Skip to Main Content
Skip Nav Destination

Nanopore electrochemistry refers to the promising measurement science based on elaborate pore structures that offer a well-defined geometric confined space to adopt and characterize single entities including single cells, single particles, and even single molecules by electrochemical technology. The electrochemical confined effect within the nanopore displays the ability to achieve single entity discrimination by focusing electron, photon, ion and magnetic fields into a small area. It converts the intrinsic properties of single entities into visible electrochemical read-outs with ultra-high temporal-spatial resolution. Therefore, the nanopore technology permits the possibility to resolve the transient signals for further revealing the single biomolecules dynamics. Nanopore electrochemistry has been applied in biophysics, chemical biology, disease diagnostics and other advanced disciplines by pushing the detecting limitation to molecular scale. In this book, we summarize the nanopore electrochemistry from the following three main areas. First, a nanopore-based single biomolecule sensing interface with chemical group precision is described in detail, including the design, instrumentation and advanced application. Then, the solid-state nanopore is summarized along with its fabrication process and application. Last, the glass nanopipette is introduced since it enables the monitoring of biomolecule interactions and the electron-transfer process in single living cells with high resolution and negligible cell damage. With the advent of advanced measurement mechanisms, instrumentation and data algorithms, the electrochemically confined nanopore is undoubtedly an exciting and promising field. We expect the next avenue for the wide application of nanopore electrochemistry in a variety of disciplines, leading us to explore the new chemistry at a much smaller scale.

This book aims to summarize the advancement of nanopore electrochemistry, from the fundamental sensing mechanism of pore–substrate interactions within a confined space to the exploration of rich single-molecule information from electrochemical signals. We present the single nanopore for single molecule sensing based on a comprehensive understanding of nanopore electrochemistry.1,2  Two decades ago, researchers developed single peptide sensing using nanopore electrochemistry. The approach was simple but the scope “beyond sequencing of DNA” was foreseeable. Then the conceptual advances occurred, making it possible for single-molecule sensing in a label-free manner via “electrochemically confined space”. Not only the biomolecule nanopore interface (e.g. protein channels) but also inorganic materials (e.g. graphene, Si, SiNx) could build such a confined space. The nanopore sensing interface could be treated as a miniaturized electrode, it is feasible to manipulate every sensing element inside the biological nanopore sensing interface with chemical group precision. Therefore, nanopore electrochemistry achieved single molecule detection in a high-throughput manner with high amperometric resolution without labeling. Moreover, other electrochemical confined effects such as the confinement of biological interactions, the electron-transfer process, and the light inside nanopores, were also demonstrated. These confined effects provide the mutual information of a single molecule from the simultaneously recording of the electric and optical signals. The advancement of instrumentation with sufficient temporal-spatial resolution is also critical to sensitive electrochemical measurement. Many studies have demonstrated that the ions inside the nanopore confined space act as the smallest probes, since their vibrations demonstrate the intrinsic properties of analytes. Therefore, the application of time-frequency analysis of ionic current contributes to the appearance of a “single-molecule ionic spectrum”, which enables the mapping of the single molecule weak interactions, such as hydrogen bonds, the Van der Waals force and the electrostatic effect. It is undeniable that nanopore electrochemistry, which shows the capability of addressing entirely new biological questions, is an exciting area for future research.

The understanding of physics, chemistry and the life sciences is determined by measurement science, deepening the precision and depth of observation. In order to process at ultra-fine resolution, for example at single-molecule level, a compatible sensing tool should be used to process the analytical methodologies. At this point, the single-molecule method has attracted increasing attention and has gradually become widespread in chemistry and biology. Single-molecule analysis, in terms of biology, has the capability to reveal brand new biological questions since it provides rich heterogeneities and stochastic information on dynamic biological systems.3–5  Fundamentally, the sensing performance is mainly determined by the sensing interface. The sensing domain comprises reactive molecules for recognition and/or functional materials for signal amplification. Commonly, the sensing domain of the nanopore provides the strongest interaction with the bypass analytes; each substrate provides a unique interaction that turns out as signature readouts. Traditionally, the macroscale sensing domain has been applied in a wide range of versatile detecting cases. However, general sensing information can no longer support exploring fundamental chemical and biological reactions at the molecular level; as a result, single-molecule detection resolution is urgently needed. In order to push the limits of detection to the single molecule level, a comparable miniaturized sensor interface is a must. Adapted from traditional macroscale sensors, micro- or even nanoscopic sensors have been developed. We hereby think to turn over and roll the conventional sensing interface into a small structure for the sake of reducing the sensing domain of the sensor (Figure 1.1). Inspired by nature where membrane proteins on either eukaryotic or prokaryotic cells, acting as nanopores that are capable of probing the surrounding substrates and then selectively uptaking what is required, an artificial system has been created to accommodate such protein nanopores. Therefore, we propose that the nanopore is one of the most suitable structures for the sensing interface for single-molecule measurements as it accommodates a single molecule inside. The nanopore technique provides a well-defined three-dimensional (3D) structure that is comparable to the size of most target single molecules. The single entity is initially fitted into a proper spatial alignment, i.e. the nanopore-based sensing interface,6  then an external stimulus is applied to the analyte, i.e. a bias voltage. Finally, the electrochemical readouts are acquired to be decoded that contain abundant single molecule information. In contrast to the conventional macroscopic strategy to dilute towards the single-molecule level, the nanopore provides a confined space to accommodate only one single molecule from the bulk solution, which eliminates the assembly effect.7,8  When a constant bias voltage is applied across the nanopore system, any accommodation in the substrate in single-molecule fashion offers unique electrochemical behavior that correlates with the spatial and temporal information. Once the sensing interface is compatible with the geometrical dimension of the bypass substrates, the biophysical properties of the analyzed substrates (i.e. physical dimension, polarity, charge state, dipole moment, etc.) can be revealed from the nanopore electrochemistry readouts. Herein, the single-molecule sensing interface provides a confinement for the analytes with different geometries and sizes.1 

Figure 1.1

(a) A typical macro sensing interface undergoing turn-over, roll and finally self-assembly into a so-called single-molecule interface. Also, single-biomolecule interface manipulation of the aerolysin interaction with poly(dA)4. (b) Wild-type aerolysin and (c) mutant I aerolysin with one amino acid at the nanopore opening modified with a negatively charged induced decreased capture rate. (d) Mutant II aerolysin with one amino acid at the nanopore lumen modified with a negative charged demonstrated stronger interaction. Reproduced from ref. 2 [https://doi.org/10.1093/nsr/nwy029] under the terms of a CC BY 4.0 license [https://creativecommons.org/licenses/by/4.0/].

Figure 1.1

(a) A typical macro sensing interface undergoing turn-over, roll and finally self-assembly into a so-called single-molecule interface. Also, single-biomolecule interface manipulation of the aerolysin interaction with poly(dA)4. (b) Wild-type aerolysin and (c) mutant I aerolysin with one amino acid at the nanopore opening modified with a negatively charged induced decreased capture rate. (d) Mutant II aerolysin with one amino acid at the nanopore lumen modified with a negative charged demonstrated stronger interaction. Reproduced from ref. 2 [https://doi.org/10.1093/nsr/nwy029] under the terms of a CC BY 4.0 license [https://creativecommons.org/licenses/by/4.0/].

Close modal

The pore-forming membrane protein, composed of several sub-units, is regarded as a single biomolecule interface (Figure 1.2), i.e. biological nanopore sensors. To achieve relatively high selectivity sensing of target analytes, it is necessary to modify the sensing domain to be specifically active to corresponding targets. Empowered by site-direct mutagenesis, one can manipulate the interface biophysical properties at each amino acid group. In general, under a constant bias voltage, each analyte is driven towards the nanopore opening, then it somehow interacts with each amino acid ladder and finally translocates/binds with the nanopore. One can manipulate a few molecules at the nanoscale interface to enhance the stochastic probabilities of analytes approaching the sensing hot spot so as to boost the electrochemical sensitivity. Each ion among the protein–molecule interaction can act as the smallest probe. The nanopore sensing mechanism involves reversible non-covalent interactions at the amino acid ladder to execute successive transport of ions and analyte into the channel, but at different rates. Such a dynamic process is dominated by ion redistribution over the sensing interface. Moreover, the handling of each amino acid inside the channel can determine the pore–analyte interaction reaching the amplification of the non-covalent interactions.9 

Figure 1.2

The sensing mechanism of a biomolecule nanopore interface. Top: the electrochemistry testing cell and the related data acquisition block diagram. Two compartments are separated by a Teflon septum with an aperture (ca. 50 μm) at the center. The aperture is further used to support an artificial lipid bilayer where a biological nanopore can be reconstituted and functionalized. Each compartment is connected via a pair of Ag/AgCl electrodes toward the headstage. The signal acquired by the headstage is first amplified and filtered at high signal-to-noise, then sampled and converted into a digital signal that can be recognized by the data processor. Middle: a cross-sectional view of the biological nanopore example illustrating that a polymer segment is brought near the nanopore “mouth”, then captured by the nanopore lumen and finally released to the exit (an example of a translocation process). Bottom: the corresponding amperometric response of the nanopore electrochemical recording. A constant open current (pA) is obtained under a constant bias voltage (mV) when the substrate approaches the nanopore “mouth” before entering the lumen. A sudden drop in the current (conductance) is observed as soon as the substrate enters the nanopore lumen. Finally, the current immediately returns back to the open current once the substrate is released from the lumen region.

Figure 1.2

The sensing mechanism of a biomolecule nanopore interface. Top: the electrochemistry testing cell and the related data acquisition block diagram. Two compartments are separated by a Teflon septum with an aperture (ca. 50 μm) at the center. The aperture is further used to support an artificial lipid bilayer where a biological nanopore can be reconstituted and functionalized. Each compartment is connected via a pair of Ag/AgCl electrodes toward the headstage. The signal acquired by the headstage is first amplified and filtered at high signal-to-noise, then sampled and converted into a digital signal that can be recognized by the data processor. Middle: a cross-sectional view of the biological nanopore example illustrating that a polymer segment is brought near the nanopore “mouth”, then captured by the nanopore lumen and finally released to the exit (an example of a translocation process). Bottom: the corresponding amperometric response of the nanopore electrochemical recording. A constant open current (pA) is obtained under a constant bias voltage (mV) when the substrate approaches the nanopore “mouth” before entering the lumen. A sudden drop in the current (conductance) is observed as soon as the substrate enters the nanopore lumen. Finally, the current immediately returns back to the open current once the substrate is released from the lumen region.

Close modal

The nanopore-based single-molecule interface modulates the sensitivity and selectivity of single-molecule sensing. Further analysis allows us to understand that a unique electrochemical signal is generated not only due to spatial occupancy but also due to being dominated by the individual non-covalent bond-making and bond-breaking step. Such weak interactions occur due to nanopore-analyte-induced dynamic conformational changes and the electron/charge transfer process. In order to amplify and acquire the slightest electrochemical responses, several approaches such as high bandwidth electrochemical instrumentation, delicate sensing tools and an advanced data algorithm have been developed. In this book, we discuss the fabrication process of a single-molecule interface. There are pore forming materials adopted from nature that are synthesized into nanopores as well as pores fabricated in inorganic substrates. The single-biomolecule composed nanopore, also dubbed single-biomolecule interface, owes unique specificity to each residue inside the pore lumen being controllable. Solid-state nanopores fabricated with the aid of advanced fabrication processes can reach any desired pore opening dimension. One of the feasible fabrications processes based on a glass capillary provides an approach of high bandwidth recording of the electrochemical redox process for single-entity analysis.

We will also discuss the electrochemical confinement effects on nanopore sensing, especially on measuring the single-molecule non-covalent reactions and the conformational dynamics of individual molecules. Besides electrochemical information being read from each individual molecule's translocation signatures, electrochemically confined effects also provide correlation with other external driving forces, such as light scattering or bipolar electrochemistry, generating new sensing mechanisms along with the application.

Taking another biological nanopore, α-hemolysin, as an example, a dsDNA (double strand DNA) whose projection area is larger than the nanopore lumen diameter experiences an unzipping process providing a partial blockage. The unzipped dsDNA transverses through the narrowest constriction zone in its single-strand formation and generates a unique electrochemical signal in accordance with each nucleotide base. Apart from the direct readout information, the weak non-covalent interactions between substrates and nanopores may be hard to observe as these small fluctuations may be submerged in the noise level. Nonetheless, the hidden dynamic information containing such weak non-covalent interactions influences the ion movement in the nanopore lumen. Fundamentally, these weak interactions contain the continuous vibration of charge and electron density from the molecule or inter-molecule. In the entire interaction process, the charged ions or electrons from the bulk solution or those introduced along with the analyte, frequently collide and interact in the nanopore lumen, i.e. the nanoscale confined space. The ions inside such a nanopore confined space form a dynamic net that correlates with the electron density. The entrance of the analyte inevitably modifies this dynamic net, which can be regarded as each ion becoming the smallest sensor. These smallest sensor elements perceive and reflect non-covalent interactions into ionic vibration, which should be observed at the frequency domain. In short, all the frequency modification contains the information that should reflect the various (i.e. weak/strong non-covalent interactions typically) biophysical interactions among the analytes, ionic dynamic nets and each amino acid group that compose the nanopore. These reflections are commonly buried in the noise of the ionic current traces in the time domain acquired with the current state-of-art hardware.

Recently, we adopted the Hilbert–Huang transform (HHT), which has the capability to obtain high precision in the frequency and time domains, to analyze the nonlinear and non-stationary nanopore data.10,11  Taking the example of the oligonucleotide poly(dA)4 interaction with the wild type (WT) and the K238E aerolysin nanopores (Figure 1.3), the WT nanopore gives a larger dispersion degree, forwarding the energy axis in normalized energy (z-axis)–probability (x-axis) spectrum compared to the K238E analog. This observation demonstrates the fingerprint spectra, instead of the fingerprint current response, for the capability to analysis the non-covalent interactions.12  By mimicking the frequency–energy spectrum of the typical ion current response into the hearable frequency, we illustrate that a nanopore sensor is composed of a single biomolecule interface akin to a tuba (Figure 1.4). When the molecule of interest flows into the “tuba” successively, the intrinsic properties of individual molecules can consequently be revealed by the dynamic modulation of the interactions (“button”) between the “molecule flow” and sensing interface. Consequently, as the translocation of individual molecules through the confined pore, the beautiful molecule music of pore–analyte weak interactions will be “played”.

Figure 1.3

Schematic diagram of the frequency analysis of ionic current traces using the Hilbert–Huang transform method. (a) The illustration for sensing poly(dA)4 using WT aerolysin and K238E analog. (b) Instance of the ionic current for the poly(dA)4 interaction WT aerolysin nanopore, with a zoomed-in figure in the sub-diagram. (c) Representation of each intrinsic mode function (IMF) from the empirical mode decomposition (EMD) analyzes the enlarged part in (b). (d) The 3D spectrum energy–frequency–time distribution of the enlarged blockade shown in (b). Reproduced from ref. 11 with permission from the Royal Society of Chemistry.

Figure 1.3

Schematic diagram of the frequency analysis of ionic current traces using the Hilbert–Huang transform method. (a) The illustration for sensing poly(dA)4 using WT aerolysin and K238E analog. (b) Instance of the ionic current for the poly(dA)4 interaction WT aerolysin nanopore, with a zoomed-in figure in the sub-diagram. (c) Representation of each intrinsic mode function (IMF) from the empirical mode decomposition (EMD) analyzes the enlarged part in (b). (d) The 3D spectrum energy–frequency–time distribution of the enlarged blockade shown in (b). Reproduced from ref. 11 with permission from the Royal Society of Chemistry.

Close modal
Figure 1.4

Single-molecule music. Ideally, transferring the frequency–energy spectrum from the ionic current into the voice frequency could possibly let us hear the rhythm from membrane channels. Reproduced from ref. 2 [https://doi.org/10.1093/nsr/nwy029] under the terms of a CC BY 4.0 license [https://creativecommons.org/licenses/by/4.0/].

Figure 1.4

Single-molecule music. Ideally, transferring the frequency–energy spectrum from the ionic current into the voice frequency could possibly let us hear the rhythm from membrane channels. Reproduced from ref. 2 [https://doi.org/10.1093/nsr/nwy029] under the terms of a CC BY 4.0 license [https://creativecommons.org/licenses/by/4.0/].

Close modal
1.
Ying
 
Y.-L.
Zhang
 
J.
Gao
 
R.
Long
 
Y.-T.
Angew. Chem., Int. Ed.
2013
, vol. 
52
 (pg. 
13154
-
13161
)
2.
Ying
 
Y.-L.
Cao
 
C.
Hu
 
Y.-X.
Long
 
Y.-T.
Natl. Sci. Rev.
2018
, vol. 
5
 (pg. 
450
-
452
)
3.
Gooding
 
J. J.
Gaus
 
K.
Angew. Chem., Int. Ed.
2016
, vol. 
55
 (pg. 
11354
-
11366
)
4.
Ha
 
T.
Nat. Methods
2014
, vol. 
11
 (pg. 
1015
-
1018
)
5.
Dufrêne
 
Y. F.
Evans
 
E.
Engel
 
A.
Helenius
 
J.
Gaub
 
H. E.
Müller
 
D. J.
Nat. Methods
2011
, vol. 
8
 (pg. 
123
-
127
)
6.
E. J. G.
Peterman
and
G. J. L.
Wuite
,
Single Molecule Analysis: Methods and Protocols
,
Humana Press
,
2011
7.
Cao
 
C.
Ying
 
Y.-L.
Hu
 
Z.-L.
Liao
 
D.-F.
Tian
 
H.
Long
 
Y.-T.
Nat. Nanotechnol.
2016
, vol. 
11
 (pg. 
713
-
718
)
8.
Wang
 
J.
Yang
 
J.
Ying
 
Y.-L.
Long
 
Y.-T.
Chem. – Asian J.
2019
, vol. 
14
 (pg. 
389
-
397
)
9.
Cao
 
C.
Long
 
Y. T.
Acc. Chem. Res.
2018
, vol. 
51
 (pg. 
331
-
341
)
10.
Alzahrani
 
H.
Antoine
 
C.
Baker
 
L.
Balme
 
S.
Bhattacharya
 
G.
Bohn
 
P. W.
Cai
 
Q.
Chikere
 
C.
Crooks
 
R. M.
Das
 
N.
Edwards
 
M.
Ehi-Eromosele
 
C.
Ermann
 
N.
Jiang
 
L.
Kanoufi
 
F.
Kranz
 
C.
Long
 
Y.
MacPherson
 
J.
McKelvey
 
K.
Mirkin
 
M.
Nichols
 
R.
Nogala
 
W.
Pelta
 
J.
Ren
 
H.
Rudd
 
J.
Schuhmann
 
E.
Siwy
 
Z.
Tian
 
Z.
Unwin
 
P.
Wen
 
L.
White
 
H.
Willets
 
K.
Wu
 
Y.
Ying
 
Y.
Faraday Discuss.
2018
, vol. 
210
 (pg. 
145
-
171
)
11.
Liu
 
S.-C.
Li
 
M.-X.
Li
 
M.-Y.
Wang
 
Y.-Q.
Ying
 
Y.-L.
Wan
 
Y.-J.
Long
 
Y.-T.
Faraday Discuss.
2018
, vol. 
210
 (pg. 
87
-
99
)
12.
Li
 
M.-Y.
Ying
 
Y.-L.
Fu
 
X.-X.
Yu
 
J.
Liu
 
S.-C.
Wang
 
Y.-Q.
Li
 
S.
Cao
 
C.
Wan
 
Y.-J.
Long
 
Y.-T.
ChemRxiv. Preprint
2018
Close Modal

or Create an Account

Close Modal
Close Modal