Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Biological Nanopores: Confined Spaces for Electrochemical SingleMolecule Analysis Chan Cao and Yi-Tao Long* Key Laboratory for Advanced Materials, School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai 200237, P. R. China
CONSPECTUS: Nanopore sensing is developing into a powerful single-molecule approach to investigate the features of biomolecules that are not accessible by studying ensemble systems. When a target molecule is transported through a nanopore, the ions occupying the pore are excluded, resulting in an electrical signal from the intermittent ionic blockade event. By statistical analysis of the amplitudes, duration, frequencies, and shapes of the blockade events, many properties of the target molecule can be obtained in real time at the single-molecule level, including its size, conformation, structure, charge, geometry, and interactions with other molecules. With the development of the use of α-hemolysin to characterize individual polynucleotides, nanopore technology has attracted a wide range of research interest in the fields of biology, physics, chemistry, and nanoscience. As a powerful single-molecule analytical method, nanopore technology has been applied for the detection of various biomolecules, including oligonucleotides, peptides, oligosaccharides, organic molecules, and disease-related proteins. In this Account, we highlight recent developments of biological nanopores in DNA-based sensing and in studying the conformational structures of DNA and RNA. Furthermore, we introduce the application of biological nanopores to investigate the conformations of peptides affected by charge, length, and dipole moment and to study disease-related proteins’ structures and aggregation transitions influenced by an inhibitor, a promoter, or an applied voltage. To improve the sensing ability of biological nanopores and further extend their application to a wider range of molecular sensing, we focus on exploring novel biological nanopores, such as aerolysin and Stable Protein 1. Aerolysin exhibits an especially high sensitivity for the detection of single oligonucleotides both in current separation and duration. Finally, to facilitate the use of nanopore measurements and statistical analysis, we develop an integrated current measurement system and an accurate data processing method for nanopore sensing. The unique geometric structure of a biological nanopore offers a distinct advantage as a nanosensor for single-molecule sensing. The construction of the pore entrance is responsible for capturing the target molecule, while the lumen region determines the translocation process of the single molecule. Since the capture of the target molecule is predominantly diffusion-limited, it is expected that the capture ability of the nanopore toward the target analyte could be effectively enhanced by site-directed mutations of key amino acids with desirable groups. Additionally, changing the side chains inside the wall of the biological nanopore could optimize the geometry of the pore and realize an optimal interaction between the single-molecule interface and the analyte. These improvements would allow for high spatial and current resolution of nanopore sensors, which would ensure the possibility of dynamic study of single biomolecules, including their metastable conformations, charge distributions, and interactions. In the future, data analysis with powerful algorithms will make it possible to automatically and statistically extract detailed information while an analyte translocates through the pore. We conclude that these improvements could have tremendous potential applications for nanopore sensing in the near future.
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promote cell lysis,1 such as α-hemolysin2 and aerolysin.3 The other type is outer-membrane protein channels, such as MspA4
INTRODUCTION
Two main pore materials embedded in a lipid membrane have been used as biological nanopores. One type is toxins, for which water-soluble monomers can self-assemble to form a nanoscale pore that could destroy the cellular osmotic balance and © XXXX American Chemical Society
Received: March 22, 2017
A
DOI: 10.1021/acs.accounts.7b00143 Acc. Chem. Res. XXXX, XXX, XXX−XXX
Article
Accounts of Chemical Research
Figure 1. Principle of biological nanopores for single-molecule analysis. The characteristic blockade currents induced by different analytes can be recorded by a current amplifier and converted into digital signals by an analog-to-digital converter. Then, with automatic data processing, the information on single molecules can be read out in real time. I is the blockade current, I0 is the open pore current, and ΔI = I0 − I.
and OmpG.5 The typical experimental instrument is composed of two chambers separated by a lipid membrane that is formed across a small orifice; the chambers are denoted as cis and trans (Figure 1).6 After addition of an electrolyte buffer into the two chambers, a constant electric current could be recorded after a biological pore inserts itself into the membrane and a voltage is applied across the pore using two Ag/AgCl electrodes. As a molecule is captured and transported through the confined space of the biological nanopore, the ions occupying the nanopore are excluded, which results in a detectable electrical signal from the transit blockade event.7 To date, biological nanopores have been used for a wide range of molecular sensing applications, including DNA sequencing,8−13 RNA detetction,14 peptide identification,15 protein analysis,16−20 poly(ethylene glycol) (PEG) discrimination,21 ion detection,22 and sensing of organic molecules.23
blockade current of poly(dT)45 but significantly accelerated the process of poly(dT)45 entry into the narrow constriction of αhemolysin. The conformational changes of nucleic acid aptamers are critical for specific binding of a target molecule. Recently, an ATP-binding DNA aptamer (ABA) was utilized as a probe to detect different target molecules (Figure 2b).28 First we measured the native folded ABA structure, which performed a typical event with a duration of 4.17 ms. Then the ABA:reporter complex (where the reporter is a 14-mer complementary ssDNA that can bind to ABA) was tested by an α-hemolysin. Many transit events were observed, and their durations were 0.50 ms. After the ABA:ATP complex was added to the cis chamber of α-hemolysin, the duration of blocked events decreased to 0.29 ms. Therefore, the conformational dynamics of the aptamer, including the folded conformation, linear strand, and complex with ATP, could be achieved at the single-molecule level by monitoring the characteristic blockade currents. Furthermore, the lightregulated interactions between the RNA aptamer and photochromic spiropyran were investigated29 (Figure 2c). The RNA aptamer exhibited a characteristic three-level blockade current that has been labeled Level 1, Level 2, and Level 3. Since the RNA aptamer could specifically bind to the closed form of spiropyran,30 we premixed the RNA aptamer and spiropyran under visible light (λ > 490 nm) with a concentration ratio of 1:2 to form RNA:spiropyran complexes. After the complexes were added to the cis chamber, three-level blockade currents were observed, which were similar to those of the RNA aptamer alone. However, the duration of Level 1 increased from 50.02 ± 1.95 to 56.49 ± 2.13 ms as a result of the enhancement of the stability of the RNA aptamer by the spiropyran molecule. When the complexes were irradiated under UV light (λ = 365 nm), the ring-closed spiropyran was photoisomerized to merocyanine, which could not bind with the RNA aptamer. The results indicated that the RNA:merocyanine complex induced a similar signal, but the duration of Level 1 (48.90 ± 0.91 ms) was consistent with that of RNA alone (50.02 ± 1.95 ms) rather than that of the RNA:spiropyran complex (56.49 ± 2.13 ms). Therefore, the two photoisomers spiropyran and merocyanine could be determined by the α-hemolysin nanopore at the single-molecule level upon tuning of the translocation process
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DNA-BASED SENSING Since the introduction of the α-hemolysin nanopore to study individual polynucleotides, it has been developed as a widely used biological pore for nucleic acid detection.10 The narrowest constriction of α-hemolysin is 1.4 nm in the stem domain, and thus, it allows only single-stranded DNA (ssDNA) to transport. However, α-hemolysin has a wide vestibule with a diameter of approximately 4.6 nm,2 which can capture varied conformations of nucleic acids such as duplexes and G-quadruplexes.24 Later, Kasianowicz demonstrated that translocation of poly(dT)100 through α-hemolysin induced a typical double-step blockade current because of the high entropic barrier as it entered the pore.25,26 To decrease the entropic barrier, an antigen binding fragment (Fab) of antibody HED10 was designed to accelerate the translocation of poly(dT).27 HED10 shows specific binding of poly(dT), which can act as a rudder to significantly decrease the energy barrier for translocation of poly(dT)45. As illustrated in Figure 2a, poly(dT)45 yielded a typical blockade current with two levels (labeled as level I and level II), which was consistent with the previous results.25 Next, the poly(dT)45:Fab HED10 complex was preincubated and added to the cis side of αhemolysin. The complexes produced a comparable double-step current trace, but the duration of level I decreased from 1.33 to 0.54 ms, indicating that the presence of Fab did not change the B
DOI: 10.1021/acs.accounts.7b00143 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Accounts of Chemical Research
Figure 2. (a) Representation of poly(dT)45-specific binding with Fab HED10 in an α-hemolysin and corresponding typical events. (b) Conformational changes of ABA and the corresponding typical events induced by different target molecules. (c) Mechanism of light-regulated RNA−spiropyran interaction and typical events caused by RNA aptamers and RNA:merocyanine and RNA:spiropyran complexes. Reproduced with permission from refs 27−29. Copyright 2011 Royal Society of Chemistry, 2011 John Wiley and Sons, and 2014 Royal Society of Chemistry, respectively.
was transported through an α-hemolysin, the typical event was divided into three segments (Figure 3b), each of which was fitted to a Gaussian distribution. It was verified that the short linker was responsible for the transient blockade as obtained in SII. To study the effect of ssDNA length on the translocation of the bioconjugate, a longer ssDNA, poly(dA)60, was conjugated to P7. The duration of separated poly(dA)60 (0.45 ms) was longer than that of the control sequence of poly(dA)20 (0.36 ms) in the assay for P7−B20. Following the interest of the current prior to SII, we enhanced the sensitivity of α-hemolysin for detecting low-molecular-weight PEG.34 Two ssDNAs were conjugated to the PEG molecule (named as DPD; Figure 3c), which could form a four-base pair hairpin structure. Therefore, as a DPD molecule transported through α-hemolysin, the
of the RNA aptamer. Moreover, the weak interaction between the short peptide of p53 DBD (p53-P) and a 40 base pair double-stranded DNA (dsDNA) (B40) has also been tested using an α-hemolysin nanopore (Figure 3a).31 In addition, the detection of conformational changes of DNA induced by heavy ions could also be achieved by an α-hemolysin.22 These observations suggested that the biological nanopore is an excellent biosensor for analyzing the conformational changes of DNA.32 Building on previous investigations of DNA conformation, the structure of a peptide−DNA conjugate was studied using an α-hemolysin.33 The collagen-like peptide (P7) and a 20-mer oligonucleotide (B20) were conjugated through a flexible disulfide linkage (denoted as P7−B20, Figure 3b). As P7−B20 C
DOI: 10.1021/acs.accounts.7b00143 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Accounts of Chemical Research
Figure 3. (a) Illustration of weak interactions between P53-P and B40 by an α-hemolysin (left) and corresponding two typical current traces (right). (b) Process of a peptide−oligonucleotide conjugate traversing through an α-hemolysin (left) and a corresponding typical event, which could be divided into SI, SII, and SIII. (c) Typical three-step blockade for the translocation of a DPD conjugate. Reproduced with permission from refs 31, 33, and 34. Copyright 2013 Chinese Chemical Society, 2012 Royal Society of Chemistry, and 2014 American Chemical Society, respectively.
P3ox was similar to that of P3 except for the duration, which increased to 649 μs. Overall, detailed structural information on the single peptide could be obtained using the biological nanopore, which is notably difficult to obtain by bulk spectroscopic methods such as circular dichroism and NMR spectroscopy. Following this finding, a series of negatively charged α-helical peptides terminated with a general formula Fmoc-DxAyKz, were measured, where Fmoc = fluorenylmethoxycarbonyl, x and z = 1, 2, or 3, and y = 10, 14, 18, or 22.15 The blockade currents induced by all of these peptides could be assigned to two types. Type I had a small blockade amplitude and a long duration, while type II exhibited a large amplitude and a short duration. According to the statistical analysis, type II represented the translocation of peptides through the pore, while type I was due to bumping interactions of peptides with the inner wall of the nanopore. Interestingly, we noticed that as the dipole moment of the peptide increases, the bumping event/translocation event ratio decreases significantly. As the dipole moment increases, it becomes less probable for a peptide to bump into the entrance of the pore rather than translocate straight through. Therefore, the results showed that both the dipole moment and the net charge had major effects on the transport characteristics, demonstrating that a biological nanopore can provide useful structural information on a single peptide/protein, including the charge, length, and dipole moment. On the basis of the preliminary investigations of peptides, we studied the conformational changes of amyloid β peptide (Aβ), which is the sticky peptide prominent in Alzheimer’s disease. After the addition of Aβ1−42 on the cis side of α-hemolysin, the majority of blockade events had a large amplitude (96.58 pA) with a long duration (10.07 ms). When an inhibitor, Congo red (CR), was premixed with Aβ1−42 for 1 h, one obvious
hairpin structure underwent an unzipping process, resulting in a prolonged duration, which significantly enhanced the temporal resolution of the biological nanopore. Furthermore, biological nanopores have been applied to detect binding of various transcription factors to target DNAs and non-covalent interactions of DNA−protein complexes in such confined channels.35
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SENSING CONFORMATIONS OF PEPTIDES AND PATHOGENIC PROTEINS As a powerful single-molecule method, biological nanopore technology has been attracting more and more interest for the investigation of proteins in addition to DNA analysis, especially after publication of the original paper in 2004, which used biological nanopores as a confined space to study the structure of a peptide.36,37 Collagen-like peptides with different repeats of [-CSA-(Gly-Pro-Pro)n]2 (n = 1, 2, and 3, denoted as P1, P2, and P3, respectively; Figure 4a) were measured by αhemolysin. For P1, there were two populations, with the minor one located at −45.0 pA and the major one at −52.3 pA (labeled 1 and 2, respectively). According to the four possible structures of the peptides, as depicted in Figure 4b, the blockade event of 1 may be caused by the translocation of a single linear peptide, while 2 can be assigned to a monomeric peptide with a folded U-shaped structure during the translocation. For P2, three populations were observed. Populations 1 and 2 ranged from −45.0 to −55.0 pA. Population 3 centered at −57.6 pA with a duration of 547 μs, which was consistent with a dimeric linear molecule. For P3, the major population of 4 has a larger blockade current centered at −75.4 pA and a longer duration of 591 μs, which was produced by the structure of a linear collagen-like triple helix. Furthermore, oxidized P3 (P3ox) was measured as a control experiment. The behavior of D
DOI: 10.1021/acs.accounts.7b00143 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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structure of Aβ35−25 through α-hemolysin. For Aβ25−35, the duration of PI decreased while PII increased with negative applied potentials and reached a minimal value at −60 mV, suggesting that the peptide strongly interacts with the αhemolysin before translocating through the cis side or exiting from the trans side. Moreover, the circular dichroism spectra revealed that Aβ25−35 contains both random-coil and β-sheet structures. Thus, we deduced that PI is ascribed to translocation of the random-coil form of Aβ25−35 while PII is caused by a strong interaction between the β-sheet structure and the trans opening of α-hemolysin. Additionally, α-hemolysin was used to investigate α-synuclein, fibril formation of which is critical in pathogenesis of Parkinson’s disease.40 As illustrated in Figure 6a, α-hemolysin achieved real-time monitoring of α-synuclein structure and its conformational changes induced by different voltages at the single-molecule level. Enzymatic reactions are involved in many fundamental biological processes,41 and studying them at the single-molecule level would reveal hidden aspects of enzyme behavior. In an αhemolysin channel, the ribosomal unfolding of an RNA pseudoknot has been successfully mimicked, which uncovered the noncanonical interaction involved in RNA tertiary structure and separated this interaction from traditional base-pairing hybridization.42 To investigate the cleavage process of exonuclease I (Exo I), we monitored the degradation of dA5 induced by Exo I using an aerolysin nanopore that has exhibited a high sensitivity for short oligonucleotide detection.43 After addition of dA5 and Exo I to the cis chamber of aerolysin, the majority of events were produced by dA5, which generated a Gaussian peak of I/I0 at 0.37 (blue peak in Figure 6b). Subsequently, the Gaussian peaks of dA4 (I/I0 = 0.49, green) and dA3 (I/I0 = 0.67, orange) arose, followed by a significant decrease of dA5, revealing the stepwise cleavage process of dA5 into dA4 and dA3 by Exo I. The probability of the dA4 and dA3 peaks continued to increase while that of the dA5 peak decreased. Approximately 8 h later, the majority of dA5 was cleaved into dA4 and dA3. Meanwhile, the peak of dA4 began to decrease, and that of dA3 continued to increase. Finally, nearly all of the species were degraded into dA3 after 20 h. Therefore, real-time monitoring of the current traces produced in the aerolysin nanopore enabled the identification of every individual cleavage intermediate.
Figure 4. (a) Representation of the translocation of collagen-like peptides through an α-hemolysin. (b) Contour plots of current transients of P1, P2, P3 and P3ox, respectively. (c) The translocation of an α-helical peptide through aerolysin nanopore and two types of blockade currents. Reproduced from refs 36 and 15. Copyright 2004 and 2006, respectively, American Chemical Society.
population at 29.0 pA with a short duration was observed, implying that CR interfered with the intermolecular hydrogen bonding of the aggregates. In contrast to CR, β-cyclodextrin (βCD) could promote the aggregation of Aβ1−42, which resulted in a reduction of the translocation events since Aβ1−42 aggregates are too big to transport (Figure 5a).16 Moreover, recent investigations have shown that lipid-bilayer-coated nanopores can directly determine the size of individual Aβ oligomers in solution without chemical modification.38 In the next step, the effects of initial structural features on the timedependent aggregation of two Aβ fragments, Aβ25−35 and Aβ35−25, were studied. These two fragments have the same sequence but possess different secondary structures because of the different charge states of the terminal residues.39 As shown in Figure 5b, the majority of blockade events of monomeric Aβ35−25 had a wide distribution with a low current amplitude and a short duration (defined as PI). However, Aβ25−35 displayed two populations, one of which was similar to PI of Aβ35−25 while the other had a concentrated current distribution at 0.63 (defined as PII). Voltage-dependent experiments indicated that the duration of PI of Aβ35−25 decreased with increasing negative potential. On the basis of its low amplitude, we attributed PI mainly to translocation of the random-coil
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EXPLORING NOVEL BIOLOGICAL NANOPORES It is well-known that pore structure plays a critical role in nanopore sensing. Therefore, researchers have explored a wide range of transmembrane protein pores to satisfy various demands in analyzing single molecules. Aside from the commonly used α-hemolysin, other pores such as MspA,4 phi29 DNA-packaging nanomotor,44 FraC,45 OmpG,5 ClyA,46 FhuA,47 lysenin,48 and CsgG49,50 have been developed. We explored the possibility of using Stable Protein 1 (SP1) to detect DNA.51 The characteristic structure of SP1 is a ringlike dodecamer with an inner diameter of 3.0 nm, which broadens the existing research areas of pore-forming biomaterials from unsymmetrical to symmetrical pores. The duration of poly(dA)45 (0.78 ms) in SP1 was twice as long as that of poly(dA)20 (0.41 ms), which demonstrated that the longer sequence of poly(dA)45 led to intensive interactions between the biopolymer and SP1. The durations of both poly(dA)20 and poly(dA)45 in the SP1 nanopore were longer than those in αhemolysin, which indicated that the geometry of SP1 could effectively slow down the translocation of ssDNA. The E
DOI: 10.1021/acs.accounts.7b00143 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 5. Biological nanopore for studying Aβ peptides. (a) Representations of typical events of Aβ42:β-CD, Aβ42, and Aβ42:CR, respectively. (b) Initial structures, typical events, and 2D contour plots of Aβ35−25 (red) and Aβ25−35 (blue). Reproduced with permission from refs 16 and 39. Copyright 2011 American Chemical Society and 2016 Royal Society of Chemistry, respectively.
signals were acquired after addition of Oligo-1 to the cis or trans side of α-hemolysin. To obtain direct evidence for the translocation of Oligo-1 through aerolysin, a control ssDNA (Oligo-2) that had been attached to a fluorescent moiety at the 5′ side of Oligo-1 was tested. After the addition of Oligo-2 to the cis side of aerolysin with continuous recording for 6 h, the solution in the trans chamber was collected to perform total internal reflection fluorescence (TIRF) experiments. The images indicated that the intensity of fluorescence obviously increased with a longer recording time compared with the control experiment in the absence of Oligo-2. Furthermore, different lengths of polydeoxyadenines (dAn) were measured using the aerolysin nanopore.43 Aerolysin performed a perfect current separation for different lengths of dAn (n = 2, 3, 4, 5, 10), and even dinucleotides could be detected directly. The average translocation speed of individual DNA bases through aerolysin is 0.3−2.0 ms/nucleotide, which is nearly 1000 times slower than that through wild-type α-hemolysin (3.3 μs/ nucleotide). An interesting phenomenon is that some plots obviously appeared in the scatter plots of dA10, which had similar distributions to dA5, dA4, and dA3, while dA10 was very pure according to mass spectrometry (Figure 7c). The plots of dA5, dA4, and dA3 might be attributed to the inevitable truncated impurities of the dA10 from the trace impurities remaining after purification via HPLC, which demonstrated that aerolysin is very sensitive for oligonucleotide detection.
symmetric geometry, stability, and engineering ability of SP1 ensure that it is ideal and suitable for further application in dsDNA analysis. In addition to the use of SP1 to broaden the application of nanopores, another toxin, aerolysin, was discovered and investigated for single-molecule sensing since its narrower diameter could improve the sensitivity for sensing of smaller molecules.52 Aerolysin, from Aeromonas hydrophila, can spontaneously assemble a heptameric transmembrane barrel with a diameter of approximately 1.0−1.4 nm3 (Figure 7a). In 2006 it was initially used as a biological nanopore for α-helical peptide analysis,15 and subsequently it was applied to study protein modification,53 protein unfolding processes,54 oligosaccharides,55 enzyme activity,56 and PEG molecules.57 Recently, we first utilized aerolysin to discriminate different lengths of oligonucleotides.58 Unexpectedly, it exhibited a high sensitivity for oligonucleotide analysis and achieved the identification of four types of nucleobases (A, T, C, and G)59 and DNA methylation in real human serum directly.60 The open-pore current of aerolysin is nearly 60 pA at 120 mV in 1.0 M KCl solution, which is identical to the value for α-hemolysin and the β-CD complex system under the same conditions (nearly 62 pA at 120 mV).61,62 A short ssDNA containing only four bases (denoted as Oligo-1, with sequence 5′-ACTG-3′) was tested using aerolysin.63 In the presence of Oligo-1, a large number of current signals were observed and showed a nearly 50% blockade (Figure 7b). In contrast to aerolysin, no current F
DOI: 10.1021/acs.accounts.7b00143 Acc. Chem. Res. XXXX, XXX, XXX−XXX
Accounts of Chemical Research
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DATA ANALYSIS AND AN IMPROVED PLATFORM FOR ELECTROCHEMICAL DETECTION In nanopore experiments, one of the main issues that hinders its development is the lack of efficient software to analyze the data. To promote the application of nanopores, our group has been developing data analysis software (Figure 8a). First, we focused on using an automatic process with a well-designed algorithm and software to extract the basic information on the events.64 The data processing introduced a second-orderdifferential-based calibration (DBC) method to correct the region of blockades and accurately measure the duration. To eliminate the influence of baseline drift, a moving-window algorithm was applied to evaluate the localized baseline at every time point. The DBC method significantly improved the accuracy for determining the start and end points of blockades. Notably, the blockade events could be affected by the bandwidth of the system, which is influenced by the membrane capacitance and the frequency response of the current amplifier or a low-pass filter in the recording instrument. This could lead to a serious problem when the duration of the blockade decreases below double the rise time (trise ≈ 0.33/fc, where fc is the cutoff frequency of the filter). The results demonstrated that with the DBC method the relative error in the duration was 71% less than that of the conventional method with the single threshold when the nominal duration was as low as 0.05 ms. Additionally, this novel integration method significantly enhanced the nominal bandwidth of the data analysis.65 On the basis of the developed method, we designed an integrated software system called “Nanopore Analysis” on the MATLAB platform.66 With this program, management of data, detection of events, and a preview of individual events could be achieved easily and conveniently. It takes 10 s to complete a 1 min data recording with a 100 kHz sampling rate. Considering the different criteria or purposes for data analysis, all of the
Figure 6. (a) Detection of α-synuclein protein and its conformational changes induced by different voltages. (b) Stepwise degradation process of dA5 induced by Exo I. Reproduced with permission from refs 40 and 59. Copyright 2013 American Chemical Society and 2017 Springer Nature Group, respectively.
Figure 7. (a) Structure of aerolysin heptamer. (b) Single-channel recording for the addition of Oligo-1, statistical analysis, and TIRF images before and after addition of Oligo-2. (c) Discrimination of different lengths of dAn and scatter plots of dA10. Reproduced with permission from refs 43 and 63. Copyright 2016 Springer Nature Group and 2016 American Chemical Society, respectively. G
DOI: 10.1021/acs.accounts.7b00143 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 8. (a) Example of data processing from a raw current trace and event statistics. (b) Circuit diagram of the capacitance-feedback current amplifier. Reproduced with permission from refs 64 and 70. Copyright 2015 American Chemical Society and 2015 Elsevier, respectively.
along with the outstanding feature of whole-band frequency hysteresis, the integrated current measurement system possesses significant abilities for nanopore detection by gaining a current resolution of
