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Nanopore-Based Protein Sequencing Using Biopores:

Current Achievements and Open Challenges
Alina Asandei, Giovanni Di Muccio, Irina Schiopu, Loredana Mereuta, Isabela S. Dragomir,
Mauro Chinappi,* and Tudor Luchian*

degradation.[7] Known limitations affecting

The synergy of life sciences discoveries, biomolecular and protein engineering these methods, are: i) mass spectrometry
advances, and groundbreaking nanofabrication technologies, has intro- often fails to reveal the complete sequence
duced over the past years the wide use of the nanopore-based investigations information of a protein;[8] ii) Edman deg-
of matter at the molecular level. This review focuses on the fundamental radation is optimally applied to sequences
of 30–50 residues long without modified
principles of α-hemolysin (α-HL) protein-based nanopores, as sensitive
or buried N-terminal amino acid;[9] iii) in
investigative tools that combine single-molecule detection with the ability to certain instances, accurate sequencing
simultaneously manipulate single molecules, in otherwise complex samples. becomes challenging with mass spec-
Herein, there are presented some of the efforts directed to control the capture trometry, since some amino acids have
dynamics and translocation speed of tailored polypeptides through the α-HL the similar mass and charge (e.g., leucine
and isoleucine);[10] iv) the fractionation
nanopore, by harnessing the electro-osmotic flow and nanopore-tweezing
step (i.e., breaking apart a longer sequence
influence on individual molecules, which are engineered to resemble mac- into many smaller peptides), preceding
rodipoles. The reported applications of this approach suggest a solution to the analysis of the mass-to-charge ratio of
enhance the temporal resolution of nanopore detection, prove the capability of each new peptide with the mass spectro­
the system in distinguishing between groups of distinct amino acids from the meter, is slow and tedious.[8,11,12]
studied poly­peptides, and propose a strategy to translate such single-molecule Built on the success of nucleic acids
detection and characterization,[13–26]
sensors in devices suitable for polypeptide sequencing at unimolecular level.
nanopores became a viable alterna-
tive for single-molecule-based, primary
sequence recognition, and conformational
1. Introduction analysis on peptides and proteins, thus precluding the need
for proteolysis or labeling.[27–51] Through an enhanced analysis
Primary structure identification of peptides and proteins is cen- throughput, the possibility of primary structure identification
tral for the advancement of proteomics, as it determines how in peptides and proteins with nanopores, opens new vistas in
these biopolymers fold and perform their biological function, deciphering the proteome, since in vitro peptide or protein
and previous work established that even subtle changes in a pro- amplification assays lack. To this end, the underlying para-
tein’s primary sequence can cause debilitating pathologies.[1–4] digm is that the polypeptide-mediated ionic current fluctua-
Traditional methods for proteome analysis and protein tions through a voltage-biased nanopore are correlated with the
sequencing include mass spectrometry[5,6] and Edman distinct physicochemical properties of individual amino acids
(Figure 1).[26,52–55]
Dr. A. Asandei, Dr. I. Schiopu, I. S. Dragomir
Within simplifying assumptions,[29] the analyte-induced
Interdisciplinary Research Department blockade amplitude on a single nanopore modeled as an elec-
Alexandru I. Cuza University trically neutral, uniform cylinder, can be correlated with the
700506 Iasi, Romania analyte volume (Figure 1I,d and the corresponding text inset),
G. Di Muccio, Prof. M. Chinappi σ buffer ∆Vδ
Department of Industrial Engineering ∆I block = I blocked − I open ≈ (σbuffer is the conductivity of
l p2
University of Rome Tor Vergata
Via del Politecnico 1, 00133 Rome, Italy the buffer, lp is the nanopore’s length, and δ quantifies the
E-mail: mauro.chinappi@uniroma2.it volume of the analyte). Note that for other shapes other than
Dr. L. Mereuta, Prof. T. Luchian cylinders, the current blockade varies with the molecule posi-
Department of Physics tion inside the nanopore.[56] The formula above embodies
Alexandru I. Cuza University also simplifications of the formalism described originally by
700506 Iasi, Romania
E-mail: luchian@uaic.ro
DeBlois and Bean[57] regarding the relative geometry of the ana-
lyte and the nanopore, impact of the extension of the electric
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smtd.201900595. field lines beyond the nanopore inner region, or distortion of
the electric field lines inside the nanopore created by the pres-
DOI: 10.1002/smtd.201900595 ence of an analyte. Such an approach has been widely used for

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Figure 1. Nanopore-based characterization of individual biopolymer primary structures. I,a) In the absence of nanopore–analyte interactions, the
ionic current through a single protein nanopore present in a reconstituted lipid membrane, and held at a constant transmembrane potential (ΔV), is
relatively constant I,b). Electrophoretic capture of a single biopolymer at the mouth of the nanopore, described by the association rate rateon, followed
by biopolymer trapping inside the nanopore I,c), are seen as reversible changes of the ionic current through the nanopore between the open state
(Iopen—free nanopore) and blocked state (Iblocked—nanopore transiently occupied by the biopolymer) I,d). In the simplest embodiment (I,d), the three
main descriptors used to characterize such analyte–nanopore interactions, are: the time intervals between consecutive blockade events (τon), blockade
duration (τoff ), and blockade amplitude (ΔIblock = Iblocked – Iopen) (see also text). The primary structure of nucleic acids and proteins II,a) may be obtained
from the degree to which the bases or amino acid residues reduce the ionic current flowing through the nanopore II,b).

quantitative and qualitative estimations of individual analytes
volume,[41,58–61] and it was successfully extended to more com-
plicated scenarios, in which the electric potential profile along
the nanopore axis assumed a piecewise linear dependence, and
the relative amplitude of blockade current events ΔIblock repre-
sented not only the volume excluded by the analyte, but mobile
cation binding to it as well.[21,62]
From an analytical standpoint and when evaluated within
the frame of a diffusion-controlled process, the analyte cap-
ture rate (rateon) by the nanopore is proportional to ΔV (see
the text inset in Figure 1),[60,63] whereas the mean transit time
of the analyte across the nanopore (τtransit), as derived from 1D
drift-diffusion models based on individual time translocating
values (τoff),[29,59] is inversely proportional to ΔV (see the text
inset in Figure 1). In these expressions, the parameters have
their usual thermodynamic and geometric meanings, namely:
kB—the Boltzmann’s constant, Tm—absolute temperature
of the buffer, Dbuffer and Dnanopore—analyte’s diffusion coef-
ficient in buffer and inside of the nanopore, respectively, z
and e−—effective valence of the analyte and electronic charge,
rp—nanopore’s radius on the cis side. For analytes comparable
in dimension to the nanopore’s inner volume, the electro-diffu-
sion-driven analyte to the nanopore mouth does not necessarily
ensures capture and translocation. In such cases, the analyte
requires climbing across the free energy barrier that involves
the entropy penalties for confining the analyte inside the nano-
pore and enthalpic contributions from the analyte–nanopore
interactions, and the capture rate is treated within the classical
Eyring’s transition state theory.[43]
It may be argued that for analytical purposes, τon time inter-
vals (Figure 1I,d) could be measured between the beginning
of successive blockade events. Within the data analysis theory
applied to Markov models as in Figure 1, open time intervals
can be analyzed in terms of exponentially distributed values,
1
leading to τon’s arithmetic average τ on = , only if the
rate on
measurement of individual τon’s starts from the moment when
the nanopore enters the “open” state and it lasts until the first
“open” → “blocked” transition occurs.[64] We therefore adopted
as a rule that quantification of τon time intervals should start
immediately when a previously bound analyte to the nanopore
dissociates from it (Figure 1I,d), as done previously in our labo-
ratory[53] and others.[16,26,31,43]
Despite successful implementation of this technique to stud-
ying structural features on polypeptides[39,42,47,65–71] and recent
efforts directed to polypeptide sequencing,[46] it still remains
an open challenge to accurately deconvolve the ion current
fluctuations to unequivocally deduce the polypeptide’s primary
sequence. In a reserved optimistic tone on this approach,
Kennedy et al.[27] have concluded that despite the use of a sub-
nanometer pore which allows the detection of post-translational
modification on a single amino acid residue, the method still
lacks the sensitivity needed to discriminate between all of the
amino acids.

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Major hurdles that limit the accurate polypeptide sequencing
at single amino acid resolution, include: i) the dimension of
the sensitivity region inside the nanopore, usually the most
constricted domain of the inner volume, does not scale with
the size of individual amino acids, implying that current fluc-
tuations schematically shown in Figure 1II,b, are not correlated
with the passage of individual residues;[53] ii) with the excep-
tion of simplified, engineered peptide constructs,[72,73] the het-
erogenous charge distribution along the polypeptide’s sequence
hinders the controlled, electrophoretic-driven translocation of
the polypeptide through nanopore. To add to this complexity,
peptide-nanopore nonspecific interactions, as well as and the
peptide length, are relevant factors during such peptide recogni-
tion attempts;[74,75] iii) the high velocity of the polymer’s passage
across the nanopore poses serious challenges to unraveling the
identity of the moving monomers. In relation to this, two chal-
lenges are critical to the subsequent statistical analysis of the
blockade events. First, due the extremely short dwell residence
of small analytes inside the nanopore (e.g., a freely diffusing
molecule as small as sucrose with the diffusion coefficient
D = 0.5 × 10−9 m2 s−1, situated in the middle of the α-HL,
spends ≈6.3 ns before escaping the nanopore) and depending
upon the applied voltage and salt concentration, the number
of charge carriers from the electrolyte solution flowing across
the transiently-blocked nanopore is very small (≈hundreds).
This entails a low signal-to-noise ratio blockade signal in the
low-frequency regime, thus degrading the signal before sup-
plementary amplifier electronics and circuitry parasitic noises
add up.[25,76–79] Second, the extremely short durations of such
events hinder their accurate resolving upon convolution with
the microsecond time-resolution of most commonly available
patch-clamp amplifiers.[77] In a recent work, authors provide a
comprehensive account on noise-and bandwidth-related chal-
lenges during recordings on ion channels and nanopores.[80]
To alleviate these shortcomings, in an alternative approach
authors described an analytical tool to characterize such short
analyte–nanopore interactions. Briefly, they modeled the tran-
sitions of the nanopore-mediated current to individual states,
as caused by reversible analyte–nanopore interactions, with an
equivalent electrical circuit. Subsequently, such a model allowed
to quantify the system response, and eventually enabled the
estimation and characterization of short-lived blockade states as
induced by polyethylene glycols with as few as 8 mono­mers,
approximately the size of sucrose.[81]
In terms of structural details, there are three major classes
of nanopores useful for single-molecule detection and recogni-
tion: i) biological (usually protein-based) nanopores; ii) solid-
state nanopores, and iii) hybrids of biological and solid-state
nanopores. They each display complementary benefits and
common drawbacks for the task outlined above.[26,53,77,82–88]
Notably, the recent description of synthetic membrane-span-
ning nanopores constructed with DNA nanotechnology, holds
the game-changing potential in many biotechnology and bio-
medicine applications.[89]
As a brief comparison on their particular benefits, solid-state
nanopores, although nontransferrable to lipid-based mem-
branes, are more stable than biological ones in extreme buffer
conditions (e.g., urea, sodium dodecyl sulfate (SDS)) and can
be used in sensing protocols that require strong denaturating
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agent.[27,29,90] On the other hand, biological nanopores with
known crystal structure are amenable to atomic-level engi-
neering and functionalization, as their surface can be finely
tuned through site mutation on individual single amino acid
groups.[85,91–94]
From an historical perspective and within the framework
described above, the homo-heptameric alpha-hemolysin (α-HL)
protein nanopore proved successful for structural identifica-
tions in polypeptides, due to intrinsic benefits, such as known
crystal structure at atomic resolution,[95] structural stability, and
relative absence of gating transitions accompanying the ion
transport through it.[76,96] In early studies, efforts were devoted
to capturing small peptides and then correlating some of their
features (e.g., conformation, length) with the transient block-
ades on the ion current through the nanopore and dwell time
statistics[43,65,97] α-HL was also shown to be a powerful tool for
analyzing the unfolding of entire proteins[98–100] and recently, it
was proposed to hold potential for the detection of post-trans-
lational modifications[28,101] and the analysis of ion–peptide
interactions.[102]
Here, we will summarize some of the theoretical insights
and experimental results obtained in our laboratories, for the
goal of unveiling new perspectives in the realm of polypeptide
identification and sequence recognition.
2. Reading the Primary Structure on Macrodipole-
Like Polypeptides
To achieve amino acid discrimination on polypeptides from
current blockade events through the α-HL with the approach
outlined above, we sought to: i) control the dynamics of poly-
peptides through the α-HL, and ii) unravel and analyze ionic
current blockades stemming from the polypeptide passage
across the nanopore’s most sensitive, constriction region.
As described, a critical challenge plaguing the nanopore-
based analysis to distinguish between and possibly identify
specific groups of amino acids, is the relatively short residence
time of analytes inside the nanopore.[25,53] The existing litera-
ture presents methods to alter the mean analyte residence time
in the nanopore, such as: changing the temperature,[51,103,104] by
increasing analyte–nanopore wall interactions,[105] or modifying
the geometry of the nanopore,[60] by modification of the trans-
locating molecule,[106,107] by altering viscosity and electrolyte
concentration of the buffer,[21,99,108,109] rapid switching or modu-
lating the transmembrane potential,[110] controlling the balance
between the electrostatic and electro-osmotic forces,[59,69,111]
using Li+ as counterions in the electrolyte solution,[112] coating
nanopores with a fluid lipid bilayer where specific mobile
ligands able to bind analytes are embedded,[58]or using optical
tweezers.[113]
A straightforward approach to control an electrically charged
biopolymer trafficking across a nanopore, is to alter the applied
voltage (ΔV). A reduced voltage across the nanopore reflects
as a correspondingly diminished electric force acting upon
a charged molecule passing through the nanopore, entailing
increased dwell times of the analyte inside the nanopore (τtransit;
see also Figure 1, text in the inset).[114] From the same physical
principles however (Figure 1, text in the inset), a reduced ΔV
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Figure 2. Schematics of the nanopore-tweezer paradigm. a1) A trans-positive voltage applied across a membrane containing a single nanopore gener-
ates an inhomogeneous electric field E (gray dashed lines) oriented toward the negative electrode. The electrical field is larger inside the nanopore and
goes to zero, far from the nanopore. A dipolar-like analyte is attracted by the maximum of the electrical field (dielectrophoresis). a2) Sketch of the free-
energy profiles Gtot as a function of the position of the dipolar analyte center. To enter the nanopore, the analyte has to overcome a capture free-energy
barrier (ΔGcp). When the analyte enters the nanopore, it gets trapped in a metastable state where the forces acting on it equilibrate. The escape from this
metastable state is characterized by an escape barrier ΔGex. a3) As the voltage increases, the capture barrier ΔGcp decreases (i.e., capture is enhanced),
while the escape barrier ΔGex increases, leading to longer dwell times of the analyte inside the nanopore. b–e) Different stages of a macrodipole-like
peptide capture and trapping inside a α-HL by dielectrophoresis. A macrodipole-like, trans-added peptide gets oriented with the positive moiety toward
the β-barrel entry of the nanopore, gets imported into the nanopore mainly under the influence of the electric force F+ b), moves across the nanopore,
and eventually protrudes to the cis side of the membrane c–e). During the course of motion, the negative moiety of the peptide will enter the β-barrel
domain of the nanopore, where it experiences an electric force F− oriented toward the trans side of the membrane, opposite to F+. At some moment
during this transit, the overall number of positive and negative residues from the peptide which experience the electric field inside the nanopore, will
become such that |F+| approximately balance |F−|. This determines an approximately net zero electric force acting on the peptide, which will assume a
metastable state inside the nanopore c). Small thermal movements of the peptide toward the cis d) or trans side e) of the membrane determine a net
electrical force which drives the peptide back to the metastable state, before it eventually escapes the nanopore to either side. A theoretical descrip-
tion of the nanopore tweezer (dielectrophoretic trapping) mechanism in term of free energy profiles can be found in previously described work.[118,119]
Panels (a1)–(a3) and (b)–(e) are adapted from[118] and,[119] respectively.

decreases the capture rate of the analyte inside the nanopore
(rateon). More specifically, for charged biopolymers (e.g., DNA),
the dependence of the capture rate and residence time with ΔV
is nonlinear. Experimental data[115] and a theoretical descrip-
tion[116] were described earlier. To a certain extent, the reduc-
tion of the analyte capture rate with decreasing ΔV’s can be
mitigated by the use of salt gradients between the cis and trans
sides of the system.[42,117] From our experience though, steep
salt gradients maintained across a lipid membrane containing a
single α-HL, have a destabilizing effect on the system.
As an alternative solution to this challenge, dubbed “the
nanopore-tweezer approach,” we used model polypeptides
consisting of 12 asparagines, whose N- and C-termini were
engineered to contain 12 patches of glutamates and arginines
(Figure 2). We demonstrated experimentally that for such
macro­dipole-like peptides, an increase in ΔV leads simultane-
ously to an increase in the capture rate by the nanopore and
of the residence time of the polypeptides inside the nanopore,
regardless of the applied potential polarity.[118,119]
From a physical perspective,[115] it is clear that increased
polypeptides capture rates by the nanopore at increasing ΔVs,
reflected the augmented electric interactions between the
polypeptide’s end and the potential drop near the nanopore
entrance (Figure 2b). On the other hand, the residence time
of the polypeptide captured transiently inside the nanopore is
enhanced with the increase in ΔV, due to an electrostatic tug-
of-war between the charges on opposite sides of the polypeptide
and the ΔV (Figure 2c–e). The strategy described above to con-
trol and slow-down the polypeptide passage across the nano-
pore, constitutes a productive solution to tackling the challenge
of correlating ionic current fluctuations through the nanopore
with the peptide’s primary structure, within a reasonable band-
width of the amplifier (≈100 kHz). From a geometrical rea-
soning, a metastable, captured polypeptide (Figure 2a,c) spans
the entire nanopore and it gets positioned nearly symmetri-
cally relative to the nanopore’s constriction domain.[95] As the
α-HL’s vestibule and β-barrel excluded volumes remain largely
constant during small position displacements of an entrapped
polypeptide, they contribute to a constant residual current
across the nanopore, Thus, the amino acid residues occupying
the geometrically-sensitive constriction region of the nano-
pore are key in affecting the ensuing ionic current blockades.
In other words, the random displacements of the polypeptide
inside the nanopore and across the constriction region gen-
erate current fluctuations sensitive to the identity of the central
amino acid residues from the polypeptide (vide infra).

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Figure 3. Representative traces showing the reversible ion current alterations induced by macrodipole-like peptides through an open α-HL nanopore.
Distinct peptide constructs, termed (Pe1 Ac-(R)12 – (A)6 – (E)12 – NH2, panel a); Pe3 Ac-(R)12 – (W)6 – (E)12 – NH2, panel b); or Pe5 Ac-(R)12 – (A)3 –
(W)3 – (E)12 – NH2, panel c), were added to the trans side of the membrane (20 × 10−6 m), in buffer containing 2 m KCl, 10 × 10−3 m HEPES, pH = 7. An
increase in the applied potential from ΔV = +40 mV (panels (a)–(c)) to + 80 mV (panels (d)–(f)), leads to the peptide capture rate enhancement (the
average of time intervals separating successive blockade events τon decreases), while the average duration of the blockade events (τoff ) increases. The
expanded views shown in panels (d)–(f) illustrate ionic current fluctuations through the α-HL, elicited by a metastable peptide inside the nanopore.
Panels (g)–(i) display the voltage-dependent, average values of the interevents time intervals (τon; “free α-HL”) and the blockade-events durations (τoff;
“blocked α-HL”) for the peptide tested, from measurements on single molecule ion-current blockades. These graphs can be qualitatively compared
with the free-energy barriers reported in Figures 2 and a3. Indeed, the larger the capture (or escape) time, the larger the free-energy barrier associated
to the capture (or escape) process. Reprinted with permission.[72] Copyright 2017 American Chemical Society.

As a first application of the nanopore-tweezer approach, we
demonstrated its potential for single-molecule interrogation of
primary structure on model polypeptides. To this end, we engi-
neered polypeptides whose residues under study presented a
marked difference in their physical size, namely six A (alanine),
six W (tryptophan), or a combination of three A and three W
residues, flanked at the N- and C-termini by oppositely charged
segments at neutral pH, each containing 12 R (arginine) and
12 E (glutamate) residues (Figure 3).[72]
In a first series of experiments, we demonstrated that the
transmembrane potential augments such dipolar-like polypep-
tides capture rate by the α-HL, and simultaneously increases
their residence time inside the nanopore (Figure 3).
The slowed-down passage of these polypeptides across the
nanopore, which is better seen at increased potentials (see
Figure 3, compare traces shown in panels (a)–(c) at ΔV = +40 mV
to those illustrated in panels (d)–(f) at ΔV = +80 mV), provides
an opportunity to measure with increased signal-to-noise ratio
ionic currents fluctuations, as in expanded traces in Figure 3d–f.
By considering the essential geometrical descriptors of the
α-HL’s constriction region situated between M-113 and alter-
nating K-147 and E-111 from each of the seven monomers of
the heptameric nanopore (length of ≈0.6 nm and diameter of
≈1.4 ÷ 1.5 nm),[120,121] and knowing that each amino acid on the
studied polypeptide spans 0.38 nm,[27] we posit that an amino
acid residue lodged precisely in the constriction as well as the
neighboring ones just outside it, govern the magnitude of ionic
current fluctuations through the nanopore, while fully occupied
by the gliding polypeptide (Figure 5I,b,c). This is consistent
with previous MspA experiments, demonstrating that current
fluctuations across the nanopore were critically affected by
≈three to four nucleotides moving in and out of the constriction
region, of approximately similar length.[122]
Within this model, we proposed that data displayed in the
expanded traces in Figure 3d–f stem from the successive traf-
ficking across the α-HL’s constriction region of at least three
residues located in the middle section of a captured poly­
peptide, situated in a balanced force regime stemming from
the tug of war between the oppositely oriented electric forces
exerted at its ends.
For a detailed view of this phenomenon, we show in Figure 4,
representative excerpts from original recordings that illustrate
the time unfolding of the fractional blockade currents ensued
by a single Pe1 (Ac-(R)12 – (A)6 – (E)12 – NH2) a), Pe3 (Ac-(R)12 –
(W)6 – (E)12 – NH2) b), or Pe5 polypeptide (Ac-(R)12 – (A)3 –
(W)3 – (E)12 – NH2) c), while assuming a metastable state inside
the α-HL. To account for the differences in the current block-
ades shown in Figure 4II,a–c, we calculated the relative ionic

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Figure 4. Testing the ability of the α-HL to distinguish between representative amino acids in a peptide, from ion current fluctuations across the
nanopore. I) A macrodipole-like polypeptide captured in a metastable state inside the α-HL, presents the amino acid residues from its middle region
to the nanopore’s constriction domain a). From this state, thermal position fluctuations of the polypeptide, leading to the partial displacement from
the α-HL’s constriction region of a group of ≈3 amino acids, followed by the re-entry of another such group b,c), generate corresponding ionic current
flickering d,e). II) In a real-life example, the oppositely oriented electric forces acting at the termini of the transiently captured Pe1 (Ac-(R)12 – (A)6 –
(E)12 – NH2) a), Pe3 (Ac-(R)12 – (W)6 – (E)12 – NH2) b), or Pe5 polypeptide (Ac-(R)12 – (A)3 – (W)3 – (E)12 – NH2) c), diminishes the analyte’s kinetics
inside the nanopore, and reveals distinct blockade events associated to the polypeptide movement along the nanopore’s constriction region. The all-
points histograms pinpoint the amplitude distribution of the sub-blockade events (“&” and “$” levels), distinct from the fully blocked substate (“#”),
suggestive of a group of ≈3 residues from the peptide’s middle region of a captured peptide trafficking across the nanopore’s constriction domain.
Reprinted with permission.[72] Copyright 2017 American Chemical Society.

  The positioning of the polypeptide inside the nanopore asso-

current blockade  ∆I blocked  , where ΔIblocked = Iblocked − Iopen,
 I open  ciated to the “#” substate is unstable, as its movement toward
the cis or trans side caused mainly by thermal fluctuations
Iblocked corresponds to either of the “#,” “$,” or “&” substates,
tends to unbalance the electric forces acting at its termini.
and Iopen measures the ionic current through the free α-HL
We proposed that the reversible current fluctuations around
in the absence of interactions (see Figure 3a–f, “free α-HL”
level “#,” to substates of lower blockade seen as “$” (Pe3) and
substate).
“&” (Pe1), respectively, reflect polypeptide occupancy states
We recall that within a volume-exclusion model (vide supra)
inside the nanopore, whereby the constriction region har-
and simplifying assumptions,[58,59] an electrically neutral ana-
bors a lesser number of amino acids as compared to the “#”
lyte captured inside a cylinder-like nanopore determines cur-
substate (see also the schematics in Figure 4I,c). This stems
rent blockades, ΔIblocked, directly proportional to the electrolyte
from the fact that α-HL’s constriction region is the narrowest
volume excluded by the analyte (δ) (see also Figure 1, inset).[29]
domain inside the nanopore (volume of ≈924 Å3), so that the
More accurate descriptions based on Poisson–Nernst–Planck
net ionic current blockade caused by a captured polypeptide is
equation,[21] Ohm law with local conductivities[123] and quasi-
critically dependent on the occupancy extent of it. Experimental
1D models for pore resistance[124,125] have been developed for
observations support this assertion, as the relative blockade
estimating the current blockade. In accordance to the scenario
amplitude of the “$” substate measured for the W-containing
depicted in Figure 4I,b, ΔIblocked estimated for the “#” substate
∆I blocked
reflects the current blockade determined by a peptide captured peptide ( ($, Pe3) = 0.93 ± 4 × 10−3), is slightly larger
I open
inside the nanopore, precisely when a group of approximately
than that of the “&” substrate measured for the A-containing
three amino acids from the middle section of it occupies the con- ∆I blocked
striction region of the α-HL. As the relative blockade amplitude peptide ( (&, Pe1) = 0.83 ± 6 × 10−3). Another worth-
I open
of the “#” substate is slightly larger for the W-containing pep- mentioning benefit of this approach lies in that the oppositely-
∆I blocked
tide Pe3 ( (#; Pe3) = 0.98 ± 9.1 × 10−4), as compared to the oriented forces acting at the polypeptide’s ends exert a confor-
I open
∆I blocked mational control over the trapped molecule, removing folds
A-containing peptide Pe1 ( (#; Pe1) = 0.95 ± 6 × 10−3), it
I open and maintaining it in a largely linear topology.[119] This has the
reflects well the fact that individual W residues displays a larger potential to augment the accuracy of volumetric identification
volume of electrolyte (≈239 Å3) than A residues (≈100 Å3) do.[126] of amino acids.

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Based on these findings, a next objective was to assess the
system’s ability to discriminate between groups of three distinct
amino acids, from fluctuations in a single blockade event. In
such experiments, we used another polypeptide construct (Pe5),
presenting in its middle section three A followed by three W
residues (Ac-(R)12 – (A)3 – (W)3 – (E)12 – NH2). The analysis of
the ionic current fluctuations accompanying such a construct
being transiently captured inside the nanopore, revealed three
distinct peaks, which were denoted in Figure 4II,c by “#,” “$,”
and “&.” Based on their relative amplitudes, which for the deep
(“#”) and, respectively, shallower events (“$” and “&”) equal
∆I block ∆I block
(#, Pe5) = 0.94 ± 10−3, ($, Pe5) = 0.89 ± 3 × 10−3,
I open I open
∆I block
and (&, Pe5) = 0.84 ± 2 × 10−3, which are close to those
I open
measured in the case of Pe3 and Pe1 polypeptides (vide supra),
these data were interpreted as evidence of the system being
able to distinguish between groups of distinct amino acids on
polypeptides containing a mixture of three A and W residues in
their middle section.
By using a similar paradigm, we proved the ability of the
α-HL nanopore to identify three-amino acids long patches
of polar S (serine) and aliphatic I (isoleucine) residues in the
primary structure of polypeptides.[73] Recently, the robustness of
our approach was also confirmed by independent experimental
results by Zhao et al.[127] and Pérez et al.[128] that employed a
FraC nanopore, and by coarse-grained computational analysis
by Ghosh and Chaudhury.[129]
If combined with click addition of positive and negative
tails to the termini of a short peptide, this approach for resi-
dues identification can be applied to any peptide construct,
regardless of its charge. In this scenario, the click addition
of a single strand DNA tail to a peptide terminal introduced
by Biswas et al.[130] can potentially foster the use of the nano-
pore tweezer method for other nanopore protein sensing
applications.
3. Insights on Protein Sequencing from Atomistic
Simulation
Molecular dynamics (MD) simulations have proved a useful
computational tool to investigate transport phenomena in bio-
logical nanopore sensing systems and to interpret experimental
data. Examples are the estimation of α-HL ionic conductance
and electro-osmotic flow,[120,131,132] investigation of single-
stranded DNA molecules inside α-HL,[133] the cotranslocational
unfolding of thioredoxin,[134] and DNA base distinguishability
in MspA.[135]
A core question in nanopore protein sequencing is the capa-
bility to distinguish among the 20 amino acids. In the sensing
approach based on ion current blockade, this amounts to check
if each amino acid can be unambiguously associated with a
specific ion current blockade level. As a preliminary step in
assessing the capability of α-HL to distinguish between all the
20 standard amino acids, we estimated the reduction of avail-
able space for ionic transport through the nanopore induced by
20 different small homopeptides, one for each standard amino
acid, using all-atom MD simulations.[124] The system, sketched
in Figure 5a, is constituted by the α-HL nanopore (blue)
embedded into a lipid membrane (gray). A 35-residues homo-
peptide (orange chain) is imported into the nanopore with the
central residue close to the nanopore constriction. The simula-
tion box is filled up by 2 m KCl electrolyte solution.
In principle, ionic fluxes can be directly sampled through
nonequilibrium MD by applying an external electric field to
the system.[136] However, due to high thermal noise, this pro-
cedure requires very long simulations to reduce the statistical
error on the estimation of the average current. Moreover, dif-
ferent metastable states of the peptide inside the channel may
give rise to significant current blockade differences. Hence,
multiple replicas of each system are needed, making the com-
putational cost even larger and impractical for the estimation of
all the 20 standard amino acids. To overcome this difficulty, we

Figure 5. Atomistic simulation of nanopore steric exclusion by homopeptides. a) System setup. The system is constituted by an α-HL blue nanopore
(blue) embedded into a lipid membrane (gray). A 35-residues homopeptide (orange chain) is imported into the nanopore with the central residue close
to the nanopore constriction. The simulation box is filled up by 2 m KCl electrolyte solution, that, for the sake of clarity, is not shown. b) Nanopore steric
exclusion estimator for all residues (Va is the amino acid volume). Gray circles correspond to hydrophobic residues, yellow squares to polar, blue up-
triangles to positively charged residues, and red down-triangles to negatively charged ones. The dashed line is the least square regression line. Error
bars represent the standard error of the mean over five independent replicas, and they are reported only when larger than symbols. Figure adapted
from Di Muccio et al.[124]

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www.advancedsciencenews.com
formulated a simple theoretical model for the estimation of the
electrical resistance of the nanopore. This approach requires
relatively short equilibrium runs that allowed us to reduce by
a factor of four the computational requirements. We tested this
formulation against direct ionic current measurements from
MD simulations, for four selected amino acids (A, Q, F, W), and
found that our nanopore steric exclusion estimator correlates
quite well with the measured current blockade,[124] allowing us
to extend this procedure to all the 20 canonical amino acids.
Figure 5b reports the nanopore steric exclusion estimator as
a function of the amino acid volume. As expected, our results
show that the amino acid volume is the main feature that rules
the nanopore steric exclusion. In addition, we also find that the
amino acid charge significantly influences the nanopore steric
exclusion estimator. Indeed, charged residues show a significant
minor steric hindrance with respect to uncharged amino acids
of similar volume as apparent, for instance, by the comparison
between D (aspartate) and N (asparagine) in Figure 5b. We also
find that ion concentration inside the nanopore is higher for
charged residues (i.e., the charged homopeptides bring a coun-
terion shell into the nanopore), and that charged homopeptides
lie in a slightly stretched conformation. Both these observa-
tions suggest that charged homopeptides leave more room for
the electrolyte passage and may tentatively explain the smaller
value of the nanopore steric exclusion estimator with respect to
residues of the same size. A similar result was recently reported
in a computational work by Si et al.,[137] where ionic currents
of different short homo- and hetero-peptides translocating
through a graphene nanopore are studied via nonequilibrium
MD. In particular, it is reported that two different protonation
states of the histidine residue show two significantly different
current signals, the larger current being related to the charged
state. Hence, for similar amino acids dimensions, the charged
residue give rise to a smaller blockade. Another recent compu-
tational work by Wilson et al.,[123] compares the blockades of all
20 individual amino acids in MspA nanopore, reporting again
volume-dependent blockades (i.e., blockade increases with
volume), but not a significant charge effect. This work employs
a reduced steric exclusion model to estimate the current block-
ades computing a 3D local conductivity map of the electrolyte
inside the channel, based on the distance of each point from the
nearest nonsolvent atom. This approach provides ionic current
blockade estimation in good agreement with nonequilibrium
runs. However, the local conductivity map does not take into
account the effects of surface charge of the biomolecules, this
may be one of the possible reasons for the different behavior
of charged residues when compared with our study,[124] and
Si et al. work.[137] These recent studies, together with the con-
stant increase in the computational performance, suggest that
in the near future MD simulations will play an important role
to elucidate the main mechanism responsible for the current
blockades.
4. Capture and Translocation Control
In Sections 1–3, we discussed mainly about the capability of
nanopore systems to distinguish the different amino acids.
However, distinguishability is only one of the requirements
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that need to be fulfilled by a working nanopore protein sensing
device. The other crucial issue is the control of protein cap-
ture and its sequential translocation.[36] In order to address the
difficulties associated with the control of protein capture and
translocation, it is instrumental to briefly recall how these prob-
lems were solved for the nucleic acids sequencing. For DNA,
capture is achieved by electrophoresis induced by the poten-
tial difference applied across the nanopore, as the molecule is
highly charged (e.g., in 1 m salt, the effective charge of ssDNA
is ≈30% of the charge of bare ssDNA.[138] Precise translocation
control, however, required more efforts.[139,140] In brief, the
most effective solution was to employ a processive enzyme.[141]
Indeed, several enzymes are naturally able to bind the DNA
molecule and to move along its chain to perform their bio-
logical function, such as DNA repairing and replication (e.g.,
polymerase). These enzymes can be adapted to push the DNA
chain through the nanopore ensuring a unidirectional single-
monomer translocation on a millisecond time scale, that, in the
DNA case, allows to recognize single bases.[140] Notably though,
electrophoretic capture and enzyme-controlled translocation
as mentioned above cannot be straightforwardly extended to
peptide chains. If we exclude extreme acidic and alkaline pHs,
proteins can be positively or negatively charged, with the charge
not being uniformly distributed along the chain. This ren-
ders electrophoresis not a robust method to capture a generic
(positive, neutral, or negative) protein on nanopores. Several
strategies were proposed to overcome this issue and achieve
an efficient electrophoresis-induced capture. One possibility
is to add a charged tail to one protein terminal.[130,142–144] In
this approach, the charged tail enters the nanopore by electro-
phoresis and it induces the translocation of the whole peptide
chain, supplementary assisted in certain cases by a molecular
motor.[143] A second strategy is to employ SDS, an anionic com-
pound that in combination with heat and reducing agents is
able to denature the protein and create a negative charged shell
around it.[27,51,90,104,145,146]
Our groups focused on a third strategy, namely adjusting the
electro-osmotic flow (EOF).[69,132] EOF refers to the net motion
of the solvent (usually water) induced by an applied voltage. If
the nanopore is selective for cations (or anions), the positive and
negative ion distributions inside the nanopore differ and, con-
sequently, in some regions of the nanopore, the electrolyte solu-
tion presents an average net charge. Consequently, the external
field induced by the applied voltage results in a net force that
acts on the solvent, thus generating the EOF, see Figure 6a,b.
For biological nanopores, it is also feasible to tune their surface
charge by specific residues mutations and/or changing the pH
of the solution. For the specific case of α-HL, we showed that at
low pHs, the EOF is so intense that it is able to capture charged
peptides against electrophoresis,[69] see Figure 6c,d. Atomistic
simulations provided a qualitative explanation of the process
and, more importantly, a quantitative estimation of the EOF.[132]
The reduction of pH from 7 to 2.8 strongly alters the charge
distribution at the β-barrel entrance, where the surface charge
switches from negative (pH = 7) to positive (pH = 2.8) see
Figure 6e,f. Hence, α-HL is more anion selective at low pH
and, consequently, the EOF flux increases (Figure 6g). The pos-
sibility to employ EOF to capture molecules irrespective of their
charge was also recently explored by other research groups for
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Figure 6. Electro-osmotic flow (EOF) and peptide capture. Nanopore surface charges alter the ion distribution in the electrolyte solution. Ions of
opposite charge with respect to surface accumulate in a thin layer close to the wall, e.g., a positively charged surface attracts a negative cloud of ions
a). The external electric field induced by the applied voltage ΔV results in a net force on the charged fluid layer that puts in motion the fluid. For positively
charged surface a), the fluid velocity ueo is opposed to the direction of the electrical field E, while, for negative charged surfaces b), ueo and E have the
same direction. c,d) EOF can be used to capture a peptide against electrophoresis. In the reported example,[69] a positively charged peptide is initially
present on the α-HL’s cis side, and the electrical field as well as electrophoretic forces Fepl are directed from the trans to cis side. The experiment is
performed at pH 2.8, where α-HL is highly positively charged. Current traces adapted from[69] show a multilevel signal that is interpreted as the move-
ment of the peptide along different regions of the nanopore, hence the capture is induced by EOF against electrophoresis. e,f) Ion concentration maps
from equilibrium molecular dynamics, MD, simulations. The regions not accessible to ions (membrane and channel) are in dark blue. The red peaks
at both pH in the Cl− density correspond to the anions accumulation caused by the presence of positive residue rings at the constriction. A marked
increase of Cl− ions in the trans region for pH = 2.8 is responsible of the enhancement of the nanopore selectivity and, consequently, of the EOF.
g) EOF from MD simulations as a function of ΔV at pH 7 and pH 2.8 for 2 m KCl, adapted from Bonome et al.[132]

FraC[147,148] and Aerolysin[149] nanopores. Even more, achieve-
ments were accomplished for the goal of the efficient use of
EOF in molecule capturing and investigation.[150,151]
Concerning translocation control, there is no direct equiva-
lent of the DNA processive enzyme (roughly speaking, there is
no protein-polymerase) that may be easily engineered to control
translocation. An interesting approach was to use an unfoldase
and in particular, the ClpX unfoldase was placed in solution
on the trans side of α-HL, while the protein to be translocated
was present on the cis side.[143] A peptide tag was added to
the protein terminal allowing ClpX to specifically bind the C
terminus of the protein, when translocating to the trans com-
partment. At this point, ClpX induces the translocation of the
protein. The approach enabled discrimination among distinct
protein domains and among variants of these protein domains
due to sequence differences.[32] With particular appeal for such
future endeavors, gold nanoparticle plasmon heating may be
employed for the purposes of small proteins unfolding and
analysis with nanopores.[51]
5. Open Challenges and Opportunities
Despite the recent progresses, the route toward nanopore pro-
tein sensing is still full of interesting open challenges. None
of the three main requirements, peptide capture, transloca-
tion control, and amino acid distinguishability can be con-
sidered, to date, fully solved. Biological nanopores can be
engineered to increase their capability to distinguish among
the different amino acids and/or to improve the tuning pos-
sibility of the electro-osmotic flow. These two requirements
can be fulfilled independently since distinguishability takes
place is mainly at the nanopore constriction, while EOF is
sensitive to surface charges alterations in wider regions of the
nanopore, such as the α-HL vestibule, the large open of the
FraC funnel,[147] and the two wide vestibules of CsgG.[152,153]
Concerning distinguishability at single amino acid level with
the α-HL, the ionic current blockade signal corresponding to
the amino acid in the constriction will be also affected by the
amino acids that occupy the β-barrel. In other biopores such
as Mpsa,[93] and the already mentioned FraC and CsgG, this
effect should be less relevant since the section of the vestibule
is much larger than the section of the constriction. Another
possibility is to modify the α-HL by cutting a portion of the
β-barrel region close to the trans side, as reported by Stod-
dart et al.[154] and numerically analyzed by Di Muccio et al.[124]
Beside the progresses in the capability to modify biological
pores, the possibility to distinguish all the 20 amino acids
(or, at least, a large subset of them) still remains a formidable
open challenge.

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Figure 7. Exopepdidase-based nanopore protein sequencing. a) A pos-
sible approach to nanopore protein sequencing is based on sequentially
cutting single amino acids from one protein terminal and then importing
them one by one into the nanopore. Three main conditions are needed:
i) an exopeptidase able to cut all the 20 amino acids, ii) a way to bind the
exopeptidase close to the nanopore entrance, and iii) a method to import
all the residues into the nanopore preserving the chain order of the trans-
locating monomers. This last requirement may be obtained via dielec-
trophoresis b). Indeed, there are no conceptual limitations that impede
the dielectrophoretic capture and trapping, described for macrodipole in
Sections 2–3, for small dipole, such as a single amino acid. Another pos-
sibility is EOF (electro-osmotic flow) (c) that proved efficient to capture
molecules irrespective of their charge.
Molecular motors, as protein unfoldase ClpX employed
in Nivala et al.,[143] can be employed for translocation control.
To be effective, these motors probably need to be stuck at the
nanopore when translocating the protein, in order to reduce
fluctuations and ensure a strictly unidirectional translocation.
Another interesting approach is to employ a molecular hopper
moved in an electric field and controlled by a chemical ratchet
as proposed by Qing et al.[94]
An alternative direction, inspired from the exonuclease
DNA sequencing approach,[155] is to preserve the chain order
of the translocating monomers sequentially cutting single
amino acids from one protein terminal and then importing
them one by one into the nanopore, Figure 7. This exopepti-
dase method requires three main ingredients: i) an exopepti-
dase able to cut all the 20 amino acids, ii) a way to bind the
exopeptidase close to the nanopore entrance, and iii) a robust
method to import all the residues into the nanopore. Con-
cerning the first requirement, computational approaches may
help in the design of such an enzyme as recently did for the
development of an Edmanase, an engineered enzyme capable
to perform the stepwise removal of N-terminal amino acids
in an Edman degradation.[156] The second requirement, trans-
location control, appears in principle slightly simpler since it
may be achieved by covalently attaching proper linkers to both
nanopore and exopeptidase. A possible method it to apply a
complementary DNA strand; indeed, the binding of DNA
strands to nanopore is quite a robust technique in the nano-
pore field.[157,158]
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Theoretical insights on the feasibility of exopeptidase
sequencing are complex since the transport of single monomers
into the nanopore is ruled by the interplay among EOF, dielec-
trophoresis, Brownian diffusion, specific chemical interactions
and, for charged residues, electrophoresis. A theoretical work
by Reiner at al.,[159] analyzed the exonuclease/nanopore-based
DNA sequencing. They studied the capture of a monomer
under the effect of electrophoresis and electroosmosis showing
limitations of the exonuclease approach for DNA sequencing.
In particular, for applied voltage typically used in biological
nanopore systems, they found a capture probability of below
0.5 for a molecule released at the pore entrance under typical
voltage employed for biological nanopores. We expect that these
limitations will be relevant also for the protein sequencing
case. A main obstacle is the Brownian motion which for small
molecules may overwhelm all the other effects, introducing a
mismatch between the protein sequence and the order (and
the number) of the residues traversing the nanopore. For this
purpose, any method that can reduce the Brownian diffusion
(e.g., increasing the viscosity of the solvent) would be highly
beneficial. In addition, pressure gradients can be exploited to
further increase the incoming solvent flow and, hence, the cap-
ture probability. Other possible strategies to limit the effect of
Brownian diffusion may involve modifications to the pore set-
up, such as an additional confinement around the exopeptidase
or different pore design. A preliminary analysis neglecting
EOF and dielectrophoresis was presented a few years ago for a
tandem nanopore set-up.[160] Further analysis in this direction
could allow to get first insights on the feasibility of exopepti-
dase nanopore sequencing.
Although not described in this review, solid-state nano-
pores may also play an important role in the development of
protein sequencers. Indeed, solid-state nanopore allows more
options in the selection of the electrolyte solution features,
such as the above cited SDS approach.[27,90,145] Moreover,
solid-state nanopore, constructed in particular from 2D mate-
rials, may be employed in tunneling sensing where the single
monomer is detected measuring the transverse electronic cur-
rent.[37] Concerning protein sequencing, recent computational
studies have shown the possibility to detect specific fingerprints
of the atoms involved in the peptide bonds,[38,161] although
experimental evidences still need to be provided. Progress on
solid-state nanopore fabrication may also open the way to com-
bined approaches that merge the longitudinal ion current and
transversal tunneling current tunneling to improve the signal
distinguishability.
As a final comment, the efforts spent on protein sequencing
may lead to innovative approaches potentially useful for other
applications on protein sensing. Examples are single molecule
protein identification,[145,162] and detection of post-translational
modifications.[28,101] Notably, during the writing of this work,
a landmark paper on the topic reviewed herein appeared in
which authors reported the single-molecule detection of all 20
proteinogenic amino acids in an aerolysin nanopore, with the
help of a short polycationic carrier.[163]
Despite all existing challenges reviewed herein, we firmly
believe that proof-of-concept experiments involving the α-HL
nanopore to demonstrate amino acids recognition of individual
polypeptides, could pave the way toward the nanopore-based
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www.advancedsciencenews.com
polypeptide sequencing in the near future, and help pinpointing
post-translational modifications or quickly and cheaply identify
the sequence of proteins which are key for the onset and devel-
opment of many crippling diseases.
Acknowledgements
A.A. and G.D.M. contributed equally to this work. The work was supported
by UEFISCDI Grant Nos. PN-III-P4-ID-PCE-2016-0026, PN-IIIP1-
1.1-TE-2016-0508, PN-III-P1-1.1-PD-2016-0737, 34PFE/19.10.2018, and
PN-III-P1-1.2-PCCDI-2017-0010/74PCCDI⁄2018 (PNCDI III).
Conflict of Interest
The authors declare no conflict of interest.
Keywords
electrophysiology, molecular dynamics, polypeptide sequencing, protein
nanopores, single-molecule sensing
Received: August 28, 2019
Revised: January 14, 2020
Published online:
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