32nd Annual International Conference of the IEEE EMBS Buenos Aires, Argentina, August 31 - September 4, 2010

An Integrated, Low Noise Patch-Clamp Amplier for Biological Nanopore Applications

Gang Wanga , William B. Dunbarb , Member, IEEE
Abstract We present an integrated, low noise patch-clamp amplier for biological nanopore applications. Our amplier consists of an integrator-differentiator architecture coupled with a novel opamp design in the CMOS 0.35 m process. The post-layout full-chip simulation shows the input referred noise of the amplier is 0.49 pA RMS over a 5 kHz bandwidth using a veried electrical model for the biological nanopore system. In our biological nanopore experiments studying protein-DNA interactions, we encounter capacitive transients with a nominal settling time of 5 ms. Our amplier design reduces the settling time to 0.2 ms, without requiring any compensation circuitry.

compensation is heuristic and involves turning knobs on the amplier box [5]. Our patch-clamp design is motivated in Section II and presented in Section III. Simulation results are given in Section IV and Section V concludes the paper. II. PATCH -C LAMP S YSTEM FOR B IOLOGICAL NANOPORE A PPLICATIONS A. Background We use the protein channel -Hemolysin (-HL) for the nanopore. When created in an ionic buffer of 0.3 M KCl, an applied voltage across the pore produces a steady open channel current signal (53 pA at 160 mV). Biological polymers, such as single-stranded DNA (ssDNA) or RNA, can then be electrophoretically driven through the nanoscale pore. The voltage across the pore creates a eld that forces polymers to translocate through the pore due to the polymers net (negative) charge. These polymers block ion ow through the pore during translocations, thus attenuating the open channel current. Modulations in the current signal can then be used to identify the translocating polymer. The patch-clamp amplier, Molecular Devices Axopatch 200B, is widely used to regulate the applied voltage and measure the ionic current through a nanopore channel [5]. Figure 1 shows the schematic diagram of the nanopore device with a patch-clamp amplier. The dynamic range of input signals is 200 pA for biological nanopore applications.
measure current

I. I NTRODUCTION The patch clamp is a fundamental technique for measuring the activity of cell membrane channels, and is commonly used in nanopore applications. Biological nanopores are an established method for studying polynucleotide and polynucleotide-binding enzyme kinetics at the single molecule level [1-3], and offer great promise for inexpensive genomic sequencing [4]. As a single molecule sensor, variations in the ionic current through the pore can be correlated with the kinetics of each molecule captured in the pore. Specically, an applied voltage controlled with a patch-clamp amplier pulls charged molecules such as DNA or RNA through the pore. Correlations between the ionic current amplitude and the nucleotide identity of individual DNA or RNA molecules translocating through the pore has been shown through various assays using -hemolysin nanopores [1]. More recently, current amplitude signatures have been identied to distinguish unbound DNA from enzyme-bound DNA in the pore, and in real-time [2]. These signatures were exploited with active voltage control to examine the timescales of enzyme binding and unbinding to single DNA molecules above the pore [3]. One limitation of the active control approach is that capacitive elements of the patchclamp amplier and nanopore system cause a transient in the current signal immediately following a change in command voltage. The transient typically lasts 1-5 ms and masks binding/unbinding events that are faster than the settling time. In this paper, we will describe an integrated, ultra low noise patch clamp amplier design. An advantage of the design is that transient settling times are reduced to 0.2 ms without requiring any compensation circuitry. Although compensation circuitry with existing ampliers can in principle achieve settling times below 1 ms, the method for
This work is supported by NIH K25 HG004035-03 and NSF CAREER ECCS-0845766. a Postdoctoral Scholar, and b Assistant Professor, Computer Engineering. University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA, 95064. Corresponding author: dunbar@soe.ucsc.edu.

Cl-

command voltage

+
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cis trans

patch-clamp amplifier

Fig. 1. Schematic diagram of the nanopore device with a patch-clamp amplier. Voltage is applied across a single -HL channel inserted in a lipid bilayer membrane. Current through the nanopore is carried by K+ and Cl- ions.

As stated, we characterized the current signatures of enzyme-bound DNA complexes captured in the nanopore, using the Klenow Fragment (KF) of DNA Polymerase I as our model enzyme [2]. Captured KF-DNA complexes result in events with two current amplitudes (Figure 2). The initial current level corresponds to the enzyme bound state (EBS) of the captured DNA, with KF on top of the pore and the single-stranded template threaded through the length of the nanopore channel (Figure 2, ii). The second current amplitude results upon voltage-promoted enzyme dissociation,

978-1-4244-4124-2/10/$25.00 2010 IEEE

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when the duplex DNA drops into the nanopore vestibule (Figure 2, iii). Subsequently, the duplex DNA dissociates and ssDNA exits into the trans compartment. (Figure 2, iv).

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lifetime of EBS events) [3], up to half of binary complexes captured may be diagnosed as unbound DNA events by the control logic because the EBS was masked by the 5 ms transient (Figure 3b). This undermines our aim of modeling the data to uncover the rates constants for binary complex formation and dissociation. An amplier that reduces the transient settling time would alleviate this limitation. B. Electrical Model of Patch Clamp System We use the resistive feedback headstage in the Axopatch 200B. Figure 4 shows the nanopore setup. It includes a nanopore model, a resistive feedback headstage, a 4-pole Bessel lter, and a parasitic capacitor Cp . Rf is the feedback resistor in the resistive feedback headstage. In the nanopore model, Rc models channel resistance, Cm models the membrane capacitance, and Ra models the electrolyte access resistance between the electrodes and the nanopore channel. The low-pass 4-pole Bessel lter in the Axopatch 200B is typically set at 5 kHz in our experiments. Our amplier needs to detect > 3 pA signals (i.e., the 21 pA to 17 pA shift shown in Figure 2).

60 pA 40 20 0 0 50 100 ms

Fig. 2. Current trace and illustration of events for KF-bound DNA capture event at 160 mV: (i) open channel, (ii) KF-bound DNA capture (21pA) into the nanopore, (iii) voltage-promoted KF dissociation from DNA causing drop in current amplitude (17pA), and (iv) DNA duplex dissociation and translocation through the nanopore, resulting in an open channel current equivalent to (i).

The two-level amplitude pattern within KF-bound DNA events was exploited in our recent work on active control of DNA in a nanopore tethered by duplex regions [3] (Figure 3). A limitation of nanopore systems is that a capacitive transient
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Fig. 4. Electrical model of the nanopore, the patch-clamp preamplier and the 4-pole Bessel lter.

Time (msec)

Fig. 3. (a) Sequence of events during automated voltage control of DNA tethered by duplex segments: (i) Trans-side negative voltage (-20 mV) exposes DNA for KF binding above the pore. Switching to transside positive voltage (160 mV), KF is (ii) bound or (iii) not bound to the DNA. Initial detection of (ii) results in sustained voltage until detection of dissociation (iii). Detection of (iii) (from state (i) or (ii)) results in return to (i). (b) Recorded current and voltage signals in which (i)-to-(iii)-to-(i) transitions were detected, but the 5 ms transient may have masked the presence of state (ii).

The electrical model in Figure 4 has been veried by comparing experimental data and simulation. The total input capacitance (Cp + Cm ) of the setup is about 6.5 pF and the channel resistance (Rc ) is 3 Gohm. Resistance Ra was measured to be 200 kohm, and therefore can be ignored. Figure 5 shows that the experimental waveforms and simulation waveforms are well matched. The responses are to a 5 mV step voltage change. III. I NTEGRATED PATCH -C LAMP A MPLIFIER D ESIGN A. Noise Analysis for TIA The transimpedance amplier (TIA) is the critical component in the patch-clamp amplier. The front-end stage of a low noise TIA can be implemented by a resistive or a capacitive feedback amplier. The simplied model of a TIA is shown in Figure 6. A popular choice for the rst stage of the amplier is to use a common-source amplier with a resistor load [7]. RF is the feedback resistor, RD is the load resistor and Q1 is the input transistor with transconductance gm1 . These elements are the possible dominate noise sources. The second stage is modeled by an amplier that has a gain of A2. The noise due to the second stage can be ignored.

is superimposed on the ionic current measurement following a change in applied voltage. The transient necessarily masks information in the measured current for a time that is proportional to the magnitude of the net voltage change. Recently, Lemay [6] identies this speed bump lifetime as the bottleneck in dening the ultimate time resolution of nanopore detection systems, i.e., ...which are limited by the speeds at which the potential can be switched and the ionic current measured. As an example, a 180 mV step change causes a 5 ms transient (Figure 3b). The transient settling time dominates the total latency of the nanopore system. Since 50% of EBSs are faster than 5.8 ms (the median

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a)

[9] was chosen in this design for the front-end amplier in standard CMOS process in order to eliminate the noise due to the feedback resistor RF . B. Patch-clamp Preamplier Design Figure 7 shows the integrator-differentiator patch-clamp preamplier. Ci is the integration capacitor, Cd is the differentiation capacitor, Rd is the feedback resistor in the differentiator. Rs and Cf d are added to improve stability and reduce high frequency noise. Adiff is the gain of the difference amplier. The transimpedance gain of the preamplier is (Cd /Ci ) Rd Adiff . Cd is chosen to be 20 pF in the design. The reset switch is used for avoiding saturation. It needs to be reset periodically. The shaded block opamp1, shown

b)

Fig. 5. Measured and simulated output, in the model, Rc = 3 Gohm and Cp + Cm =6.5 pF. (a) The bandwidth of the lter is set at 5 kHz. (b) The bandwidth of the lter is set at 1 kHz.

Fig. 7.

Integrator-differentiator patch-clamp preamplier, based on [5, 9].

in Figure 7, is the critical component in the amplier. Both icker noise and thermal noise of the opamp have to be reduced. Also, the opamp needs to drive a large capacitive load (Cd ). The opamp proposed is designed to meet these specications. The schematic of the opamp is shown in Figure 8. The opamp has three parts, a resistive load input stage, a folded cascade 2nd gain stage and a class AB output stage. The 2nd gain stage is connected to the class AB output stage with Miller compensation.
Fig. 6. A simplied model of a TIA.

Cin is the total capacitance including Cp and Cm in Figure 5. It is about 6.5 pF for biological nanopore applications, as detailed above. With the following equations (1)-(3), the input referred noise can be estimated for our applications: 4kT RF 8 kT 2 IQ 1,in (f ) = 3 4kT 2 IR (f ) = D ,in RD
2 IR (f ) = F ,in

(1)
2 2 Cin gm1 2 2 Cin gm1

Fig. 8.

Schematic of the Opamp.

(2) (3) C. The Patch-clamp Amplier The patch-clamp amplier is shown Figure 9. It includes the patch-clamp preamplier shown in Figure 7. The sample/hold and the multiplexer are used to avoid the glitches due to the resets. The sample/hold circuit is composed of a simple switch and capacitor. The large hold capacitor is chosen to reduce noise and for charge injection. High frequency noise due to charge injection is suppressed by the

In order to achieve the noise specication of 1 pA RMS at 5 kHz bandwidth using resistive feedback, the feedback resistor has to be large (> 100 Mohm). A recent design by Weerakoon et al. [8] uses a 25 Mohm feedback resistor and a silicon-on-sapphire process that achieves 5 pA RMS noise at 10 kHz bandwidth. The integrator-differentiator architecture

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Fig. 9.

Schematic of the patch-clamp amplier.

4-pole low pass lter. With existing patch-clamp ampliers, step voltage changes induce amplier saturation for large voltage changes (> 15 mV using the amplier settings in nanopore experiments), and generate the capacitive transient. Compensation circuitry is the standard approach for reducing the transient settling time, e.g. [8]. In our design, the reset is synchronized with command voltage steps, which eliminates amplier saturation even for large (180 mV) voltage changes. Moreover, the transient settling time is dictated by the rise time of the lter (0.2 ms for 5 kHz bandwidth), and no compensation circuitry is required. IV. S IMULATION R ESULTS We designed the custom patch-clamp amplier in National Semiconductor 0.35m process. The supply voltage is 2.5V. The total transimpedance gain of the patch-clamp amplier is 1.88 gohm. The integrator needs to be reset every 1 ms and the reset period is 20 s. Detailed post-layout transient noise simulations in Cadence software show that the input referred noise of our amplier is 0.49 pA rms, using a 4-pole 5 kHz bandwidth lter and a realistic electrical model of the biological nanopore. With the command voltage step, the output can settle within 5% range in 200 s with a 4-pole 5 kHz bandwidth low pass lter as shown in Figure 10. The layout of the patch-clamp amplier is shown in Figure 11.

Fig. 11.

Layout of the patch-clamp amplier.

pA rms over a 5 kHz bandwidth has been described. State-ofthe-art amplier technology allows for control and detection response in 1-5 ms. Our amplier will reduce the response time to 0.2 ms (>5 times faster), without requiring any compensation circuitry. The integrated design will also permit multiplexing and therefore use in devices with multiple pores. In future work, we will also design the amplier which is suitable for both biological and solid state nanopore applications. VI. ACKNOWLEDGMENTS The authors would like to thank Bijoy G Chatterjee and Dilip Risbud at National Semiconductor, and Prof. Kenneth Pedrotti, Daniel Garalde and Patrick Ellis for their contributions. R EFERENCES
[1] M. Akeson, D. Branton, J. Kasianowicz, E. Brandin and D. Deamer. Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys. J., vol. 77, p. 322733, 1999. [2] S. Benner, R. J.A. Chen, N. A. Wilson, R. Abu-Shumays, N. Hurt, K. R. Lieberman, D. W. Deamer, W. B. Dunbar, and M. Akeson. Sequence-Specic Detection of Individual DNA Polymerase Complexes in Real Time Using a Nanopore. Nature Nanotechnology, 2(11):71824, 2007. [3] N. Wilson, R. Abu-Shumays, B. Gyarfas, H. Wang, K. R. Lieberman, M. Akeson, and W. B. Dunbar. Electronic control of DNA polymerase binding and unbinding to single DNA molecules. ACS Nano, 3(4):9951003, 2009. [4] D. Branton, et al. The potential and challenges of nanopore sequencing. Nat Biotechnology, 26:11461153, 2008. [5] Axopatch 200B, Molecular Devices Corporation, Sunnyvale, CA. [6] Serge G. Lemay. Nanopore-based biosensors: The interface between ionics and electronics. ACS Nano, 3(4):775779, 2009. [7] D. Johns and K. Martin. Analog Integrated Circuit Design. John Wiley and Sons, Inc. 1996. [8] P. Weerakoon, K. Klemic, F.J. Sigworth, and E. Culurciello. An integrated patch-clamp amplier for high throughput planar patchclamp systems. In IEEE International Symposium on Circuits and Systems, Seattle, WA, May 2008. [9] F. Offer and B. Clark. An improved amplier for patch-clamp recording. Biophys. J., vol. 47, p. 142a, 1985.

Fig. 10. Post-layout simulation results. Top trace is command voltage; the middle trace is the measured current with a 4th order 5 kHz low pass lter and the bottom trace is the reset signal. Here the reset is synchronized with command voltage steps to avoid the amplier saturation and improve the settling time.

V. C ONCLUSIONS An integrated CMOS patch-clamp amplier for biological nanopore applications with an input referred noise of 0.49

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