Long Y T. Analyst, 2014, 139: 3826‒3835
[2]
Si W, Zhang Y, Wu G S, Sha J J, Liu L, Chen Y F. Chin. Sci. Bull., 2014, 59(35): 4929‒4941
4
Conclusions
[3]
Ying X H, Zhu X Y, Gu J, Zhang X, Shao Y H. Chinese J. Anal. Chem., 2013, 41(5): 632‒640
In conclusion, a novel amplifier system was presented for
[4]
Guo S J, Wang E K. Accounts Chem. Res., 2011, 44: 491‒500
YAN Bing-Yong et al. / Chinese Journal of Analytical Chemistry, 2015, 43(7): 971–976
[5]
Cherf G, Lieberman K, Rashid H, Lam C, Karplus K, Akeson M. Nat. Biotechnol., 2012, 30(4): 344‒348
[6]
Manrao E, Derrington I, Laszlo A, Langford K, Hopper M, Gillgren N, Pavlenok M, Niederweis M, Gundlach J. Nat. Biotechol., 2012, 30(4): 349‒353
[7] [8] [9]
Cracknell J, Japrung D, Bayley H. Nano Lett., 2013, 13(6):
[18] Goyal G, Freedman K, Kim M. Anal. Chem., 2013, 85(17), 8180‒8187 [19] Wang J R, Ma J, Ni Z H, Zhang L, Hu G Q. RSC Adv., 2014, 4(15): 7601‒7610 [20] Yang C, Liu L, Zeng T, Yang D W, Yao Z Y, Zhao Y L, Wu H C. Anal. Chem., 2013, 85(15): 7302‒7307
2500‒2505
[21] Meller A, Branton D. Electrophoresis, 2002, 23(16): 2583‒2591
Wang Y, Zheng D L, Tan Q L, Wang M, Gu L Q. Nat.
[22] Uram J, Ke K, Mayer M. ACS Nano, 2008, 2(5): 857‒872
Nanotechnol., 2011, 6(10): 668‒674
[23] Smeets R, Keyser U, Dekker N, Dekker C. Proc. Natl. Acad.
Zhang X, Zhang J J, Ying Y L, Tian H, Long Y T. Chem. Sci., 2014, 5(7): 2642‒2646
[10] Sutherland T, LongY T, Stefureac R I, Bediako-Amoa I, Kraatz H B, Lee J. Nano Lett., 2004, 4(7): 1273‒1277 [11] Wang H Y, Ying Y L, Li Y, Kraatz H B, Long Y T. Anal. Chem., 2011, 83(5): 1746‒1752 [12] Ying Y L, Zhang X, Liu Y, Xue M Z, Li H L, Long Y T. ACTA Chim. Sinica, 2013, 71(1): 44‒50 [13] Nivala J, Marks D, Akeson M. Nat. Biotechnol., 2013, 31(3): 247‒250 [14] Rotem D, Jayasinghe L, Salichou M, Bayley H. J. Am. Chem. Soc., 2012, 134(5): 2781‒2787 [15] Zhang S Q, Ting S, Wang E K, Wang J H. Chin. Sci. Bull., 2014, 59(35): 4946‒4952 [16] Ying Y L, Zhang J J, Meng F N, Cao C, Yao X Y, Willner I, Tian H, Long Y T. Sci. Rep., 2013, 3: 1662 [17] Meng F N, Yao X Y, Ying Y L, Zhang J J, Tian H, Long Y T. Chem. Comm., 2015, 51(7): 1202‒1205
Sci. U. S. A., 2008, 105(2): 417‒421 [24] Gu Z, Ying Y L, Cao C, He P G, Long Y T. Anal. Chem., 2015, 87(2): 907‒913 [25] Zhang N, Hu Y X, Gu Z, Ying Y L, He P, Long Y T. Chin. Sci. Bull., 2014, 59(35): 4942‒4945 [26] Gao R, Ying Y L, Yan B Y, Long Y T. Chin. Sci. Bull., 2014, 59(35): 4968‒4973 [27] Sakmann B, Neher E. Single-Channel Recording, Plenum Press New York, 1995, vol. 362 [28] Rosenstein J, Wanunu M, Merchant C, Drndic M, Shepard K. Nat. Methods, 2012, 9(5): 487‒492 [29] Balan A, Machielse B, Niedzwiecki D, Lin J, Ong P, Engelke R, Shepard K, Drndić D. Nano Lett., 2014, 14(12): 7215‒7720 [30] Goldstein B, Kim D, Xu J, Vanderlick T, Culurciello E. IEEE Trans. Biomed. Circuits Syst., 2012, 6(2): 111‒119 [31] Mancini R. Op Amps for everyone, Elsevier, 2003 [32] Cao C, Ying Y L, Gu Z, Long Y T. Anal. Chem., 2014, 86(24): 11946‒11950
Cite this article as: Chin J Anal Chem, 2015, 43(7), 971–976.
RESEARCH PAPER
A Low Noise Amplifier System for Nanopore-based Single Molecule Analysis YAN Bing-Yong, GU Zhen, GAO Rui, CAO Chan, YING Yi-Lun, MA Wei, LONG Yi-Tao* Department of Automation& Key Laboratory for Advanced Materials, East China University of Science and Technology, Shanghai 200237, China
Abstract: A novel amplifier system was designed and prepared for low-noise recording of pico-ampere current in nanopore experiment (< 100 pA). As an example, the amplifier system was applied in α-hemolysin based nanopore detection of DNAPEG-DNA conjugate to record the signals of translocation and bumping events in buffer solution (1 M KCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The amplified current signal was filtered by a 3 kHz Bessel filter and sampled by a 100 kHz analog-digital convertor. As a result, the presented amplifier system could lower the noise in recording the current. Current blockages (< 10 pA) of single molecules with low amplitude were recovered due to the high signal-to-noise ratio. Key Words: Nanopore; Single molecule detection; Current amplifier; Denoising
1
Introduction
In the last two decades, studies based on monitoring the current fluctuation across nanopore received a great deal of attention, due to their promising applications for rapid, sensitive and label-free detection of individual molecules[1‒4]. Most of the studies were carried out by using the nanopores for resistive-pulse sensing experiments. Transient current change induced by a single molecule could inform their unique properties which were hardly obtained in ensemble measurements. For example, the sequence of DNA was directly read out as the bases passed through the nanopore one after another, inducing different degrees of current blockages[5,6]. In addition, the versatility of nanopore was demonstrated in the studies such as sensing the structures of RNA[7‒9], peptides[10‒12] and proteins[13‒15], detecting the interactions between supramolecules[16,17], measuring the size of nanoparticles[18,19] and concentrations of metal ions[20]. However, the ionic current across nanopore was extremely low (picoampere to nanoampere) owing to their high resistance. For example, the open pore current of α-hemolysin
nanopore was as low as 100 pA at a voltage bias of 100 mV (1 M KCl), and the blockage of current was less than 20 pA at some cases[21]. Therefore, those small current signals could be submerged by the noise in the recording process[22,23]. Though the development in data analysis enables extracting the blockage information from current trace at condition of high noise, the missing of short-lived events is still inevitable[24,25]. Physically suppressing the noise in signal remains a feasible and direct way to improve the performance of nanopore measurement. Conventionally, the current was measured and converted into a voltage signal by using a resistor-feedback amplifier[26]. The resistance of the feedback resistor on the order of 0.1‒10 GΩ controls the gain of current, whereas it also contributes to the majority noise in the signal. Though using a large resistance can lower the noise, the dynamic range for current measurement will also be narrowed due to the saturation of amplifier[27]. Other methods for lowering noise have been developed. For example, Rosenstein et al[28] fabricated an integrated nanopore sensing platform to improve the signal-to-noise ratio at high bandwidth. Balan et al[29] suppressed the current noise by reducing the nanopore chip
________________________ Received 2 March 2015; accepted 4 May 2015 * Corresponding author. Email: [email protected] This work was supported by by the National Natural Science Foundation of China (Nos. 21125522, 21327807, 51407078) and the Fundamental Research Funds for the Central Universities of China (Nos. 222201313004, WH1514049). Copyright © 2015, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(15)60843-X
YAN Bing-Yong et al. / Chinese Journal of Analytical Chemistry, 2015, 43(7): 971–976
capacitance. In this study, a novel design of the circuit of amplifier system was presented for low-noise current measurement (Fig.1a). The amplifier system features in a capacitorfeedback amplifier (A1) and an inverting differential integrator (A3). The advantages of capacitor-feedback amplifier were demonstrated by Goldstein et al[30], and the thermal noise on the absence of feedback resistor was effectively reduced. The differential integrator could highly suppress the commonmode noise between the current signal and reference voltage. Furthermore, a drift compensation was introduced for the reference voltage.
2 2.1
Experimental Materials
α-Hemolysin and decane (≥ 99%) were purchased from Sigma-Aldrich (St.Louis, MO, USA). 1,2-diphytanoly-snglycero-3-phosphocholine (chloroform, ≥ 99%) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Ultrapure water (18.2 MΩ·cm at 25°C) was obtained by the Milli-Q System (EMD Millipore, Billerica, MA, USA.). Buffer solution (1 M KCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was prepared before use. All other chemicals were of analytical grade. The detected DNA-PEG-DNA conjugate
Fig.1
(5’-GTCACGATGGCCCAGTAGTT-HPO4-(CH2-CH2-O)9-T TGATGACCCGGTAGCACTG-3’) was synthesized and HPLC-purified by Sangon Biotech (Shanghai) Co. Ltd. (China). 2.2 Nanopore experiments As described previously, 1,2-diphytanoly-sn-glycero-3phosphocholine in decane (30 mg mL‒1) was applied to form a bilayer across a 150 μm orifice in a lipid bilayer chamber (Warner Instruments, Hamden, CT, USA) which was filled with buffer solution. The solution of α-hemolysin was injected to the cis chamber proximal to the bilayer. Then, seven monomers of α-hemolysin would assemble to form a hydrophilic channel in the bilayer. The two compartments of the bilayer cell were termed cis and trans (shown in Fig.1). A pair of Ag/AgCl electrode was inserted into the two compartments, respectively. After a single nanopore was formed on the bilayer, analyte was injected into the cis chamber. The voltage was set to +120 mV during the experiments. The presented amplifier and a commercial resistor-feedback amplifier system (ChemClamp, Dagan Corporation, Minneapolis, MN, USA) were used to amplify and measure the ionic current flowing through the nanopore, respectively. The amplified currents were filtered at 3 kHz and converted to digital signals by DigiData 1440A hardware (Axon Instruments,
Schematic (a) and photograph (b) of the setup of the nanopore experiment and the amplifier circuit
YAN Bing-Yong et al. / Chinese Journal of Analytical Chemistry, 2015, 43(7): 971–976
Forest City, CA, USA) at 100 kHz sampling rate. Nanopore data were recorded by PClamp software (Axon Instruments, Forest City, CA, USA). A homemade program called NANOPORE ANALYSIS was used to recognize the current blockages, evaluate and distribute the current amplitudes of blockages[24]. As the current traces had different signal-noise rate, the thresholds were adapted as 4σ, where σ was the standard deviation of the whole current trace. For pulse-like events, the current amplitude was evaluated by using integration method as described previously. For multi-level events, the minimum point was chosen for measuring the current amplitude.
3 3.1
Results and discussion Circuit analysis of the amplifier system
During detection, the amplifier system worked as a voltage clamp to measure the ionic current across the nanopore while holding the reference voltage at a certain level. The first stage integrated the current, and its output voltage (V1) was related to the quantity of electric charge poured into the feedback capacitor (Cf). The V1was evaluated by using the Equation (1). dt (1) where, a 0.1 pF feedback capacitor is selected to achieve a high bandwidth for the amplifier. Then, the signal of V1 is differentiated by differential amplifier (A2). The output voltage (V2) can be calculated as Equation (2). (2) Since reference voltage was not changed during recording, it was applied at the positive lead prior to a RC circuit for drift compensation. The differential integrator (A3) integrates the difference between V2 and reference voltage, whose output follows Equation (3). (3) It is notable that the common-mode noise from the reference voltage is eliminated owing to the advantage of differential integrator[31]. To avoid the saturation of Cf and C2, CMOS switchs (S1, S2 and S3, ADG452, Analog Devices) are used to discharge the capacitors, which have low leakage current and introduce limited noise. V1 and Vout are sampled by the analog-to-digital chip (AD7626, Analog Devices). The final differentiated output is linear to the input current i(t) as in Equation (4). (4) According to the value of the elements as shown in Table 1, the current was evaluated based on the final output voltage as i(t)/Vout = 1 pA mV‒1. The dynamic range of the amplifier was as large as ±10 nA with this configuration.
3.2
Noise of the amplifier system
To testify the presented amplifier system, α-hemolysin nanopore was fabricated and detected at 120 mV. As shown in Fig.2a, the open pore current measured (gray) by a resistor-feedback amplifier system (Fig.2b) had higher noise than that detected by the presented amplifier system (blue). The noise spectral density of the two current traces indicated that the high frequency noise was suppressed by the presented amplifier. 3.3
Application for detecting single molecule
Subsequently, the presented amplifier was applied for recording the current trace in real experiment. The detected molecule was a DNA-PEG-DNA conjugate, which could translocate through the α-HL nanopore, and induce obvious current blockage. Besides the translocation events, bumping of the molecules with the nanopore was common in the nanopore studies due to the nature of single molecule behavior, which appeared to be a pulse with low blockage current[32]. However, this part of events was often missed or not concerned, when locating the blockages through a strong threshold (20 pA) due to the high noise level[24]. In comparison with conventional amplifier, the presented amplifier had lower noise in recording the current trace for single molecular sensing than the conventional amplifier. Blockages with low current amplitude could be easily distinguished from the baseline by applying a low threshold. Examples of individual blockages with different current amplitude were depicted in Fig.3b. Table 1
Typical value of capacitors and resistors for amplifier systema
Capacitor (PF)
C0 105
Cf 0.1
C1 10
C2 10
Resistor (kΩ)
R0 100
R1 100
R2 100
R3 100
C3 10
a
This configuration of capacitors and resistors were used in the following nanopore measurements.
Fig.2
(a) Current trace measured by resistor-feedback amplifier (gray) and the proposed amplifier (blue); (b) Noise spectral density of the two current trace in (a), respectively
YAN Bing-Yong et al. / Chinese Journal of Analytical Chemistry, 2015, 43(7): 971–976
Fig.3
(a) Current trace measured in the real detection experiment by resistor-feedback amplifier (gray) and the proposed amplifier (blue); (b) Examples of the blockage events detected in the experiment with different current amplitude. The current amplitudes measured by resistor-feedback amplifier system (c) and the presented amplifier system (d) were distributed into histogram, respectively. The peaks were fitted by Gaussian function
Obviously, with lower threshold (10 pA) used in the data analysis, blockages lower than 20 pA were recognized. The detailed structures in the blockage such as multi-level blockage could be distinguished by the amplifier system. Furthermore, we distributed the current amplitude of the blockages into histograms. The distribution of current amplitudes measured by the conventional amplifier system was a single Gaussian peak (P0) centered at 0.58 (I/I0), which suggested that there was only translocation events existing during the detection (Fig.3c). However, two Gaussian peaks emerge in the histogram of current amplitude measured by the presented amplifier system (Fig.3d). The first peak (PI) with low current amplitude (0.21, I/I0) was related to the bumping blockages, while the second peak (PII) centered at 0.55 (I/I0) was consistent with the P0 in Fig.3c, therefore this part of events was also corresponding to the translocation event. It was clear that the recovery of PI contributed to the less noise in the current trace as measured by the presented amplifier system.
monitoring the momentary fluctuation of ionic current across nanopore. Electrical noise was suppressed by the following strategies: (1) using capacitor-feedback amplifier; (2) employing a differential integrator to eliminate the common-mode noise in current trace; (3) introducing drift compensation for the reference voltage. We characterized the performance of the amplifier system in real nanopore measurement. As a result, thermal noise and noise at high frequency were highly reduced. The example study for detecting DNA-PEG-DNA conjugate in real nanopore experiment demonstrated the significant improvement of the presented amplifier system for recovering the blockages with low current amplitude.
References [1]
Ying Y L, Cao C, Long Y T. Analyst, 2014, 139: 3826‒3835
[2]
Si W, Zhang Y, Wu G S, Sha J J, Liu L, Chen Y F. Chin. Sci. Bull., 2014, 59(35): 4929‒4941
4
Conclusions
[3]
Ying X H, Zhu X Y, Gu J, Zhang X, Shao Y H. Chinese J. Anal. Chem., 2013, 41(5): 632‒640
In conclusion, a novel amplifier system was presented for
[4]
Guo S J, Wang E K. Accounts Chem. Res., 2011, 44: 491‒500
YAN Bing-Yong et al. / Chinese Journal of Analytical Chemistry, 2015, 43(7): 971–976
[5]
Cherf G, Lieberman K, Rashid H, Lam C, Karplus K, Akeson M. Nat. Biotechnol., 2012, 30(4): 344‒348
[6]
Manrao E, Derrington I, Laszlo A, Langford K, Hopper M, Gillgren N, Pavlenok M, Niederweis M, Gundlach J. Nat. Biotechol., 2012, 30(4): 349‒353
[7] [8] [9]
Cracknell J, Japrung D, Bayley H. Nano Lett., 2013, 13(6):
[18] Goyal G, Freedman K, Kim M. Anal. Chem., 2013, 85(17), 8180‒8187 [19] Wang J R, Ma J, Ni Z H, Zhang L, Hu G Q. RSC Adv., 2014, 4(15): 7601‒7610 [20] Yang C, Liu L, Zeng T, Yang D W, Yao Z Y, Zhao Y L, Wu H C. Anal. Chem., 2013, 85(15): 7302‒7307
2500‒2505
[21] Meller A, Branton D. Electrophoresis, 2002, 23(16): 2583‒2591
Wang Y, Zheng D L, Tan Q L, Wang M, Gu L Q. Nat.
[22] Uram J, Ke K, Mayer M. ACS Nano, 2008, 2(5): 857‒872
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