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Advancement of Next-Generation DNA Sequencing through Ionic Blockade and Transverse Tunneling Current Methods

May 2024
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May 2024

DOI:10.1002/smll.202401112

Authors:

Rameshwar L. Kumawat

Northwestern University

Milan Kumar Jena

Indian Institute of Technology Indore

Sneha Mittal

Indian Institute of Technology Indore

Biswarup Pathak

Indian Institute of Technology Indore

DNA sequencing is transforming the field of medical diagnostics and personalized medicine development by providing a pool of genetic information. Recent advancements have propelled solid‐state material‐based sequencing into the forefront as a promising next‐generation sequencing (NGS) technology, offering amplification‐free, cost‐effective, and high‐throughput DNA analysis. Consequently, a comprehensive framework for diverse sequencing methodologies and a cross‐sectional understanding with meticulous documentation of the latest advancements is of timely need. This review explores a broad spectrum of progress and accomplishments in the field of DNA sequencing, focusing mainly on electrical detection methods. The review delves deep into both the theoretical and experimental demonstrations of the ionic blockade and transverse tunneling current methods across a broad range of device architectures, nanopore, nanogap, nanochannel, and hybrid/heterostructures. Additionally, various aspects of each architecture are explored along with their strengths and weaknesses, scrutinizing their potential applications for ultrafast DNA sequencing. Finally, an overview of existing challenges and future directions is provided to expedite the emergence of high‐precision and ultrafast DNA sequencing with ionic and transverse current approaches.

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REVIEW

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Advancement of Next-Generation DNA Sequencing through

Ionic Blockade and Transverse Tunneling Current Methods

Rameshwar L. Kumawat, Milan Kumar Jena, Sneha Mittal, and Biswarup Pathak*

DNA sequencing is transforming the ﬁeld of medical diagnostics and

personalized medicine development by providing a pool of genetic

information. Recent advancements have propelled solid-state material-based

sequencing into the forefront as a promising next-generation sequencing

(NGS) technology, oﬀering ampliﬁcation-free, cost-eﬀective, and

high-throughput DNA analysis. Consequently, a comprehensive framework

for diverse sequencing methodologies and a cross-sectional understanding

with meticulous documentation of the latest advancements is of timely need.

This review explores a broad spectrum of progress and accomplishments in

the ﬁeld of DNA sequencing, focusing mainly on electrical detection methods.

The review delves deep into both the theoretical and experimental

demonstrations of the ionic blockade and transverse tunneling current

methods across a broad range of device architectures, nanopore, nanogap,

nanochannel, and hybrid/heterostructures. Additionally, various aspects of

each architecture are explored along with their strengths and weaknesses,

scrutinizing their potential applications for ultrafast DNA sequencing. Finally,

an overview of existing challenges and future directions is provided to

expedite the emergence of high-precision and ultrafast DNA sequencing with

ionic and transverse current approaches.

1. Introduction

The genome refers to the complete set of genetic material (DNA

and RNA) present in an organism. It encompasses all the genes,

regulatory elements, and non-coding regions that determine

an individual’s traits, functions, and hereditary information.[1]

These genetic materials carry a broad range of biological infor-

mation and genetic instructions at the molecular level, which

is crucial for the growth, development, and reproduction of a

living being. A DNA molecule consists of four kinds of nu-

cleotides: deoxyadenosine monophosphate (dAMP), deoxythymi-

dine monophosphate (dTMP), deoxyguanosine monophosphate

(dGMP), and deoxycytidine monophosphate (dCMP).[2]Each nu-

cleotide is composed of a nucleoside (nucleobase +deoxyribose

R. L. Kumawat, M. K. Jena, S. Mittal, B. Pathak

Department of Chemistry

Indian Institute of Technology (IIT) Indore

Indore, Madhya Pradesh 453552, India

E-mail: biswarup@iiti.ac.in

The ORCID identiﬁcation number(s) for the author(s) of this article

can be found under https://doi.org/10.1002/smll.202401112

DOI: 10.1002/smll.202401112

sugar) and a phosphate group. The

dAMP and dGMP are known as purine-

based nucleotides, whereas dTMP and

dCMP are called pyrimidine-based nu-

cleotides. The speciﬁc order of these

four nucleotides in DNA essentially

decodes all the biological phenomena.

DNA sequencing is the extraction of

precise and accurate order of nucleotides

in a DNA strand.[3]This sequencing in-

formation is vital for early disease de-

tection and facilitating eﬃcient ways of

maintaining human health. However, the

complexity of genomes, genetic variations,

and large sizes (for example, the hu-

man genome consists of ≈3.2 billion

nucleotide pairs) are quite challenging

and require capable sequencing methods

to identify each nucleotide accurately.[4]

The evolution of high throughput next-

generation sequencing (NGS) technology

and increasing demand for personalized

medicine development have revolution-

ized the ﬁeld of genomics and disease

diagnostics.[5–8]The NGS technology can

provide an opportunity to extract genomic

information to prevent, diagnose, and cure human diseases that

would lead medical research and medical care to a new era.

It continuously motivates the scientiﬁc and research com-

munity to develop a controlled, rapid, and cheap nanotechnol-

ogy that can sequence the whole human genome accurately.

During the COVID-19 pandemic, NGS sequencing has been a

boon for humanity as it helped in gathering crucial information

about the SARS-CoV-2 virus and its deadly variants, which are

vital for vaccine production and delivering fast and cost-eﬀective

testing methods like real-time reverse transcription-polymerase

chain reaction (RT-PCR).[9–12]However, the NGS method is a

very time-consuming and expensive process. Hence, the devel-

opment of inexpensive and faster DNA sequencing techniques

is an immediate requirement. Currently, scientists are more fo-

cused on electrical detection methods to achieve fast and cost-

eﬀective DNA sequencing with high precision. Figure 1rep-

resents the schematic depiction of the latest electrical DNA

sequencing methods along with their applications in various

ﬁelds.

The electrical detection methods address various challenges of

previous traditional sequencing methods, namely, the use of haz-

ardous chemicals, scalability, and technicalcomplexity.[13]Among

the two electrical detection methods, the ionic current method

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Figure 1. Schematic illustration of electrical detection methods for DNA sequencing and related applications in the ﬁelds of biology, agriculture, and

health science. The electrical detection methods are mainly divided into two categories: ionic current and transverse tunneling current methods. The

ionic current method is based on the blockade of longitudinal ionic current as DNA passes through the nanopore. In contrast, the transverse tunneling

current method detects the variations in the transverse electrical signals resulting from interactions between the nucleotides and nanoelectrodes. The

ionic current method is broadly divided into three parts: biological nanopore, solid-state nanopore, and hybrid nanopore and heterostructure. Similarly,

the transverse current method is also divided into four parts: nanopore, nanogap, nanochannel, and heterostructure.

with biological nanopores has been extensively explored and

successfully commercialized with industrial scalability.[14–16]

However, the lack of long readability inhibits its routine ap-

plication in healthcare and medical science. To address the

challenges of the ionic current technique, a complemen-

tary approach, known as the transverse tunneling current

method, is proposed, which detects the speciﬁc molecular ﬁn-

gerprints of each translocating nucleotide through variations in

the transverse tunneling current. It considers solid-state two-

dimensional (2D) nanomaterials with four types of potential

DNA sequencing devices: nanopore, nanogap, nanochannel, and

heterostructures.

While numerous reviews have delved into DNA sequencing,

there remains a gap in systematically documenting the breadth

of developments and breakthroughs in this ﬁeld, encompass-

ing both experimental and theoretical reports.[17–26]This review

seeks to address this by providing a comprehensive overview

of the evolution and advancements in single-molecule DNA

sequencing, speciﬁcally focusing on electrical detection meth-

ods. Our review aims to encapsulate the historical progres-

sion, various developmental stages, and potential challenges en-

countered in DNA sequencing methodologies. Given the sig-

niﬁcant strides made and the promising applications of elec-

trical detection methods utilizing solid-state materials, our fo-

cus lies in elucidating the key milestones in this area. We

underscore the potential of the transverse tunneling current

approach, particularly with solid-state nanodevices, as an ad-

vanced NGS method. This method holds promise in overcom-

ing challenges associated with existing techniques, particularly

the ionic current method. By consolidating the advancements,

challenges, and future prospects in single-molecule DNA se-

quencing with electrical detection methods, this review en-

deavors to provide valuable insights for researchers in the

ﬁeld.

2. History

Since the pioneering discovery of three-dimensional double helix

DNA by Watson and Crick in 1953, there has been a rapidly grow-

ing interest in developing a potential DNA sequencing technique

capable of decoding all the biological phenomena occurring in

the human body at the molecular level.[2]The advancement in

DNA sequencing techniques has a diverse and rich history with

several paradigms. On the basis of diﬀerent principles, DNA

sequencing methods are mainly divided into four categories:

ﬁrst-generation (basic), second-generation, third-generation, and

next-generation sequencing.[27–29]Below, we provide a brief sum-

mary of key milestones of DNA sequencing technologies.

In 1965, for the very ﬁrst time, Holley et al. isolated the com-

plete nucleotide sequence of transfer RNA from yeast. That was

the ﬁrst nucleic acid for which the structure was known.[30]In

the same year, Sanger et al. determined the related sequence

of nucleotide by partial digestion.[31]Later, Wu et al. intro-

duced a primer extension method to determine twelve bases

of bacteriophage lambda.[32]Maxam and Gilbert reported a

method to sequence RNA transcription copies of 24 base pairs

of lactose repressor binding sites.[33]In 1975, Sanger and co-

workers developed a rapid and straightforward chain termina-

tion method for the determination of the sequence of single-

stranded DNA by Escherichia coli DNA polymerase I and DNA

polymerase from bacteriophage T4.[3]In an another study, they

reported a similar strategy based on chain termination using

2′,3′-dideoxy, and arabinose nucleoside analogs of the normal de-

oxynucleotide triphosphates, acting as speciﬁc chain-terminating

inhibitors of DNA polymerase.[34]Further, a sequencing method

based on nucleobase-speciﬁc chemical modiﬁcation and subse-

quent termination has been demonstrated that partially breaks

a terminally labeled DNA molecule at each base repetition.[35]

First-generation DNA sequencing is mainly about these two

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above-discussed methods known as Sanger’s chain-termination

method and the Maxam–Gilbert chemical cleavage method. One

common thing in both these methods is that they both used the

distances of a radioactive label along a DNA molecule to deter-

mine the nucleotide order at each base position. At that time,

these two methods were instantaneously used for sequencing.

However, ﬁrst-generation sequencing methods suﬀer from sev-

eral key challenges from the perspective of cost, speed, and in-

dustrial scalability.[13,27]

After that, a massive improvement in advanced DNA se-

quencing techniques came into existence based on the Shotgun

method, which utilizes the libraries of cloned, randomly frag-

mented DNA.[36]This method was used in complete sequencing

of DNA strands with 4257 base pairs (bp). Here, the advantage is

that, unlike early sequencing methods, this method was capable

of analyzing longer fragments. Such generation DNA sequencing

techniques also include Illumina sequencing and pyrosequenc-

ing methods. The former method is based on reversible-dye ter-

minators, which enable the identiﬁcation of single nucleobases

having diﬀerent ﬂuorescent properties,[37]and later is based on

the sequencing by synthesis principle, including the detection of

nucleotide incorporated by DNA polymerase.[38]Such sequenc-

ing methods are used for single-molecule resolution without

the necessity for ampliﬁcation or labeling. Both these methods

are based on nanotechnology approaches that lead to low-cost

and procedure simpliﬁcation without the necessity for ampli-

ﬁcation or labeling. In 2005, Roche 454 GS20 technology de-

veloped a second-generation sequencing method that can se-

quence the DNA fragments equivalent to 1 billion nucleobases

in a single day (that is 1/3 of the human genome) and is much

cheaper than the methods incorporated in the “Human Genome

Project”.[39–41]This method was based on in vitro ampliﬁcation of

DNA strands and sequencing via synthesis of micro-beads, DNA

clusters, and DNA nanoballs. The above-discussed Shotgun, Il-

lumina, and pyrosequencing methods fall under the umbrella of

second-generation sequencing or massively parallel sequencing

(MSP) technology.

Third-generation sequencing techniques include stepwise

and real-time single-molecule sequencing by synthesis, Raman

scattering-based sequencing, molecular force microscopy, elec-

tron microscopy, single-molecule motion sequencing, and so

on.[42–44]Several brands like Illumina, Ion Torrent (Thermo Fis-

cher Scientiﬁc), Beijing Genomics Institute (BGI), pacBio, and

Oxford Nanopore Technologies are among the top DNA se-

quencing companies that build the ‘third-generation’ sequenc-

ing technologies with the promise to deliver human genomes

sequence in three minutes.[45]However, one of the third-

generation sequencing machines made by “Helicos Biosciences

of Cambridge,” Massachusetts, has been plagued by sequencing

errors.

All the recently developed sequencing methods, including

ionic current and transverse current methods, fall into the cat-

egory of next-generation sequencing (NGS) techniques. The

nanopore sequencing utilizing the ionic current method is cur-

rently a gold standard as it is trusted to provide reliable, cost-

eﬀective, and high-throughput DNA sequencing.[20,21]There

are several companies, such as Oxford Nanopore Technolo-

gies, Electronic BioSciences, Genia, NABsys, Life Sequencing,

and Roche and IBM, actively involved in nanopore sequencing

techniques.[20]An overview of the development of DNA sequenc-

ing techniques till 2021 has been shown in Figure 2.

It is also equally important to understand how the shift in

the diﬀerent paradigms of the development of DNA sequenc-

ing techniques aﬀects the cost. In the very beginning, the cost

of whole human genome sequencing was ≈$100 million. The

US government launched the “Human Genome Project” in 1990,

led by international scientiﬁc research with the goal of determin-

ing all 3.2 billion sequences of nucleotides building the human

genome. The project was announced to be completed on 14th

April 2003.[39,40]In Figure 3, we show the reduction of sequenc-

ing cost as a measure of sequencing technology progress with

time and compared with the curve of Moore’s Law, which signi-

ﬁes a long-term trend of doubling transistor number every two

years.[46]

Undoubtedly, Moore’s law is speciﬁcally applied to the comput-

ing and semiconductor hardware industry, which predicts that

the computing power and number of transistors will double ev-

ery 2 years and that the cost will be halved. DNA sequencing

technology evolved at a similar pace and pattern until 2007. How-

ever, thanks to the rapid development of new NGS technologies,

a stiﬀ reduction in the cost is observed from 2008 in both prices

per mega base of DNA sequence and total cost per genome.

There is a tremendous reduction in the cost of DNA sequenc-

ing from ≈$100 million in 2001 to ≈$1000 in 2021. Despite all

these achievements, the adoption of these technologies into clin-

ical practice has yet to be achieved. Scientists are hopeful that in

the near future, we can perform a whole-genome sequence in a

desktop sequencer. For a better understanding of advancement

in DNA sequencing technology, the strengths and weaknesses of

each ﬁrst, second, third, and next-generation DNA sequencing

are tabulated in Table 1.

3. Electrical Detection Techniques

The electrical detection techniques involve the identiﬁcation of

DNA nucleotides by analyzing the variations in their correspond-

ing electrical signals, also known as “electrical ﬁngerprints,”

which are measured using sequencing devices.[47–49]The basic

principle is that when single-stranded DNA (ssDNA) translocates

through the sequencing device, it leads to a change in the electric

current signals. Electrical detection techniques are broadly clas-

siﬁed into two categories: ionic current and transverse tunneling

current methods. The ionic current method detects the blockade

of the longitudinal ionic current ﬂux. The diﬀerent geometrical

shapes and sizes of each nucleotide alter the depth and width of

the ionic current read-outs diﬀerently, and on that basis, the iden-

tiﬁcation of each nucleotide is made possible. Figure 4ashows the

schematic of the ionic blockade current method. The nucleotide

sequence is determined by monitoring the distinctive ﬂuctua-

tions (both magnitude and duration) of ionic current when a

ssDNA passes through a nanopore. Nanopore devices consid-

ered for ionic current-based DNA sequencing can be divided into

three parts: biological, solid-state, and hybrid/heterostructure

nanopores, which have been discussed in section 3.1.

On the other hand, the transverse current method deter-

mines the modulation in the transverse conductance and cur-

rent signals while ssDNA is translocating through the device.

The variations in the tunneling current signals depend on the

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Figure 2. An overview of advancement in DNA sequencing technology with the timeline. Here, we have highlighted all the notable and revolutionary

events in DNA sequencing techniques reported so far.

Figure 3. A graphical representation illustrates the decrease in the cost of whole-genome sequencing vs. time with the development of diﬀerent se-

Figure 4. Schematics of electrical detection techniques: a) the ionic blockade current method-based nanopore that detects changes in the magnitude and

duration of blockade current signals as single-stranded DNA (ssDNA) molecules traverse through a nanopore. The transverse current method measures

the change in the transverse current signals of a trapped single nucleotide as a result of interactions between the nucleotide and the nanoelectrodes in

b) nanogap, c) nanopore, and d) nanochannel devices.

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Tabl e 1 . Comparison of each ﬁrst-generation, second-generation, third-generation, and next-generation DNA sequencing.

Parameters First generation Second generation Third generation Next-generation sequencing

Development Developed by Frederick Sanger in the

1970s

Introduced in the mid-2000s Introduced in the late 2000s to

early 2010

Introduced in the mid 2015

Working Principle Utilizes chain termination method Utilizes massively parallel sequencing

techniques

Sequencing is performed directly

on single DNA molecules

without ampliﬁcation

Sequencing through electrical

detection techniques, including

ionic current and transverse

current methods

Read Length Relatively long reads (less than 1kb) Short reads (≈0.075–0.15 kb) Longer reads (≈10 – 20 kb) from short to ultra-long (longest >

4Mb)

Throughput Rate per ﬂow cell Low throughput Low to Moderate throughput

(≈16–30 Gb)

Moderate to high throughput

(≈30 Gb)

High throughput up to 48 Gb

Data Analysis Complexity Moderate High Moderate to high Moderate to high

Cost˜ ˜≈13000 USD ≈50–63 USD ≈43–86 USD ≈21–42 USD

Speed Relatively slow Faster than ﬁrst-generation Real-time or near real-time

sequencing

Real-time sequencing

Sample Preparation Complex sample preparation Less complex sample preparation

compared to ﬁrst-generation

Reduced sample preparation

requirements

Simpliﬁed sample preparation

Platforms Sanger sequencing (capillary

electrophoresis)

Illumina, Ion Torrent, PacBio, etc. PacBio SMRT sequencing, etc. Oxford Nanopore sequencing

platforms (e.g., MinION)

Instrument Size and

Portability

Relatively large and less portable Varies oﬀer compact, portable devices

suitable for ﬁeldwork and

point-of-care applications

Compact, portable device suitable

for various applications (<450

Base Modiﬁcation Detection Not applicable Limited Capable of detecting DNA base

modiﬁcations during sequencing

Capable of detecting DNA base

modiﬁcations during sequencing

Accuracy (%) High accuracy, suitable for precise base

calling (>99.9%)

error rates can vary between platforms

and may require sophisticated

bioinformatics tools for error

correction

(>99.9%)

higher error rates compared to ﬁrst

and second-generation

sequencing, particularly for raw

reads (>99%)

Variable accuracy, aﬀected by

various factors such as

base-calling algorithms and

signal quality

Applications Targeted sequencing, small genomes whole-genome sequencing,

transcriptomics, epigenomics, and

metagenomics

sequencing various-omics Complex genomic regions,

structural variations, phasing,

real-time applications

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chemical and electronic properties of nucleotides. Based on the

sequencing device architectures, this method can be broadly sub-

divided into three categories: nanogap, nanopore, and nanochan-

nel. Figure 4b,c shows a schematic illustration of nanogap and

nanopore devices, respectively. In a nanogap, there exists a gap

between the two device electrodes, whereas, in the nanopore, a

nanometer size pore is created over the nanomaterial membrane

through which a ssDNA can be translocated. The nanochannel

device is a pristine nanomaterial surface over which a ssDNA

strand can be driven by an external electric ﬁeld, as shown in

Figure 4d. The variation of the interactions between the aromatic

groups of nucleotides with pristine nanochannel surface modu-

lates the electric current and conductance signals of the device,

which helps in the identiﬁcation of each DNA nucleotide. Apart

from these architectures, heterostructures (with two or three

solid-state materials) have also emerged as promising devices for

DNA sequencing. Further discussion of transverse tunneling cur-

rent techniques utilizing nanopore, nanogap, nanochannel, and

heterostructure devices has been provided in Section 3.2.

3.1. Ionic Current Blockade Technique

As mentioned above, DNA sequencing can be realized by mea-

suring ionic blockade current signals. In this technique, when

a longitudinal external voltage is applied perpendicular to the

nanopore, an ionic current is generated across the nanopore. By

application of an electric ﬁeld, the charged ions of the salt solu-

tion migrate in opposite directions through the nanopore, cre-

ating an ionic current. However, when a DNA strand is placed

in the cis chamber of the device, the negative charge drives it

in the head-to-tail fashion. The presence of DNA nucleotide in-

side the nanopore exhibits a sudden drop in the ionic current

signals known as ionic blockade current. The volume of each nu-

cleotide inside the nanopore determines the blockade height of

the ionic current signal. The temporal decrease time of the ionic

current depends on the interaction strength of the DNA molecule

with the nanopore, also called the translocation/dwell time of the

DNA molecule. The nucleotide sequence of the DNA strand can

be calculated by statistically analyzing the duration and height of

the ionic blockade current signals.

Among the developed NGS techniques, nanopores are the

most explored architectures for DNA sequencing, a comprehen-

sive discussion of which is provided below.

3.1.1. Biological Nanopores

In nature, the biological cell controls the movement of ions and

molecules into and outside of the cell membrane. Biological

nanopore sequencing mostly depends on the protein membrane,

which is coined as “porins.” Porins are embedded in the lipid

membranes and liposomes to create size-dependent porous sur-

faces with nanometer-scale “pores” distributed across the mem-

branes. Biological nanopore technology delivers high-resolution-

based sequencing with transmembrane protein channels. Suf-

ﬁciently low translocation velocity can be attained through the

incorporation of various proteins that facilitate the movement

of DNA through the pores of the lipid membranes.[50,51]Ma-

jor advantages include ease in reproducibility and modiﬁcation

that can be done at pace with modern molecular biology tech-

niques. Among biological nanopores, 𝛼-Hemolysin (HL), My-

cobacterium smegmatis (MspA), and Bacteriophage Phi29 are

among the most exclusively studied nanopores, as shown in

Figure 5.

𝛼-Hemolysin (HL) Pore: In 1996, for the very ﬁrst time, the

capture and translocation of ssDNA and RNA were demonstrated

through an 𝛼-HL biological nanopore (Figure 5a).[52,53]When the

oligomers of polyuridylic acid translocate through such a chan-

nel/pore, a distinct ionic current signal is observed. Such an

ionic current signal varies as a function of nucleotide length.

The polynucleotides cause short-lived ionic current blockade sig-

nals. In principle, based on such ionic current blockade signals,

DNA/RNA sequence can be obtained. The pyrimidine bases (C

and T for DNA or U for RNA) provide light signals compared to

purine bases (A and G). Later, Akeson and co-workers conﬁrmed

the same by studying the signal diﬀerences of DNA and RNA

polymerase translocation through 𝛼-HL nanopore device.[54]For

instance, in the presence of purine polyadenylic acid molecules,

there is less blockage in ionic current compared to that of poly-

cytidylic acid and polyuridylic acid. The secondary structure of a

homopolymer is accountable for the observed ionic current sig-

nals, not its primary structure. Because polyadenylic acid forms

a bulkier secondary structure, and the structure is unstable in-

side the nanopore, and therefore, it must disassemble. However,

polycytidylic acid and polyuridylic acid have narrower secondary

structures, and thus they block more ions than polyadenylic acid.

Meller et al. demonstrated the translocation speed of ssDNA

based on the ionic blockade current and time distributions.[57]

The translocation speed varies non-linearly with the applied elec-

tric ﬁeld. These studies were ﬁrst theoretically supported by in-

troducing a statistical physical model of polymer translocation

through a pore in the membrane.[58]In an 𝛼-HL biological pore,

there are three recognition sites for distinguishing DNA nucle-

obases, and to improve the base identiﬁcation ability, it is manda-

tory to enhance recognition at one or two points.[59,60]It has been

reported that amino acid side chain-assisted R1 site-directed mu-

tagenesis is helpful in improving base recognition, as shown in

Figure 5b.[55]The 𝛼-HL biological pore is suﬃciently stable to se-

quence genomic dsDNA at pH (<11.7), a value at which dsDNA is

denatured.[61]One of the studies on 𝛼-HL biological pores reveals

the dynamics of ssDNA in a protein pore at a single molecule

level.[62]Two diﬀerent time-scales have been noticed, and a longer

timescale has been found to be associated with the binding and

unbinding of DNA from the pore.

𝛼-HL combined with molecule adapter is reported to increase

the recognition ability of nanopore with accuracy averaging

99.8%.[63,64]Later, the translocation of ssDNA and ssRNA with

secondary structure was achieved through 𝛼-HL pore by adding

urea.[65]Nanopore force microscopy has been demonstrated to

identify oligonucleotides of equal length and sequence diﬀered

by a single nucleotide.[66]In a recent report, the eﬀect of molec-

ular crowding on the translocation behavior of DNA through 𝛼-

HL pore is studied as given in Figure 5c.[56]It has been noticed

that translocation event frequency has a nonmonotonic depen-

dence on the size of the crowding agent. However, both event

frequency and translocation time increase monotonically with in-

creasing crowder concentration. 𝛼-HL has shown great potential

in stochastic sensing of various analytes, including small organic

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Figure 5. Schematic of 𝛼-HL biological nanopore utilizing ionic current. a) 𝛼-HL biological nanopore and ionic current blockade signals caused by

schematic illustrating the eﬀect of crowding agent on translocation event of DNA translocation through 𝛼-HL biological nanopore;[56]Reproduced with

molecules, metal ions, RNA, DNA, and proteins, among others.

However, 𝛼-HL suﬀers from structural limitations such as a lim-

ited pore size of ≈1.4 nm and 𝛽-barrel, which takes a very long

to identify individual nucleotides.[67]Hence, to improve the sen-

sitivity and exploit the application of polynucleotide detection,

some other biological pores have also been explored.

MspA Channel Nanopore: Mycobacterium smegmatis porin

A (MspA) is a potential alternative to 𝛼-HL pore, which has gen-

erated a great deal of interest from both theoretical and experi-

mental aspects. It is more robust under experimental conditions,

varying pH from 0 to 14, and under high temperatures, it keeps

the channel active.[68]MspA is a funnel-shaped octameric chan-

nel nanopore that was ﬁrst prepared by the E. coil prokaryotic

expression system, which allows the transport of water-soluble

biomolecules across bacterial cell membranes.[69]This ﬁnding

has enabled all the follow-up reports with MspA, including the

site-speciﬁc mutagenesis that has enabled DNA nucleotide dif-

ferentiation using the MspA pore (Figure 6a).[70]

The authors demonstrated the protein pore MspA has great

potential in advancing next-generation DNA sequencing with im-

proved spatial resolution. They considered hairpin duplex DNA

for their study. During translocation, the sole presence of the

hairpin tail inside the pore measured the residual ionic current,

which majorly originates from the size and composition of the

ssDNA conﬁned inside the pore. Just after ≈10 ms on applying

the driving voltage of 180 mV, the hairpin duplex started dissoci-

ating to facilitate the translocation event. The translocation rate

of DNA through the MspA pore is too high to clearly identify a

single nucleotide without error. For that, DNA-cutting enzymes

were added to the system, which further enhanced the complex-

ity of DNA sequencing experiments. A mutated form of MspA

with phi29 DNA polymerase has also been reported to control

the translocation speed of the pore.[74]Here, phi29 DNA poly-

merase synthesizes DNA and acts as a motor to pull the single-

stranded template. Another report on MspA nanopore shows that

it can sequence the phi X 174 genome precisely up to 4500 nu-

cleobases in length.[75]It was the ﬁrst nanopore quadromer map

where ionic current signals of all possible 256 quadromers of four

DNA nucleotides were determined, enabling the sequencing of

long, complex, natural DNA strands.

Apart from this, several theoretical studies have also been re-

ported on polynucleotide detection through the MspA nanopore.

A report based on an MD simulation study reveals that sev-

eral arginine mutations can lead to ≈10–30-fold reduction in

the translocation speed of DNA through the MspA pore that too

without eliminating the nucleotide-induced ionic current block-

ade (Figure 6b).[71]Based on this, Bhattacharya et al. have re-

ported a study to determine the physical mechanism enabling

ionic current blockade in the MspA pore.[76]The mechanism re-

veals that the displacement of water molecules inside the MspA

nanopore determines the ionic current, while the steric and

base stacking nature of DNA determines the amount of wa-

ter displaced via the steric exclusion eﬀect. To provide more

insight into the experimental phenomenon and other possible

sensing positions of MspA nanopore, the underlying mecha-

nism of current variation for ssDNA translocation through the

MspA nanopore has further been investigated through all-atom

MD simulations (Figure 6c).[72]It has been observed that the

region below constriction is more promising for single nu-

cleotide resolution, while ssDNA translocation is in the base-

constriction-base meshing and ratcheting across the nanopore

constriction.

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Figure 6. Schematic of MspA biological nanopore utilizing ionic current. a) DNA translocation through MspA nanopore and resulting ionic current

Chemical Society, c) schematic illustration of MspA pore with residual current data for each nucleobase;[72]Reproduced with permission.[72]Copyright

2021 American Chemical Society and d) schematic illustration of machine learning integrated MspA pore with residual current data for identiﬁcation of

Tabatabaei et al. reported that the diﬀerent combinations

and ordered sequences of natural and chemically modiﬁed nu-

cleotides in custom-designed oligomers can be distinguished by

using a MspA nanopore, as shown in Figure 6d.[73]It is ob-

served that MspA pore can also detect 77 combinations and or-

dering of monomers with chemical diversity. The group further

reported a deep-learning neural network to classify combinatorial

patterns of natural and modiﬁed nucleotides that operate on raw

current signals generated by GridION of Oxford Nanopore Tech-

nologies. The machine learning technique facilitates denoising,

spike recognition, extracting, and analyzing the complex multidi-

mensional information of electric signals. With recent advance-

ments, the integration of machine learning tools with biologi-

cal nanopores is an emerging area of research that is needed to

achieve reliable and high throughput DNA sequencing. Despite

the signiﬁcant achievements of MspA nanopore in the ﬁeld, it

suﬀers from certain limitations. Here, the major limitation is

its narrow channel that allows only ssDNA and ssRNA chains

to pass through them. Thus, alternative nanopores are required

for sequencing both ssDNA and dsDNA.

Bacteriophage phi29 Nanopore: Bacteriophage Phi29 biologi-

cal nanopore is another potential candidate for sequencing DNA

through ionic current blockade measurements. Here, the advan-

tage is that compared to 𝛼-HL and MspA nanopores, the phi29

pore is more ﬂexible for biochemical modiﬁcations and has a

larger diameter, allowing the translocation of molecules larger in

size, such as dsDNA, DNA complexes, and proteins.[67]In 2009,

for the very ﬁrst time, the channel of the phi29 connector mo-

tor protein was implanted into lipid bilayers inside a microﬂu-

idic channel array for DNA sequencing purposes, as shown in

Figure 7a.[77]The bacteriophage phi29 DNA-packaging motor

acts as a path for the translocation of double-stranded DNA. The

length of the connector is ≈7 nm, with a cross-sectional area of

the channel of 10 nm2(3.6 nm in diameter) at the narrow end

and 28 nm2(6 nm in diameter) at its wider end.[78]Phi29 DNA-

packaging motor has the largest diameter, facilitating the translo-

cation of dsDNA, but cannot identify ssDNA under experimental

conditions.

According to an experimental report, channel-size conversion

of the phi29 motor can facilitate the translocation of both ssDNA

and dsDNA (Figure 7b).[79]Reengineering of the phi29 motor

channel is reported to result in two classes: one with the same

size as the wild-type channel and the other with a cross-sectional

area that is 60% of the wild-type. The smaller channel can de-

tect ssDNA at the single-nucleotide level, the wild-type connector

channel allows dsDNA translocation, and the loop-deleted con-

nector is reported to translocate both ssDNA and ssRNA with

equal competencies from both terminals.

CsgG Pore: In 2016, for the very ﬁrst time, Oxford Nanopore

Technologies (ONT) reported the Escherichia coli curli trans-

port channel CsgG nanopore for sequencing, which utilizes he-

licases as the motor enzyme to control the translocation rate of

DNA.[82]The CsgG nanopore has a well-deﬁned ≈1 nm wide con-

striction centrally located in a 4 nm wide channel.[83,84]Mutants

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Figure 7. Schematic of bacteriophage phi29 biological nanopore utilizing ionic current. a) Schematic of phi29 DNA packaging motor connector

Chemical Society, and d) Alpha-hederin nanopore and corresponding normalized histograms of conductance blockade for homopolymers and each

of CsgG mutants present several beneﬁts, including enhanced

interactions with analytes, an extended range of electrical

currents, and improved precision in sequencing modiﬁed

nucleobase.[85]The nanopore is commercially available for nucle-

obase modiﬁcation sequencing. Later, the prototype CsgG–CsgF

protein pore is reported for improved signal and base calling ac-

curacy (≈25–70%) of DNA molecules for homopolymer region

due to the double constriction eﬀect.[86]

Other than that, aerolysin pore (diameter ranges from 1.0 to

1.7 nm) has been reported as a prospective candidate to de-

tect nucleic acid at a single nucleotide level, as can be seen in

Figure 7c.[80]Aerolysin nanopore has several advantages over

𝛼-HL pore, such as high spatial resolution, slow translocation

speed, high current sensitivity, and prolonged duration time for

polynucleotide detection. It is demonstrated that the detection of

unlabeled random ssDNA is feasible through wild-type aerolysin

pore with high temporal resolution and current. The transloca-

tion of ssDNA through the pore is conﬁrmed by total internal

reﬂection ﬂuorescence (TIRF). Besides, alpha-hederin nanopore

is also reported for single nucleotide identiﬁcation, as shown in

Figure 7d.[81]Here, the advantage is its intrinsic pore-forming

properties in the cellular membrane. Spontaneous formation of

alpha-hederin nanopore in a lipid membrane with a determin-

istic lipid composition facilitates the identiﬁcation of single nu-

cleotides with high spatial and temporal resolution. Owing to

the small diameter and adequate thickness of the alpha-hederin

nanopore, identiﬁcation of ssDNA homopolymer and each DNA

nucleotide is found to be feasible.

Undoubtedly, biological nanopores oﬀer several advantages

for single molecule-based DNA sequencing. These advantages

include biocompatibility of set-up, easy and large production

of nanopores with atomic-level precision, uniform nanopore

structure, and easy tailoring of physical and chemical proper-

ties through genetic engineering techniques. With recent ad-

vancements, biological nanopores utilizing the ionic current

method have now been commercialized. The MinION nanopore

sequencer based on biological pores has ﬁnished sequencing a

human genome.[16]However, despite high sensitivity and easy

production of biological nanopores, there are certain inarguable

intrinsic disadvantages: i) low-stress durability, ii) strict tempera-

ture, pH, electrolyte concentration, and controlled environmen-

tal conditions to keep up biological pore active, iii) easy break-

down at high voltages, iv) incompatibility with the semiconduc-

tor fabrication process, and v) sensitivity to ambient conditions

and pore functionalization. In vitro studies reveal that the speed

of DNA through the nanopores is quite high (≈1 bp/1–10 μs),

which means very few ions are present inside the nanopore while

the nucleotide is passing. The challenges in biological nanopores

motivated scientists to search for new types of materials for

high-throughput DNA sequencing. In the next section, we have

discussed the potential usages of solid-state-based robust and

durable materials for the sequencing of canonical nucleobases.

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Figure 8. Solid-state Si3N4and graphene nanopore-based DNA sequencing with the ionic current approach. a) Schematic of the experimental setup

ionic current blockade method and current trace signals of DNA translocation through graphene nanopore at unfolded, partially folded, and fully folded

Academy of Sciences.

3.1.2. Solid-State Nanopores

Shortcomings of the biological nanopores motivated researchers

to explore the applicability of solid-state materials toward the de-

velopment of advanced nanopore devices for DNA sequencing.

With the advancement of nanofabrication technologies, the solid-

state nanopore has developed as a potential alternative to the bi-

ological nanopore. Solid-state materials oﬀer a range of advan-

tages, including cost-eﬀectiveness, robustness, and high durabil-

ity. They also possess superior mechanical, chemical, and ther-

mal properties.[87–89]In 2001, Li et al., for the very ﬁrst time, fabri-

cated the silicon nitride (Si3N4) solid-state nanopore as an alterna-

tive to biological nanopore by drilling with the focused ion-beam

sculpting technique.[90]This fabrication of Si3N4nanopore inves-

tigated the measurement of ionic current blockage measurement

and monitored the translocation events of DNA molecules, as

shown in Figure 8a.[91]The Si3N4nanopore membrane is sep-

arated by two chambers that conduct an electrolyte solution. The

authors demonstrated that the Si3N4nanopore is capable of ob-

serving individual molecules of dsDNA and their folding behav-

ior. In 2004, Chen et al. reported that the fabrication of an atomic

layer of Al2O3on Si3N4nanopore modiﬁes surface properties,

which leads to a reduction in the electrical noise and enhances

the DNA capture rate.[92]

Heng et al. demonstrated the relationship between DNA

translocation and blocking current and reported that the mag-

nitude and duration of ionic blockade current can lead to the

identiﬁcation of DNA nucleotides.[95]In 2009, Venkatesan et al.

demonstrated a mechanically robust and highly sensitive Al2O3

nanopore for DNA analysis purposes.[88]The major advantages of

this nanopore are easy pore size control, chemical modiﬁcation,

and improved signal performance. Subsequently, the solid-state

Al2O3nanopore sensor with enhanced surface properties is stud-

ied for real-time analysis and identiﬁcation of DNA molecules.[96]

Owing to high surface-charge density and enhanced surface

properties, an order of magnitude reduction in the transloca-

tion speed is observed relative to other solid-state Si3N4and

SiO2architectures. Later, they studied the Al2O3coated hybrid

nanopore sensor.[97]A lipid bilayer coated with Al2O3shows ex-

cellent electrical properties and enhanced mechanical stability.

Moreover, compared to black lipid membranes, Al2O3-coated hy-

brid nanopores are found to be more stable in ionic solution with

a ﬁvefold increased lifetime.

Besides silicon nitride nanopores, the DNA translocation

events through ‘kinked’ silica nanopores (SiO2) have also been

demonstrated with nearly perfect selectivity for ssDNA over

dsDNA.[98]By application of a gate electric ﬁeld, a reduction in the

DNA translocation speed at a rate of ≈55 μms

−1per 1 mV nm−1

is observed.[99]Even though the solid-state nanopores oﬀered

several advantages and achieved a similar nanopore dimen-

sion to the biological nanopore, their higher thickness prevents

achieving the required high-precision identiﬁcation of DNA

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nucleotides. The thickness of silicon nitride is usually higher

than ≈10 nm, which is equivalent to ≈30 DNA molecules, a

big challenge for silicon nitride nanopores to achieve single nu-

cleotide identiﬁcation. The challenges, such as incapability to sin-

gle nucleobase resolution, low signal-to-noise ratio, and weaker

capacitive coupling, have pushed researchers to explore other 2D

solid-state nanomaterials for DNA sequencing.

Graphene is regarded as a highly promising substrate for

solid-state pores due to its membrane thickness of 0.34 nm,

which is equivalent to the height of a nucleotide. This fea-

ture enables the possibility of discriminating between individ-

ual nucleotides.[100,101]In 2010, three diﬀerent groups, Merchant

et al., Schneider et al., and Garaj et al., independently reported

the measurement of the ﬂuctuations of ionic current when

a ssDNA translocates through an atomically thick graphene-

based nanopore.[93,102,103]Figure 8b shows the schematic set of

graphene nanopores with a dsDNA translocation through it. In

the absence of DNA, the ionic current signal remains uniform

and continuous. However, a sharp drop in the current signal

(black color signals) can be observed as soon as the dsDNA

starts translocating through the nanopore. Interestingly, the cur-

rent signals of DNA translocation through graphene nanopores

are larger than the earlier reported Si3N4nanopores with the

same diameter due to the atomic thickness of the graphene

membrane.[93]Golovchenko and co-workers demonstrated that

the average translocation time is also slightly larger for graphene

nanopore devices compared to other solid-state nanopores due

to DNA-graphene interactions.[103]Garaj et al. studied the mono-

layer graphene nanopore with a diameter equivalent to the di-

ameter of the dsDNA molecule and observed a highly sensitive

ionic current with a small change in translocating DNA molecule

diameter.[94]Additionally, they found that when the nanopore

length (L) is less than the diameter (D), a strong ionic current

distribution peak is observed near the nanopore perimeter. How-

ever, there is a uniform current distribution around the pore for

L≥D, as shown in Figure 8c. A linear increase in ionic current

measurement with an increase in the graphene nanopore diam-

eter is observed, as shown in Figure 8d.

Drndi´

c and co-workers coated the graphene monolayers with

TiO2, which acts as an insulating layer and has signiﬁcantly re-

duced the signal noise.[93]The TiO2layer over the graphene sur-

face provides a hydrophilic surface to the DNA strand for ex-

ploring low entropy dynamics during translocation through the

nanopore. However, coating of the layer increases the graphene

nanopore thickness and decreases the single-base resolution ca-

pability of graphene.

In an MD simulation study with a graphene nanopore device,

Wells et al. demonstrated that the hydrophobic interactions with

the graphene membrane lead to a dramatic reduction in the con-

formational ﬂuctuations of the nucleotides in the nanopore.[104]

The ssDNA was found to adhere to the surface of graphene mem-

branes due to the strong hydrophobic interaction between ssDNA

and graphene. They also demonstrated that the adhesion of ss-

DNA over the multilayer nanopore leads to control of the translo-

cation speed with stepwise translocation in a single-nucleotide

manner. However, Schneider et al. reported that the strong in-

teractions between the graphene and DNA nucleotides are re-

sponsible for the irreversible pore closure.[105]Additionally, the

adhesion of ssDNA over the nanopore surfaces restricts the DNA

conformational ﬂuctuations to a few typical stable conﬁgurations

only, depending on the nucleotide type. A recent MD study from

our group shows that the identiﬁcation of DNA nucleotide is pos-

sible with the graphene nanogap device using an ionic current

approach.[106]The major concern with the nanogap-type setup is

controlling the translocation of ssDNA and the overlap of ionic

current signals due to high current ﬂux through the nanogap.

However, there are certain drawbacks in graphene, such as

strong DNA–graphene hydrophobic interactions, limited per-

meation events of DNA, clogging of the pore, the require-

ment of additional functionalization of graphene surface to

facilitate controlled DNA translocation, and physisorption of

nucleobases on graphene edges (𝜋-𝜋interaction) making the

single-base resolution more diﬃcult. Hence, graphene may not

be the most promising material for ionic current-based DNA

sequencing.

To resolve the above-mentioned issues with graphene

nanopore, other 2D solid-state materials that have promising

characteristics for cheap, reliable, and eﬃcient DNA sequencing

are being explored. Yu and co-workers have reported h-BN

as a competitive candidate for DNA sequencing other than

graphene by the ﬁrst electronic measurement of DNA transloca-

tion events through atomically thin insulating h-BN nanopores

(Figure 9a,b).[107]DNA translocation signals are observed to be

much larger compared to the similar-sized Si3N4nanopores, in-

dicating higher detection accuracy. The single-atomic thickness

(1.1 nm) of the h-BN membrane has a signiﬁcant opportu-

nity to achieve high spatial resolution similar to a graphene

nanopore device. However, like graphene, the h-BN surface is

also hydrophobic in nature, which could be a reason for the easy

clogging of the pore in an experimental condition. Later, Zhou

et al. successfully enhanced the hydrophilicity of h-BN nanopores

through UV-ozone treatment.[111]They also observed suﬃcient

DNA translocation events with no easy pore clogging. In addition

to insulating nature and durability, another important feature is

the possibility of pore creation in the triangular shape, enabling

control over the nanopore geometry. The translocation speed

is found to be faster near the pore center than at the corner of

the triangle. In this regard, the well-designed triangular-shaped

h-BN pore exhibits two diﬀerent translocation pathways, and the

translocation speed of DNA can be tuned across the membrane.

In 2014, Farimani et al. investigated molybdenum disulﬁde

(MoS2) nanopore, which is found to be a promising alterna-

tive to graphene due to the comparatively high signal-to-noise

ratio (Figure 9c).[112]The results show that DNA translocation

exhibits signal amplitude ﬁve times higher than the solid-state

Si3N4nanopores and improved signal-to-noise ratio (SNR >10).

Moreover, unlike graphene nanopore, no special surface treat-

ment is needed to avoid strong 𝜋–𝜋interaction between DNA

and the surface, and the hydrophobic interaction is also less in

the case of MoS2nanopore.[108]However, the challenge of the

MoS2nanopore is that the membrane can suﬀer from breakdown

under electrical and mechanical stress. To counter the break-

down issue of the MoS2and control the translocation speed of

DNA, Zou et al. reported a study with graphene-MoS2bilayer

heterostructure.[113]Gu et al. reported the eﬀects of KCl salt and

Bmim+ionic liquid on the translocation speed of ssDNA though

the MoS2nanopore. A strong interaction between Bmim+and

ssDNA is noted, which oﬀers a considerable dragging force to

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Figure 9. Experimental and theoretical studies of 2D solid-state nanopore-based DNA sequencing with the ionic current approach a) Schematic of the

device setup for DNA translocation through BN nanopore, b) the scattering diagram showing the depth versus length for DNA translocation events

membrane for DNA translocation, where the monolayer MoS2is suspended on a Si3N4supporting membrane and TEM image of nanopore drilled by

cation through a single-layer Ti2CTxnanopore, e) concatenated ionic current spikes shown at diﬀerent voltages of 250 mV (blue), 225 mV (black), and

of graphene, MoS2,andTi

3C2MXene nanopore and single-strand DNA passing through graphene nanopore;[110]Reproduced with permission.[110 ]

decelerate the electrophoretic motion of ssDNA in the BmimCl

solution.[114]

In 2019, Wanunu and co-workers experimentally studied DNA

transport through the Ti3C2TxMXenes nanopore (Figure 9d).[109]

From Figure 9e, the concatenated negatively pointing spikes of

ionic current signals caused by the temporary position of DNA

molecules at diﬀerent voltages. The MXene nanopore demon-

strated lower ionic current leakage and similar noise of the mem-

brane compared to other reported 2D solid-state nanomateri-

als. Recently, Farimani and co-workers performed an MD-based

study on the Ti3C2MXene and found that it could be a promising

candidate for a single-nucleotide resolution with a high signal-to-

noise ratio due to its hydrophilic surface, greater electrical con-

ductivity, and stability under electrical forces.[115]

Though a variety of 2D solid-state nanopores (graphene, hexag-

onal boron nitride, MoS2, MXenes) have been extensively studied

for DNA sequencing, each material comes with its own merits

and demerits. In this regard, a comparative study with graphene,

MoS2,andTi

3C2MXene nanopore is demonstrated for DNA se-

quencing with the ionic current approach (Figure 9f).[110]Fro m

the ionic current and residence time results, it is concluded that

graphene nanopore still stands out as a better nanopore device

for the identiﬁcation of all four DNA nucleotides, whereas MoS2

and Ti3C2MXene nanopores were found to be selective.

Currently, the integration of machine learning techniques with

NGS technologies has emerged as an active area of research to

achieve ultrafast, cheap, and high throughput DNA sequencing.

Machine learning tools resolve several issues associated with the

DNA sequencing process. Diﬀerentiating single-stranded DNA

nucleotides is challenging due to the broad distribution of ionic

current signals, compounded by errors from thermal ﬂuctua-

tions, counterions, electric ﬁeld, and DNA dynamics. These re-

strictions impede the high-precision identiﬁcation of nucleotide

sequences. The machine learning protocols translate the raw se-

quencing data into base calls by feature extraction and real-time

signal processing of long-read sequencing data.

Fyta and co-workers demonstrated unsupervised learning

studies to classify diﬀerent molecular topologies from the ionic

current translocation events of MoS2nanopores without the use

of labels (Figure 10a).[116]They proposed a new eﬃcient feature

of ionic blockage height compared to traditional dwell time and

mean ionic current feature and observed that this approach is

able to identify distinct types of the most probable molecular con-

formations of the DNA threading through the MoS2nanopore. In

a follow-up study, by utilizing deep neural networks and convo-

lutional neural networks (CNN) on the experimental ionic block-

ade current data, the authors tried to increase the classiﬁca-

tion accuracy and read-out eﬃciency of MoS2pores with varying

diameters.[117]The ionic blockade height combined with dwell

time, mean ionic current, and the number of levels helped the

CNN and XGBoost models to classify all four nucleotides with

better accuracy (Figure 10b).

3.1.3. Hybrid Nanopores and Heterostructures

Afterward, hybrid nanodevices came into existence, which is a

coupling of both biological and solid-state entities. Here, the role

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Figure 10. Machine learning studies with solid-state nanopore-based ionic current blockade method. a) concatenated events of dAMP translocation

through MoS2nanopore, where the red block highlights each event and the right box shows the extracted four features for a single nucleotide translocation

nucleotide compared to the predicted identity (label) for the LSTM, XGBoost, DNN, and CNN models;[117]Reproduced with permission.[117 ]Copyright

2021 AIP Publishing Group.

of the biological nanopore is to oﬀer an atomically precise struc-

ture capable of genetic engineering, and solid-state nanopore

provides durability, size, and shape control, which is well-suited

for wafer-scale devices. In addition, biological nanopores can

attain control of the DNA translocation time and its orienta-

tion, and solid-state nanopores can provide enhanced robust-

ness and durability. The hybrid nanostructures have the poten-

tial to quickly identify the nucleotides during the DNA sequenc-

ing process.[97,118–123]It has also been noticed that the hybrid

nanopores are easier to fabricate and stable under experimental

conditions, showing low peripheral leakage allowing sensing and

discrimination among DNA nucleobases.

In 2010, Hall et al. ﬁrst introduced the hybrid pore forma-

tion by directed insertion of hemolysin into Si3N4solid-state

nanopore, as shown in Figure 11a.[123]Upon protein insertion,

the hybrid nanopore is noticed to be functional for 30–40% of

their attempts and, thus, oﬀers a platform to create wafer-scale

device arrays for genomic analysis, including sequencing. In

principle, hybrid nanopores exploit the excellent addressability of

biological macromolecules to provide controllable chemical func-

tionality for solid-state nanopores. It shows both the robustness

of the device and the biological nanopore remaining unaﬀected

while providing better control of DNA translocation velocity.

According to another experimental report, the hybrid

nanopore formed by the insertion of non-modiﬁed 𝛼-hemolysin

at the entrance of biomimetic nanopore allows polynucleotide

distinguishability with increased dwell time.[124]Here, the main

ﬁnding is that the insertion of 𝛼-hemolysin can increase the

polynucleotide dwell time without perturbing the current block-

ades and losing the nucleotide’s distinguishing ability of protein

nanopore.

Recently, DNA-based technology is also become quite popu-

lar for functionalizing nanopores.[128–131]The coolest emerged

technique among other techniques and methods is DNA

origami,[132–134]which acts as a toolbox for engineering complex

nanostructures.[135–138]Inspired by the pioneering work of Hall

et al., Keyser and co-workers experimentally fabricated a sin-

gle artiﬁcial nanopore based on DNA origami and detected 𝜆-

DNA successfully.[139]Here, the advantage is that DNA origami

nanopores can be continuously inserted and ejected from the

solid-state nanopore and can be used as resistive pulsed sensors.

Later, Farimani et al. performed MD simulations to analyze the

ionic conductivity of DNA origami-graphene hybrid nanopore, as

showninFigure11b.[125 ]In the case of bare graphene, the same

dwell time is noticed for DNA nucleobases, while the use of DNA

origami hybrid nanopore signiﬁcantly improved the dwell time

of all four nucleobases, which further increased distinguishabil-

ity. Moreover, the speed of DNA translocation is also noted to be

reduced in the presence of DNA origami due to the friction of

nucleobase near the mouth of the graphene pore. To control the

DNA translocation speed, the authors removed the bases from

the origami sheet near the nanopore. However, the eﬀect of the

number and spatial distribution of the baits, also referred to as

overhangs (genomic DNA fragments of interest for sequencing),

was not addressed properly. Later, to encounter that, MD simula-

tions were performed on a hybrid nanopore comprising a single-

layer graphene and DNA origami layer with diﬀerent numbers,

lengths, and spatial distribution of overhangs, as can be seen in

Figure 11c.[126]It has been noticed that diﬀerent customizations

give rise to base-speciﬁc translocation times.

Cressiot et al. experimentally reported the stable and easy fab-

rication of lipid free-hybrid nanopore comprising a natural DNA

pore and portal protein as adapter inserted into a Si3N4nanopore

for sensing biomolecules (peptide, globular protein, ssDNA, and

dsDNA) as shown in Figure 11d.[127]The interesting thing is

that the reported hybrid pore allowed sensing and distinguisha-

bility among diﬀerent biomolecules with low peripheral leak-

age. The work further motivates the researchers to ﬁnd out what

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Figure 11. Schematic representation of hybrid nanopores-based DNA sequencing utilizing ionic current approach. a) Schematic illustrating the translo-

graphene hybrid nanopore and the plot showing translocation rate through both bare and hybrid nanopore;[125]Reproduced with permission.[125 ]Copy-

right 2017 American Chemical Society, c) schematic of translocation of DNA through a hybrid DNA origami-graphene nanopore with ions and current-

properties of 𝛼-hemolysin are retained by the lipid-free hy-

brid pore. The theoretical assessment of polynucleotide dif-

ferentiation based on a lipid-free hybrid nanopore functional-

ized with 𝛼-hemolysin promotes the stability of protein inside

the biomimetic nanopore and its potential for sequencing the

biomolecules.[140]

Apart from hybrid nanopore, heterostructures (with two

or three solid-state materials) have also been widely stud-

ied through the ionic current method. In a framework

of MD simulations, Luan and co-workers ﬁrst studied the

stacked graphene/MoS2heterostructure nanopore, as shown in

Figure 12a.[141]The proper attraction between the negatively

charged phosphate group of DNA nucleotide and positively

charged Mo atoms provides a slow nucleotide-by-nucleotide

transport, which is crucial for precise and accurate sequencing

of DNA. Another report studied the geometry and shape eﬀects

of stacked graphene/MoS2heterostructure nanopore in a theoret-

ical framework of MD simulations, as shown in Figure 12b.[113]

The results show that among the considered circular, square, and

triangular nanopores, the circular nanopore facilitates the fastest

translocation, and triangular nanopores show the slowest translo-

cation of ssDNA. Here, the diﬀerence in the translocation speed

arises from the diﬀerence in the electrostatic attraction. In the

diﬀerent geometries of heterostructure nanopore, the number of

exposed Mo atoms is diﬀerent, which means the electrostatic at-

tractions between the positively charged Mo atoms and the nega-

tively charged phosphate group of DNA nucleotides are diﬀerent,

which signiﬁcantly aﬀects the translocation time.

Hexagonal boron nitride (h-BN) has a similar atomic struc-

ture and has been extensively studied for DNA sequencing from

both theoretical and experimental aspects.[144,145]These ﬁndings

motivated the researchers to use graphene/h-BN heterostruc-

tures for DNA sequencing. Luan et al. further checked the po-

tential of an in-plane graphene/h-BN heterostructure nanopore

for sensing ssDNA, as given in Figure 12c.[142]Both the DFT

and MD calculations revealed that due to the stronger van

der Waals attraction, ssDNA prefers to stay in the h-BN do-

main than graphene. This change in the conformation of ss-

DNA on the graphene/h-BN surface enables the sensing of

DNA through scanning tunneling microscopy (STM) and atomic

force microscopy (AFM) in the dimension perpendicular to

the heterostructure surface. Another theoretical report studied

the translocation of dsDNA through graphene/h-BN nanopore

and found that graphene/h-BN layer order is of crucial im-

portance for nucleobase-speciﬁc electrical signal variability.[146]

Here, the interesting observation is that h-BN allows the translo-

cation of dsDNA parallel to the electric ﬁeld, and this vertical

conﬁguration is the key to signal readability and single-base

sensing.

Another theoretically studied heterostructure is BC3/C3N

nanopore, where the authors reported spontaneous translocation

(without the need for an electric ﬁeld) of DNA from BC3to the

C3N side with a strong aﬃnity of C3N towards DNA bases in

comparison to the BC3surface, as given in Figure 12d.[143]In

the case of BC3/C3N nanopore, the translocation speed of ss-

DNA is noticed to be 2-fold slower than that of graphene/MoS2

heterostructure nanopore. For a better understanding, year-

wise documentation of ionic current studies reported on hy-

brid/heterostructure nanopores for DNA Sequencing is given in

Table 2.

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Figure 12. Schematic representation of solid-state heterostructures based DNA sequencing utilizing ionic current approach. a) Schematics illustrating

the translocation of ssDNA through a graphene/MoS2heterostructure nanopore and the number of transported nucleotide vs. time plot;[141]Repro-

showing translocation rate through both diﬀerent geometries of graphene/MoS2heterostructure nanopore;[113]Reproduced with permission.[113 ]Copy-

right 2020 American Chemical Society, c) schematic of MD simulations for ssDNA translocation through graphene/h-BN heterostructure nanopore;[142]

3.2. Transverse Tunnelling Current Approach

Recent advancements in the ﬁeld of 2D solid-state nanomateri-

als and ﬁeld-eﬀect transistor domain have paved the path for a

new potential technique for DNA sequencing named as trans-

verse tunneling current method. During tunneling current mea-

surement, the temporary molecular junction is created between

the electrodes, and the subsequent current is analyzed.[148]The

idea of transverse electrodes originated from the need to diﬀer-

entiate between DNA nucleotides using tunneling current sig-

nals, which could be a promising alternative to the longitudinal

ionic current approach. In principle, the tunneling current DNA

sequencing promises to exhibit suﬃciently distinct electrical cur-

rent readouts for each base, which in turn helps in deducing the

nucleotide sequence in a DNA strand. In comparison to the ionic

current technique, this technique is capable of sequencing faster

(by orders of magnitude).[20,48]

This method consists of four types of devices: nanopore,

nanogap, nanochannel, and heterostructure. In a nanopore de-

vice, the interaction of ssDNA with the edges of the nanopore

allows for the measurement of both tunneling current and trans-

verse conductance of the membrane concurrently, aiding in the

diﬀerentiation among the four nucleotides. A nanogap device

consists of a set of nanoelectrodes placed on the membrane sur-

face on both sides of the nanopore aperture. The transverse elec-

trodes allow for the measurement of electronic tunneling cur-

rent ﬂowing through the gap between those electrodes. In a

nanochannel device, the ssDNA is pulled over the surface to mea-

sure the change in the tunneling current signal due to the interac-

tion of stacked nucleotides over the nanochannel surface. In the

last decade, various 2D solid-state nanomaterials have been in-

vestigated for DNA sequencing using transverse tunneling mea-

surements, the details of which are provided in the subsequent

sections.

3.2.1. Solid-State Nanopore

Regardless of major developments in the ionic current method

with the solid-state nanopore, the high signal-to-noise ratio (SNR)

and lack of single-base resolution are the major challenges

causing hindrances in the further advancement of the ﬁeld.

Moreover, the technique requires consumables, which makes

it computationally and experimentally more expensive. The re-

searchers have developed a potential alternative to ionic current

nanopore sequencing, i.e., transverse tunneling nanopore se-

quencing, which is purely a physical method and has a high SNR.

The underlying principle of transverse tunneling nanopore se-

quencing is when a ssDNA molecule is electrophoretically driven

through a nanoscale pore (diameter of ≈2–5 nm), diﬀerent nu-

cleotides (diﬀerent in shape and size) cause diﬀerent variations

in the transverse conductance and current readouts and based

on that DNA sequencing is made possible.[149]The modulation

of transverse conductance arises from the nucleotide-speciﬁc

interaction between DNA and solid-state nanopore, which af-

fects the local density of states and characteristic nucleotide-

speciﬁc coupling strength with the nanopore edges. In these tech-

niques, DNA molecules transverse through the nanopore embed-

ded in closely placed semiconducting/semi-metallic electrodes,

and current modulation is measured between the left and right

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Tabl e 2 . Year-wise documentation of ionic and transverse current studies reported on hybrid/heterostructures for DNA sequencing.

Materials Nanodevice Molecular carrier Study Comments Year Refs.

𝛼HL +Si3N4Nanopore dsDNA Experimental The hybrid nanopore is noticed to be

functional for 30–40% of the attempts

2010 [123]

𝛼HL +biomimetic nanopore Nanopore ssDNA Experimental Discrimination of poly[A] and poly[C] with

increased dwell time

2013 [124]

DNA origami-graphene Nanopore ssDNA MD simulations Diﬀerent residence time and ionic

current signals; decreased

translocation speed

2017 [125]

Graphene/MoS2Nanopore ssDNA MD simulations The chemical potential diﬀerence on

graphene and MoS2surfaces directly

drives ssDNA

2018 [141]

Natural DNA pore- Si3N4Nanopore dsDNA and ssDNA Experimental Lipid-free hybrid nanopore exhibiting low

peripheral leakage

2018 [127]

DNA origami-graphene Nanopore ssDNA MD simulations Speciﬁc interactions between overhangs

and ssDNA results in base-speciﬁc

residence time

2019 [126]

𝛼HL +biomimetic nanopore Nanopore ssDNA MD simulations The ionic current blockade depends on

the type of polynucleotide and its

conformation

2020 [140]

Graphene/MoS2Nanopore ssDNA MD simulations Reduced translocation speed in the

order: circular nanopore <square

nanopore <triangular nanopore

2020 [113]

Graphene/h-BN Nanopore ssDNA MD +DFT Manipulation of ssDNA conformation

through diﬀerential van der Waals

interaction

2020 [142]

eOmpG/MoS2Nanopore Single-stranded

polyadenine (dA30)

Experimental 32% lower noise levels with nearly 1.9%

improved signal-to-noise ratio and 6.5

times longer dwell time

2021 [147]

BC3/C3N Nanopore ssDNA MD simulations Spontaneous translocation from BC3to

C3N and reduced nucleotide

translocation

2021 [143]

Graphene/h-BN (vertically

stacked)

Nanopore dsDNA MD simulations Heterostructure layer order plays crucial

role in controlling the base-speciﬁc

signal variability

2021 [146]

electrodes through the scattering region. A bias voltage is applied

across the 2D nanopore device to obtain a ﬁnite current of the de-

vice, and a gated voltage is applied to determine tunneling con-

ductance.

Among the 2D materials, graphene has a single atomic thick-

ness (≈0.34 nm thick, equivalent to the spacing between two

bases in a DNA strand) and extraordinary mechanical and elec-

tronic properties complementary to single-base resolution. In

2010, Nelson and co-workers studied the interaction of four DNA

nucleobases with the graphene nanopore by calculating the elec-

tronic structure and density of states of a graphene nanorib-

bon with the ab initio density functional theory (DFT), as shown

in Figure 13a.[150]Further, they also studied the transverse con-

ductance and electrical current using the Landauer–Bϋttiker for-

mula, and the result analysis concluded that single nucleotide

detection can be achieved via measurement of the in-plane cur-

rent signals and conductance of the semiconducting graphene

nanoribbons with armchair edges. Electrical current and conduc-

tance spectra provided suﬃcient information for the identiﬁca-

tion of DNA molecules inside the nanopore. Moreover, the oper-

ating current of the nanopore was noted to be insensitive to the

rotated conﬁgurations of a speciﬁc DNA nucleobase, which is a

desirable requirement for the feasibility of the device for experi-

mental realization.

However, the bare graphene nanopore has lower coupling

strength with DNA nucleotide due to the much smaller space ex-

tension of carbon outer orbitals. As the coupling strength drops

exponentially with decreasing overlap, the transverse tunneling

conductance using bare graphene nanopore will be reduced dra-

matically, which consequently deteriorates the SNR ratio. There-

fore, there occurs a need to introduce functionalized electrodes

that could improve the coupling strength between the graphene

nanopore and nucleotide.

He et al. studied the merits of hydrogen functionalization

of graphene nanopore edges on DNA sequencing with elec-

tronic transport studies (Figure 13b).[151]They demonstrated that

compared to the bare graphene nanopore, the hydrogen atom

functionalized nanopore accelerates the formation of temporary

H-bonds between nucleotide and translocating DNA molecule,

which leads to an increase in the average conductivity (3 orders

of magnitude) while exhibiting signiﬁcantly reduced statistical

variance. The results also determined that the hydrogenation of

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Figure 13. Electrical detection of DNA nucleobases by 2D solid-state nanopore devices with the transverse tunneling current approach. a) Illustra-

tion of a proposed graphene nanopore device for identiﬁcation of single nucleobases with transverse tunneling current method;[150]Reproduced with

d) schematic illustration of the Ti2C(OH)2MXene nanopore device for measuring the conductance of translocating DNA nucleotides;[153]Reproduced

graphene edges reduces the translocation speed of DNA and con-

siderably improves the accuracy of the measurement of individ-

ual nucleotides.

In a similar report, Saha et al. investigated the graphene

nanoribbon with zigzag edges (ZGNR) for DNA nucleobase iden-

tiﬁcation with the transverse tunneling current method.[148]In-

terestingly, the tunneling current across the nanopore is observed

in the range of microampere (μA) at low bias voltage (0.1 V),

which is of several orders higher magnitude than that of

graphene nanopore with armchair edges. The higher current of

the ZGNR nanopore is predominated by the local current den-

sity around the edges. Therefore, drilling a nanopore in the cen-

ter of the device, away from the edges, will not reduce the con-

ductance. Additionally, the operating current is anticipated to be

suﬃcient to yield a rich SNR and expected to be much larger

than the thermal vibration of the graphene device, orientational

ﬂuctuation of the translocating DNA strand, and solvent inﬂu-

ence on the electronic properties of the nucleotides. The ZGNR

nanopore has also been successful in identifying the epigenetic

nucleotides from the natural DNA nucleotides.[154]However, the

above-discussed reports have excluded the environmental condi-

tions and solvent eﬀects.

To get insight into the impact of environmental conditions on

tunneling current signals, Feliciano et al. studied the graphene

nanopore with the QM/MM electronic transport method, in-

corporating the eﬀect of structural ﬂuctuations of the nu-

cleotides (Figure 13c).[152]The results show that nucleotide-

speciﬁc charge ﬂuctuation is responsible for the characteris-

tic conductance modulation of the device. However, they also

pointed out that the conductance proﬁles of all four nucle-

obases are similar due to higher conducting pathways across

the graphene nanopore membrane.[155]Despite extensive stud-

ies with graphene nanopores, the high translocation velocity of

DNA, noise, and pore-clogging issues hinder individual DNA

base identiﬁcation. An extensive molecular dynamics simula-

tion followed by the non-equilibrium Green’s function (NEGF)

study demonstrated that an intrinsic stepwise translocation of ss-

DNA strand by mechanically stretching the ssDNA perpendic-

ular to the graphene sheet as it passes through the nanopore

improved the signal quality to ensure high ﬁdelity detection of

nucleotides.[156]The stepwise translocation not only increased

the dwelling time of each nucleotide inside the nanopore but also

suppressed the signal noise by escaping the hydrophobic interac-

tion between the nucleotide and graphene.

Several theoretical strategies and architectures have also

been proposed with graphene membranes to amplify the high-

resolution identiﬁcation of DNA nucleotides and address the ex-

isting challenges. For example, Ouyang et al. proposed that the

position of nanopores has a signiﬁcant eﬀect on their DNA nu-

cleobase detection ability.[157]The current signature read-outs

of each nucleobase are found to be remarkably distinguishable

upon designing a device with a nanopore at the edges of the

metallic graphene nanoribbon membrane. Girdhar et al. intro-

duced a peculiar concept of quantum point contact (QPC), where

the nanopore device is designed with irregular edge-shaped

graphene nanoribbons.[158]This QPC device is reported to ex-

hibit superior electrical sensitivity of DNA to the earlier reported

uniform armchair geometry by tuning the graphene carrier con-

centration. The proposed device can also be modeled by sand-

wiching the solid-state graphene QPC between two dielectrics,

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which will isolate the electrically active graphene layer from the

electrolyte and restrain its mechanical instability. Additionally,

the QPC carrier tunability can be achieved by the application

of simultaneous gate voltage similar to the ﬁeld-eﬀect transistor

(FET) during DNA translocation. However, the above reports did

not include the translocation velocity of DNA, which has been

a key stumbling block to obtaining error-free sequencing using

nanopores.

Another proposed alternative strategy was to use a bilayer

graphene nanopore instead of a monolayer and compute the

probability distribution of electrical signals for DNA nucleotide

identiﬁcation.[159,160]The motivation behind this is that the bi-

layer graphene nanopore is more stable and persistent than the

monolayer counterpart, which changes shape over time. Addi-

tionally, bilayer graphene has more conductance channels due

to the presence of higher electronic density of states compared

to monolayer graphene. In a similar report on graphene bi-

layer nanopore, interlayer conductance (top and bottom layer of

graphene) is present in the bilayer, while DNA nucleotides are

translocated through it.[161]The results revealed that this pro-

posed device gives rise to better identiﬁcation of the individ-

ual DNA nucleotide conductance signals compared to the iso-

lated nanopore. Moreover, the OH group functionalization of

graphene nanopore edges improved the interaction with translo-

cating ssDNA and controlled the dynamical ﬂuctuation of nu-

cleotides. Cuniberti and co-workers theoretically demonstrated

that the speed of DNA passage can be controlled by adding extra

graphene sheets on the top of the parent graphene nanopore.[162]

It should be mentioned that the dimensions of the pore con-

sidered for this study are in accordance with the earlier experi-

mentally reported graphene nanopore for DNA translocation pur-

poses. This report pointed out the reduction of the transmission

signal by the graphene pore as the DNA translocates through

the nanopore with a diameter similar to the experimental report.

Further, a hybrid nanopore with a so-called graphene nano-road

embedded in a hexagonal boron nitride (h-BN) sheet was devel-

oped, and the possibility of controlling the local current pathway

through the proposed device was demonstrated by applying a spe-

ciﬁc gate voltage.[163]

In recent years, the discovery of more solid-state 2D materials

beyond graphene has opened up a new possibility to exploit the

ﬁeld of DNA sequencing by improving the high precision detec-

tion eﬃciency of solid-state nanopores. Some studies have also

been reported on DNA sequencing as an alternative to graphene

nanopore. Prasongkit et al. investigated the potential of a topo-

logically defective graphene nanopore for DNA sequencing with

the QM/MM approach and observed that the nucleotide conduc-

tance is associated with transport across speciﬁc molecular states

near the Fermi level and their coupling with the pore edges.[164]

Pathak and co-workers investigated the single-atom-thick solid-

state 2D graphdiyne nanopore setup for DNA sequencing. From

the conductance and current sensitivity analysis, it is clear that

all four nucleotides can be distinctly identiﬁed improved sensitiv-

ity compared to graphene nanopore.[165]Semi-metallic graphene

and metallic borophene nanopores are considered for the study

to compare the preference of semi-metallic and metallic 2D ten-

dency towards single nucleotide identiﬁcation.[166]It is noted that

the borophene nanopore with robust condition channels across

the membrane doesn’t get aﬀected by the presence of nucleotide

inside the nanopore, which is evident from their current-voltage

characteristics.

Recently, MXene, a new family of 2D transitional metal car-

bides, has been poised as a potential candidate for nanopore-

based DNA sequencing with a transverse tunneling current

method due to relatively easy fabrication, which can resolve pore

clogging issues and versatile electronic properties. Mojtabavi and

co-workers have experimentally demonstrated the applicability of

Ti2CTxand Ti3C2TxMXene nanopore membranes toward sin-

gle DNA nucleotide identiﬁcation and found that, unlike other

2D materials, the MXene membranes have low noise levels and

less ionic current leakage compared to standard graphene and

MoS2nanopores.[109 ]Scheicher and co-workers have employed

combined MD simulation and DFT-NEGF analysis for single nu-

cleotide identiﬁcation with OH functionalized Ti2C MXenes, i.e.,

Ti2C(OH)2nanopore, as shown in Figure 13d.[153]The Ti2C(OH)2

nanopore was reported to help in the stabilization of DNA nu-

cleotides as DNA passes through the nanopore and allowed con-

ductance measurements by sweeping the gate voltage at several

orientations. The OH functionalized pore allowed better interac-

tion and distinct transverse conductance properties at the Fermi

level.

Several experimental studies have also been reported in par-

allel to validate the feasibility of the above-discussed theoreti-

cal predictions of solid-state nanopores for DNA sequencing.

In 2012, Lieber and co-workers investigated a silicon nanowire

ﬁeld-eﬀect transistor (FET) for single molecule DNA sequencing

utilizing integrated silicon nanowire-nanopore FET on a Si3N4

membrane, where the signal is generated through the localized

change of electric potential during DNA translocation across the

nanopore.[167]In contrast to a Si3N4membrane, the graphene

membrane acts as a conductor and thus allows transversal cur-

rent ﬂow through the membrane. Therefore, the nanopore FET

can be extended to atomically thin graphene membranes, which

have the potential to achieve single-base resolution. In 2013,

Traversi et al. fabricated the graphene nanoribbon of ≈100 nm

width for the nanopore drilling on the top of the Si3N4mem-

brane (Figure 14a).[168]Here, simultaneously, both the longitudi-

nal ionic current blockage across the nanopore with the translo-

cation of DNA molecules and the transverse tunneling current

across the graphene device are measured (Figure 14b).

In a similar report with a solid-state nanopore embedded with

a graphene nanoribbon (GNR) transistor, Puster et al. examined

the GNR nanopore composed of 50 nm (width) and 600 nm

(length) over the Si3N4membrane and noticed a signiﬁcant re-

lationship between the ionic current and nanoribbon current

due to the capacitive coupling during the DNA translocation,

which is termed as “cross-talk.”[171]In another experimental re-

port, Heerema and co-workers experimentally fabricated 30 nm ×

30 nm square-shaped graphene nanoribbon (top layer was cov-

ered with h-BN membrane) with a nanopore of 5 nm diameter

and investigated the possibility of DNA sensing with in-plane

currents by using a custom-made diﬀerential current ampliﬁer

that discriminates between the capacitive current signals and

the resistive response in graphene.[169]An important advantage

of this approach is the transverse current magnitude through

graphene nanoribbons is relatively large (μA), which facilitates

the measurements at much higher bandwidths, which opens up

the possibility to sequence DNA information at the translocation

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Figure 14. Experimental studies of electrical detection of DNA nucleobases by 2D solid-state nanopore devices with the transverse tunneling current

approach. a) Schematic representation of the device setup. A DNA molecule is translocating through a nanopore fabricated in a graphene nanoribbon

in-plane current (through a graphene nanoribbon) and ionic current (through the nanopore) signals are measured, while a DNA molecule translocates

MoS2FET-nanopore device with selective insulation around the electrodes avoids cross-talk and insulates the metal from the electrolyte while keeping

speed that is typically observed with solid-state nanopores. From

the above experimental studies of graphene nanopore for DNA

sequencing with the transverse tunneling current method, it is

noted that sculpting graphene nanoribbon can open a bandgap,

which is essential to become ambipolar FET. However, the fab-

rication of nanoribbons with smaller nanopore is challenging.

Moreover, the measurement of simultaneous ionic current and

the transverse sheet current through the graphene membrane

led to capacitive cross-talk, which has severely restricted the in-

dustrial scale realization (Figure 14c).[171]

Several other 2D materials have been computationally ex-

plored for the transverse detection of DNA nucleotides. How-

ever, there are limited experimental studies. In 2014, Feng and

co-workers experimentally established that MoS2nanopore can

complement graphene nanopore membranes and oﬀer poten-

tially better performance in transverse detection. The major ad-

vantages are that it doesn’t need any additional surface treatment

to avoid strong surface interaction, unlike graphene, and oﬀers

an improved sensitivity compared to Si3N4nanopores (SNR >

10).[108]Recently, the translocating DNA nucleotides have also

been detected from the readouts of both the ionic and transverse

current modulation through the MoS2nanoribbon, as shown in

Figure 14d.[170]Here, the authors implemented single-layer MoS2

nanopore-based FET (500 nm ×2μm) on top of an aperture in a

Si3N4membrane. The area selective insulation around the elec-

trodes is incorporated to avoid cross-talk and insulate the metal

from the electrolyte while keeping the ribbon exposed to the sol-

vent. In comparison to graphene FET, the MoS2is associated with

a reduction of low-frequency 1/fnoise with a decrease in salt con-

centration.

Recently, Pathak and co-workers explored the potential of ma-

chine learning in the ﬁeld of nanopore technology with the trans-

verse tunneling current method. By employing the supervised

ML regression tools with the quantum transport method, sin-

gle DNA nucleotide prediction is made possible using a model

monolayer gold nanopore (Figure 15a).[172]Training the XGBR

model with single nucleotide transmission data sets, including

electronic and chemical features, transmission spectra of other

nucleotides are predicted. Interestingly, dCMP nucleotide data

sets were successful in predicting all three other nucleotides

(dTMP, dGMP, and dAMP). On comparing both the DFT-NEGF

and ML-predicted transmission sensitivity values, it is observed

that though the sensitivity order of the nucleotides decreases,

their qualitative trend remains the same (Figure 15b).[173]More-

over, the local and global interpretable analysis by the SHapley

Additive exPlanations (SHAP) method shed light on the complex

relationship between the molecular properties of nucleotides and

their transmission function. Except for the energy feature, the

electronegative, ionization energies, and electron aﬃnity were

the dominant features in the prediction of the output. Notably, the

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Figure 15. Combined machine learning and transverse tunneling approach with nanopore for DNA sequencing. a) An interpretable ML prediction of the

transmission functions of single DNA nucleotides. Trained with the dCMP nucleotide data set, the XGBoost regression model predicted dAMP, dGMP,

American Chemical Society.

successful prediction of nucleotide conﬁguration was restricted

to only the most stable conﬁgurations.

In a follow-up study, the authors developed an artiﬁcial intel-

ligent nanopore with combined machine learning and quantum

transport method, where four diﬀerent XGBR models were built

that are trained on dAMP, dTMP, dGMP, and dCMP transmis-

sion function data of graphene nanopore (Figure 15c).[173]The

trained models were able to predict not only the most stable

conﬁguration but also all the considered rotated orientations in-

side the nanogap. Further, the overlapped signals of all four nu-

cleotides are classiﬁed with the support vector machine (SVM)

classiﬁer with an accuracy of up to 96.01%. Both combinations

of regression and classiﬁcation methods boosted the eﬃciency of

the graphene nanopore toward the high-precision identiﬁcation

of DNA nucleotides. Table 3below summarizes year-wise impor-

tant contributions from the experimental and computational re-

ports on nanopore-based DNA sequencing.

3.2.2. Solid-State Nanogaps for DNA Sequencing

Solid-state nanogap device has emerged as another wonderful al-

ternative technique for DNA sequencing with several advantages,

such as enabling single-molecule resolution owing to inherent

superiorities of electrical transduction methods, good compati-

bility with advanced ﬁeld-eﬀect transistor technology, and pro-

viding a suﬃcient level of sensitivity with long readability and

ﬁdelity.[49,174–176]Solid-state nanogap devices are made up of two

electrodes separated by a distance in the magnitude of a nanome-

ter or sub-nanometer. The transverse tunneling current is moni-

tored from the left electrode to the right electrode across the gap

for single-molecule identiﬁcations.

When a DNA molecule strand traverses through the nanogap,

the electronic structure, chemical composition, and induced

dipole-moment of each nucleotide located inside the nanogap

modulate the electron tunneling current of the device with dis-

tinct magnitude and time duration. That modulation in the

tunneling current allows the identiﬁcation of DNA nucleotides

with good sensitivity. Also, it is important to note that, unlike

nanopore devices, the interaction can be controlled for molecules

of diﬀerent sizes by relatively easy and ﬂexible adjustment of the

gap size to detect molecules more eﬀectively, as some molecules

have varying interactions with the nanoelectrodes depending on

the size of the gaps.[18]In comparison to other nano-devices, a

big advantage of nanogap detection systems is that they detect

the biomolecule at a single molecular level, making it highly

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Tabl e 3 . Year-wise documentation of DFT-NEGF and experimental studies reported on solid-state nanopore-based DNA sequencing.

Materials Nanopore Molecular carrier Study Comments Year Refs.

Graphene Armchair graphene nanoribbon nanopore

with diameter 1.45 nm

Single nucleobase DFT +NEGF Modulation of conductance and

current-voltage (I-V) spectra can identify

DNA bases

2010 [150]

Graphene Hydrogen functionalized nanopore

electrodes with diameter 1.1 and 1.3 nm

Single-stranded DNA MD +NEGF Hydrogen termination increases the

conductance by three orders than the bare

nanopore

2011 [151]

Graphene Graphene nanoribbon-based nanopore

with a diameter of 1.5 nm

Single nucleobase DFT +NEGF Distinguishability of the nanopore current

depends on the nanopore position on the

device

2011 [157]

Graphene graphene nanoribbon with zigzag edges

(ZGNR) and nanopore (≈1.2 nm)

Single nucleotide DFT +NEGF Transverse current in the microampere range

is achieved with an edge termination

2012 [148]

Silicon Silicon nanowire-nanopore (2 nm) Double-stranded DNA Experimental Simultaneous ionic current and FET

conductance measured

2012 [167]

Graphene Graphene pore with four sheets over the

membrane (≈1.6 nm)

Double-stranded DNA MD+DFT-NEGF The addition of extra graphene sheets

around the graphene nanopore reduced

the DNA translocation speed

2013 [162]

Graphene Bilayer nanopore with a diameter (of 1.2

and 3 nm)

Single nucleotides MD+DFT-NEGF A distinct change in the current signal for

bilayer nanopore noted than monolayer

during nucleobase detection

2014 [159]

Graphene Nanopore

(0.5 nm)

Natural and epigenetic

nucleobase

DFT +NEGF Graphene nanopore can detect both natural

and methylated nucleotides

2014 [154]

MoS2MoS2is suspended on a Si3N4(2-20 nm) Double-stranded DNA Experimental Unlike graphene, there is no strong surface

interaction with DNA SNR higher than 10.

2014 [108]

Graphene Nanopore diameter (1.3 nm) Single nucleobase QM/MM Charge ﬂuctuations in nucleotides are

responsible for the conductance

modulation

2015 [155]

Graphene Quantum point contact-edged nanopore

with a diameter (1.6 nm)

Single-stranded DNA MD+NEGF Vertically stretched stepwise translocation of

ss-DNA improved detection ability with

reduced noise

2015 [156]

Graphene Graphene nanopore over Si3N4Double-stranded DNA Experimental The cross-talk originating from capacitive

coupling between the ionic current and

device membrane current

2015 [171]

Graphene The OH group terminated graphene

bilayer nanopore with a diameter (2 nm)

Single-stranded DNA MD+DFT-NEGF The OH group improved interaction and

reduced translocation speed with a better

conductance signal

2015 [161]

Graphene and h-BN Graphene with h-BN at top layer

Nanoribbon nanopore diameter (5 nm)

ssDNA Experimental Exceedingly challenging due to low yields in

device fabrication

2018 [169]

Topologically defected

graphene

Hydrogen functionalized nanopore with

diameter (≈1.2 nm)

Single nucleotide MD and DFT +NEGF Conductance is associated with transport

across speciﬁc molecular states near the

Fermi level and their coupling to the pore

2018 [164]

MoS2Nanopore diameter (1.3 nm) Single and

double-stranded

DNA

Experimental Better FET device with a high signal-to-noise

ratio

2019 [170]

Graphdiyne Hydrogen functionalized nanopore with

diameter (≈1.4 nm)

Single nucleotide DFT +NEGF The distinction between purine- and

pyrimidine-type nucleobases

2021 [165]

Graphene and Borophene Hydrogen functionalized graphene and

borophene nanopores with diameters

≈1.3 and ≈1.2 nm, respectively

Single nucleotide DFT +NEGF Metallic borophene nanopore is showing

poor distinguishability

2021 [166]

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Figure 16. Theoretical studies of solid-state nanogaps-based DNA sequencing with transverse tunneling current approach. a) Schematic representation

of the translocation of single DNA nucleotides through a graphene nanogap (left) and illustration of transverse conductance calculation with numerical

c) current variation due to nucleotide rotation about the y-axis and translation along the z-axis at a bias of 1 V;[179]Reproduced with permission.[179 ]

of DNA sequencing through phosphorene nanogap device via transverse current approach and current sensitivity of four nucleotides calculated at the

sensitive. Furthermore, the size of the gaps can be adjusted rela-

tively easily, allowing the system to detect molecules of diﬀerent

sizes.

Lagerkvist et al. reported the modality of nanopore sequenc-

ing by exploiting the quantum mechanical phenomenon of

electron tunneling, allowing the identiﬁcation of individual

nucleobases.[177]Postma proposed the concept of nanogap gap-

based DNA sequencing by using the numerical simulation con-

cept with graphene electrodes (Figure 16a).[178]It is demonstrated

that the single base resolution of DNA nucleotide can be ob-

tained by measuring their electrical conductance signals while

translocating between two graphene electrodes. Scheicher and

co-workers have calculated the transmission function of each nu-

cleotide and transverse tunneling current variation at ﬁnite bias

with the DFT-NEGF approach (Figure 16b).[179]The results show

that the electrode-nucleotide coupling eﬀect is vital for change in

the magnitude of conductance and tunneling current signals, as

shown in Figure 16c. This study with a graphene nanogap device

for DNA sequencing was crucial in addressing the technical prob-

lem of achieving single-base resolution. Additionally, the report

highlighted that the nucleotide with a HOMO peak close to the

Fermi energy of the graphene electrodes can increase the current

magnitude.

Though graphene nanogap results were promising, the low

sensitivity and high translocation speed remained an important

concern. To address these issues of graphene nanogap, the idea of

functionalizing the electrodes with diﬀerent molecules or atoms

other than hydrogen was proposed. Prasongkit et al. functional-

ized the phosphate-group-grabbing guanidinium ion on the right

electrode and a reader-nucleobase (cytosine) on the left electrode

of the graphene nanogap and studied the electronic transport

properties of DNA nucleotides (Figure 16d).[180]Double function-

alization of nanogap edges created a temporary hydrogen bond-

ing that decreased their orientation ﬂuctuations, which further

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stabilized the nucleotide inside the nanogap and improved the

electronic coupling, leading to better distinguishability in the

current–voltage (I–V) characteristics (Figure 16e). Moreover, the

improved interactions between nucleotides and functionalized

electrodes can slow down the high translocation speed of DNA

and provide more time for each nucleotide measurement. Com-

pared to non-functionalized graphene electrodes, the function-

alized electrode shifts the molecular orbitals closer to the Fermi

level, delivering better and unambiguous electrical recognition of

nucleobase entities.

However, the functionalization of nanoelectrodes with molec-

ular groups will undoubtedly add another layer of complexity to

the fabrication process. Further, eﬀorts have also been made to

enhance the DNA recognition sensitivity of graphene nanogap

devices through nitrogen edge functionalization.[182]On compar-

ison of both hydrogen and nitrogen functionalization graphene

electrodes, it is noticed that nitrogen-functionalized electrodes

lead to increased sensitivity by up to 3 orders of magnitude with-

out aﬀecting the qualitative trend. The higher sensitivity could

be due to strong coupling and the contribution of nitrogen-

functionalized edges to resonance states close to the Fermi level,

resulting in increased conductance.

Additionally, De et al. performed a QM/MM coupled with the

NEGF study with a nitrogen-functionalized graphene nanogap

setup and demonstrated that water plays a major role in the elec-

tronic transport of the nanogap tunneling device (Figure 16f).[181]

The local current analysis showed the solvent molecule pro-

vides localized electronic states that signiﬁcantly increase the

conductance in nitrogen-functionalized nanogap devices. On

the other hand, Jung et al. also studied the potential of het-

eroatom substitution to control the dynamics of DNA translo-

cations with MD-DFT-based study on the pristine carbon nan-

otube (CNT) and nitrogen-doped carbon nanotube (capCNT)

electrodes.[183]It is observed the nitrogen doping of capCNTs

stabilized nucleotide conﬁguration by the ‘edge on’ interaction

rather than the original ‘face-on’ type interaction in the pristine

CNT electrodes. Moreover, the ssDNA translocation and confor-

mations were found to be dominated by the 𝜋–𝜋interactions of

nucleobases-CNT caps and nucleotide dipole moments-external

electric ﬁeld interaction. The capCNT facilitated the entrance

of ssDNA by dominant ‘edge on’ conformation and control of

DNA translocation speed by establishing hydrogen bonds be-

tween the N dopant atoms and nucleotides by increasing the

translocation time by ≈300%. Instead of doping, when the left

electrode of the CNT gap is functionalized with the guanine

nucleobase and attempted to detect with the quantum trans-

port method, each nucleotide orientation is stabilized by forming

temporal hydrogen bonding with the functionalized molecule.

The continuous molecular orbitals at speciﬁc transmission peaks

represent an improved coupling eﬀect with the CNT, which

is reﬂected in the distinctive electrical current signal for each

nucleotide.[184]

Fyta and co-workers investigated the diamondoid-

functionalized Au (111) electrodes for the detection of diﬀerent

nucleotides through quantum tunneling measurements and

noted that the sensitivity of the device at diﬀerent gate volt-

ages depends upon the coupling or decoupling eﬀect of the

functionalized electrodes with the targeted nucleotide, which is

conﬁrmed from the presence or absence of underlying local cur-

rent and scatter transmission channel.[185]In another report with

the ﬁrst principle study, Zou et al. studied sulfur-functionalized

gold electrodes and the eﬀect of nucleotide orientation inside

the nanogap towards single nucleotide identiﬁcation.[186]The

transport properties of nucleotides increased due to better inter-

action with the terminated sulfur atoms. The report highlighted

that sulfur atom functionalization substantially enhanced the

distinguishability of nucleotides compared to the bare gold

electrodes.

Beyond graphene, the applicability of the other 2D solid-

state materials towards nanogap-based DNA sequencing with

the transverse tunneling method has also been demonstrated.

Pathak and co-workers reported a study on phosphorene

nanogap for single-molecule DNA sequencing, as shown in

Figure 16g.[176]Phosphorene oﬀers remarkable unique proper-

ties such as biocompatibility and experimentally proven stabil-

ity in both air and water. From the DFT-NEGF study, it is ob-

served that phosphorene can eﬀectively identify all nucleotides

at both low (0.2 V) and high (1.6 V) voltages.[187]The authors

also tried to explore the potential of BC3nanoelectrodes in the

identiﬁcation of all four DNA nucleotides.[188]The results suggest

that unique identiﬁcation of all four nucleotides can be achieved

in the bias region 0.3–0.4 V. A comparative study of the interac-

tion of poly-guanine (dGMP dimer) with BC3and graphene edges

shows lower interaction energy of BC3electrodes in comparison

to graphene electrodes. Additionally, BC3is polar compared to

graphene, thus providing better electronic coupling with the po-

larized nanogap edges that resulted in distinct current readouts

by one order of diﬀerence, even at low bias.

Apart from exploring alternative materials to graphene, Mit-

tal et al. theoretically introduced the labeling eﬀect to amplify

the DNA recognition sensitivity.[189]The simplest amino acid,

glycine, is tagged to the 5’ position of all four nucleotides,

and their quantum transport properties are investigated with

graphene nanogap electrodes. The results show improved con-

ductance sensitivity, which enables better resolution. In addition,

labeling of nucleotides has the potential to control the transloca-

tion speed and dynamic ﬂuctuations while translocating through

the nanogap. However, except for the dTMP nucleotide, the elec-

tric current of all other nucleotides is found to be similar. Apart

from glycine, three other amino acids: (basic) arginine, (neutral)

tyrosine, and aspartic acid (acidic), have also been studied for la-

beling of DNA nucleotides, and results show that arginine and

glycine labels are promising to achieve distinct current signa-

tures for nucleotide identiﬁcation.[190]

The topologically defected graphene (octagons and a pair

of pentagon sp2-hybridized carbon rings embedded in a pure

graphene nanosheet) electrodes are also reported for DNA se-

quencing application with DFT-NEGF approach and found to

exhibit high order distinguishability in their current character-

istics at a wide bias range.[191]Rocha and co-workers demon-

strated the potential of diﬀerent phases of MoS2electrodes for

nanogap-based DNA sequencing. The results indicate that the

1T’-phase of MoS2is selective and sensitive toward nucleobase

identiﬁcation.[192]The eﬀect of metallic 2D (single atomic thick-

ness) borophene nanogap is also explored to check the via-

bility for nucleotide identiﬁcation.[193]The nanogap results of

borophene are observed to be better than their nanopore coun-

terpart.

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Figure 17. Experimental identiﬁcation of DNA nucleotides through nanogap device with transverse tunneling method. a) Illustration of Pt electrode-

based tunneling junction located at the bottom of a nanopore, where DNA electrophoretically translocated through the nanopore and the tunneling

mental setup of nanosized bare gold gap electrodes for detection of a nucleotide with quantum tunneling approach and chemical modiﬁcation strategy

Other than traditional architectures, a new architecture called

“semi/hybrid” nanogap with graphene membrane is also pro-

posed to distinguish DNA nucleobases.[194]The semi/hybrid

nanogap has resulted in better conductance and current sensi-

tivity for single nucleotide identiﬁcation. Moreover, this architec-

ture could be expected to reserve the advantage of both nanopore

and nanogap systems that will make this type of device more ro-

bust.

However, all these proof-of-concept studies with MD and DFT-

NEGF methods look promising. Analyzing experimental results

is extremely important to understand the diﬃculties and eﬀec-

tiveness of the demonstrated strategies for real sequencing ap-

plications. In 2010, Tsutsui et al. experimentally demonstrated

the ﬁrst evidence of trapping and identiﬁcation of single nu-

cleotides between tunneling electrodes.[195]However, this proof

of principle only restricted single nucleotides in the presence

of an STM environment. To develop a potentially much faster

tunneling current-based device, a nanopore combined tunneling

junction can help in both unfolding the DNA strand and precise

measuring of variations in the tunneling currents. In this regard,

Ivanov et al. experimentally reported nanopore-integrated Pt elec-

trodes and calculated concurrent tunneling and ionic current sig-

nals, as shown in Figure 17a.[196]The DNA with a translocation

speed greater than 1 ms was able to detect both ionic and tunnel-

ing current signals. Additionally, they also plotted the I−Vcurves

for the Pt nanogap devices with diﬀerent gap sizes and observed

a nonlinear relationship between current and voltage, as shown

in Figure 17b.

Further, Tsutsui et al. experimentally demonstrated single-

molecule detection using a nucleotide-sized nanoelectrode

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Figure 18. Machine learning studies of solid-state nanogap for DNA sequencing with transverse tunneling method. a) Detection of modiﬁed RNA

nucleotides with recognition tunneling (RT) current with combined scanning tunneling microscopy (STM) and ML techniques. The SVM, a supervised

ML method, was able to identify the individual RNA nucleotides and distinguish them from their DNA counterparts with a reasonably high accuracy of

with a high degree of precision via a one-electric current pulse method using Random Forest and positive unlabeled classiﬁcation (PUC) method;[205]

integrated with a solid-state nanopore device to identify DNA

oligomers.[199]Fanget et al. experimentally reported a high-

throughput fabrication of sub-10 nm nanogap electrodes com-

bined with solid-state nanopores that allowed concomitant

tunneling and ionic current detection of translocating DNA

molecules (Figure 17c,d).[197]Pang et al. fabricated a layered ﬁxed

tunneling junction with functionalized molecules to detect DNA

nucleotides by ‘recognition tunneling’ measurements.[200]Patel

and co-workers experimentally demonstrated the translocation

of double-stranded DNA through a graphene nanogap device.[201]

The conductance and translocation signatures are found to be dif-

ferent than other solid-state nanopores owing to the unique and

ﬂexible geometry of nanogap. However, the accurate identiﬁca-

tion of nucleotides was seriously aﬀected by the similar tunneling

molecular conductance among the nucleotides.

Furuhata et al. experimentally proposed a chemical strategy

that ampliﬁed the diﬀerence in molecular conductance among

four nucleotides by replacing the 2′-deoxyadenosine (dA) with an

analog 7-deaza dA (dzdA) (Figure 17e).[198]This study suggested

that the nucleotide with a HOMO level closer to the electrodes

has higher conductivity compared to other nucleotides. A quan-

tum tunneling probe based on double-barrelled capillary nano-

electrodes has also been reported to identify single molecules at

low concentrations.[202]

Besides computational and experimental studies, there have

been reports on the application of the ML method, which can

address the characteristic challenges of nanogap-based DNA se-

quencing. The overlap of tunneling conductance signals due to

similarity in the shape/size of nucleotides and frontier molecular

orbital is a major problem in nanogap-based DNA sequencing.

Lindsay and co-workers ﬁrst reported the use of ML methods with

the recognition tunneling (RT) current method to identify sin-

gle protein molecules.[203]The single amino acid molecule can

bind to the recognition molecules at diﬀerent orientations when

trapped between two electrodes. In this case, the support vector

machine (SVM) algorithm helped to distinguish between the sets

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Tabl e 4 . Year-wise documentation of DFT-NEGF and experimental studies reported on solid-state nanogaps for DNA sequencing.

Materials Nanodevice (gap width) Molecular carrier Study Comments Year Refs.

Gold Nanogap

(≈1.0 nm)

Nucleotides dissolved in

Milli-Q solution

Experimental Tunneling current depends on the

electrode distance and the

nucleotide-electrode coupling

2010 [195]

Graphene Nanogap

(1.6 nm)

ssDNA DFT +NEGF Signiﬁcant diﬀerences in the

conductance spectra with

single-base resolution

2010 [178]

Gold (Au) electrodes embedded in

nanopore(SiO2) consisting a

multilayer junction(SiO2/Au/SiO2)

Electrode-embedded

nanopore

(sub-nanometer

electrode gap with a

15 nm sized pore)

DNA Oligomer Experimental Electrode-embedded in-plane

nanopore oﬀers label-free

electrical identiﬁcation of

nucleobases in a DNA oligomer

2011 [199]

Graphene Nanogap

(1.47 nm)

Single nucleotide DFT +NEGF Hydrogen-terminated edges

improved tunneling current

variation by several orders of all

four nucleotides

2011 [179]

Doule-functionalized graphene

electrodes

Nanogap

(2.38 nm)

Single nucleotide DFT +NEGF Temporal hydrogen bonds and

electronic coupling enhanced

the diﬀerence in conductance

and current signal of

nucleotides

2013 [180]

Pd nanowire with Si3N4window Nanogap

(1.8 and 2.0 nm)

Solution of single

nucleotides

Experimental When functionalized with

recognition, molecules can

capture individual DNA

nucleotides

2014 [200]

Graphene Nanogap

(1.38 nm)

Nucleobase DFT +NEGF Nitrogen functionalization

boosted the sensing ability of

the device by increasing the

transverse conductance of the

device

2016 [182]

Diamondoid-functionalized gold (111) Nanogap

(1.9 nm)

Natural, mutated, and

epigenetic nucleotide

DFT +NEGF Diﬀerent nucleobases and their

modiﬁcations can be identiﬁed

by quantum tunneling

measurements across the

electrodes

2016 [185]

Graphene electrodes placed above the

SiO2substrate

Nanogap

(−)

Double-stranded DNA Experimental Graphene-DNA interaction

important for DNA

translocation time

2017 [201]

Phosphorene Nanogap

(1.45 nm)

Single nucleotide DFT +NEGF From the I–V signals, all four

nucleotides can be identiﬁed.

2018 [187]

(Continued)

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Tabl e 4 . (Continued)

Materials Nanodevice (gap width) Molecular carrier Study Comments Year Refs.

Nitrogen-capped carbon nanotube

(CNT)

Nanogap

(1.20 nm)

ssDNA MD and DFT +NEGF Controlled translocation of ssDNA

strand due to formation of

hydrogen bond between the

N-dopant atom of CNT and

nucleobases

2017 [183]

Gold (111) Nanogap

(−)

Natural and epigenetic

nucleotides

MD and DFT +NEGF The presence of water does not

hinder the detection of DNA

nucleotides

2019 [206]

Graphene Nanogap

(1.90 nm)

Single nucleotide MD and DFT +NEGF Water plays a major role in

electronic transport and

increases the conductance of

the nanogap device

2019 [181]

BC3Nanogap

(1.26 nm)

Single nucleotide DFT +NEGF The lower interaction energy of

BC3electrodes than graphene

electrodes with DNA

nucleotides

2020 [188]

Functionalized carbon nanotube (CNT) Nanogap

(1.90 nm)

Single nucleotide DFT +NEGF Current traces diﬀer by at least 1

order of current magnitude for

all the four target nucleotides

2020 [184]

MoS2Nanogap

(1.35 nm)

Single nucleotide DFT +NEGF 1T′-phase MoS2sensitive and

towards DNA sequencing

2020 [192]

Defected Graphene Nanogap

(1.47 nm)

Single nucleotide DFT +NEGF Tunneling electric current signals

that vary by more than one

order of magnitude electric

current

2021 [191]

Gold (Au) tip of two coplanar carbon

electrode

Nanogap

(<5 nm)

Mononucleotides, DNA

oligomers, and proteins

Experimental 5-orders of magnitude increase in

event detection rates and

sub-femtomolar sensitivity

2021 [202]

Graphene Nanogap

(1.56 nm)

Labeled nucleotides DFT +NEGF Labeling of nucleotides ampliﬁes

the conductance sensitivity and

selectivity

2022 [189]

Gold Nanogap

(1.89 nm)

Nucleobase DFT +NEGF Sulfur-functionalized gold

electrodes increase

distinguishability than bare gold

electrodes

2022 [186]

Borophene Nanogap

(1.26 nm)

Single nucleotide DFT +NEGF The current was in the

picoampere (pA) range, which

was fairly higher than the

electrical background noise

2022 [193]

(Continued)

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Tabl e 4 . (Continued)

Materials Nanodevice (gap width) Molecular carrier Study Comments Year Refs.

Graphene Semi/hybrid nanogap

(1.30 nm)

Single nucleobase DFT +NEGF Good conductance and current

sensitivity

2022 [194]

Graphene Nanogap

(1.56 nm)

Aspartic acid, arginine, and

tyrosine labeled single

nucleotides

DFT +NEGF Arginine and glycine amino acids

are better for labeling

2023 [190]

of electronic ‘ﬁngerprints’ associated with each binding of that

amino acid molecule. In a similar report, the modiﬁed RNA nu-

cleotides are classiﬁed by the SVM classiﬁer model to the recogni-

tion tunneling current signal of molecules (Figure 18a).[204]The

classiﬁcation of recognition tunneling current signal of DNA nu-

cleotides with the support vector classiﬁer (SVC) is noted to have

signiﬁcant (>90%) accuracy.

In another experimental report, Taniguchi et al. classiﬁed the

DNA nucleotides from the collected current signals with the ran-

dom forest classiﬁer and the positive unlabeled classiﬁcation,

as shown in Figure 18b,c.[205]The authors also studied the ef-

fect of inter-electrode distance on the electronic signal of the nu-

cleotides. With the increase of the gap between the electrodes,

the electrode-nucleotide coupling decreases, which is reﬂected in

their signature electric signals. The ML model classiﬁcation ac-

curacy (F1 score) also dropped signiﬁcantly. Hence, models are

able to extrapolate the coupling information between the elec-

trode and nucleotide. Moreover, the experimental fabrication of

solid-state nanoelectrodes is easier and more economical than

nanopores. Thus, from computational and experimental studies,

we assume that the solid-state nanogap could be realized for DNA

in the future. In Table 4, we have highlighted important reports

on DNA sequencing with the solid-state nanogap device.

3.2.3. Solid-State Nanochannels

Among the NGS methods, nanopore sequencing is the most

promising technique for achieving atomic-scale resolution with

high ﬁdelity and long readability. However, the technique does

not address several issues that need to be resolved for the ex-

perimental purpose, such as controlling DNA translocation rate,

suppression in stochastic nucleobase motions, and controlling

orientational ﬂuctuations of individual nucleobases.[25]Thus, re-

searchers are more focused on ﬁnding potential alternatives for

nanopore techniques capable of resolving the existing challenges.

Nanochannel is found to be a promising alternative to

nanopore sequencing. Here, the advantage is that the technique

can be used to detect long chains of DNA with controlled ori-

entational ﬂuctuations, unlike solid-state nanopore or nanogap

architecture.[207–209]The identiﬁcation process of nanochannel

devices relies on the phenomenon of adsorption at the inter-

face between DNA nucleotides and nano-scale solid-state mate-

rials. In this technique, the DNA strand is dragged through a

nanochannel membrane, and the detector reads the correspond-

ing change in the electric current. On account of diﬀerent chem-

ical and electronic properties, the strength of the interactions

varies for diﬀerent nucleobases.[144,207]The unique adsorption

properties of each molecule cause distinct changes in the elec-

tric current signal, enabling sequencing to occur.

Similarly, nanoribbons (1D nanochannel) are also potential

candidates for ultrafast DNA sequencing. In nanoribbon-based

DNA sequencing, the identiﬁcation of individual nucleobases

is achieved by the distinction in the transmission dip. The lo-

cation of the dips is determined by the alteration in conduc-

tance when a nucleobase interacts with a nanoribbon. According

to Landauer’s theory, the ﬂow of electrons through a molecule

occurs through elastic scattering, which involves a variety of

paths. This results in either constructive interference, where

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Figure 19. Graphene nanochannel-based DNA sequencing with the transverse tunneling current approach. a) DNA base stacking on graphene

transmission is ampliﬁed through resonant enhancement (Breit-

Weigner resonance), or destructive interference that reduces

transmission (Fano resonance).[210,211]The Fano resonance is

caused by the interference between paths formed between the

ground state and a discrete excited state, as well as the ground

state and the continuum of excited states. This Fano resonance

allows the identiﬁcation of targeted molecules through a nanorib-

bon device.

In 2011, Min et al. ﬁrst utilized the graphene nanochan-

nel technique to electrically distinguish the DNA nucleobases

(Figure 19a).[212]Based on molecular Fano resonance, which

acts as a signature of each individual, the identiﬁcation of a

single molecule is found to be feasible. A structural examina-

tion revealed that each base formed a stable stacked conﬁg-

uration throughout the entire transit. This stacking is much

stronger than the H–𝜋interactions between water molecules and

graphene. Thus, the stacked structures remain unaltered by sol-

vent eﬀects. In this regard, the proposed nanochannel technique

is found to be promising for experimental purposes.

In another report, the authors introduced two-dimensional

molecular electronics spectroscopy for DNA and cancerous

methylated DNA nucleobase detection with armchair graphene

nanoribbons at atomic resolution (Figure 19b).[213]As the DNA

molecule passes through the nanoribbon, each nucleobase is

stacked through 𝜋–𝜋interactions and exhibits diﬀerent trans-

port characteristics depending on the spatial orientations and

molecular orbital energy levels. The dip in transmission origi-

nates due to Fano resonance, and the resultant 2D conductance

map at bias voltages (Vb) and electron channel energy (E-EF) allow

each nucleobase to be diﬀerentiated at spatiotemporal resolution.

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Figure 20. Solid-state 2D materials-based nanoribbons for DNA sequencing utilizing transverse tunneling current approach. a) Schematic of armchair

ican Chemical Society and b) schematic of atomically thin black phosphorene nanoribbon for DNA sequencing and related zero-bias transmission

Graphene nanoribbons are of two types, armchair and zigzag,

and results suggest that zigzag nanoribbons may be more bene-

ﬁcial for DNA sequencing.[148,214]Based on complex graphene-

DNA binding, various mechanisms have been reported, such

as electrostatic, van der Waals, 𝜋–𝜋stacking, and hydropho-

bic interactions.[215]The 𝜋–𝜋stacking interaction between DNA

nucleobases and graphene nanoribbons allows controlled ori-

entational ﬂuctuations, which is important for single molecule

recognition.[215,207]

Furthermore, Ahmed et al. demonstrated that the local

density of states of DNA nucleotides deposited on graphene

can also serve as electronic ﬁngerprints for the recognition

of individual bases in scanning tunneling spectroscopy.[216]

The origin of dips from graphene nanoribbon devices is also

investigated, and it is found that dips originate from the

interference between the molecular orbitals of nucleobases

and graphene nanoribbons.[217]Water-immersed nucleobase-

functionalized graphene nanoribbon is reported for selective

and high-speed detection of DNA nucleotides.[218]Apart from

this, boron (B), nitrogen (N), and sulfur (S) doped graphene

nanoribbons have also been explored for DNA nucleobase

identiﬁcation.[219]B and S-doped graphene nanoribbons act as p-

type semiconductors, and N-doped graphene nanoribbons act as

n-type semiconductors. It is noticed that doping of heteroatoms

in armchair graphene nanoribbon at diﬀerent sites causes dif-

ferent dips in the transmission curve, which in turn leads to

enhanced sensitivity. Comparative analysis of aforementioned

doped nanoribbons reveals doping of B at diﬀerent sites can lead

to more distinct conductance. Rocha and co-workers theoreti-

cally studied the environmental eﬀects of DNA detection on the

graphene surface with a hybrid QM/MM and NEGF study.[220 ]

In the presence of solvent, a signiﬁcant decrease in the trans-

mission of pristine graphene is observed below the Fermi level.

Moreover, the horizontal orientation of ssDNA and dsDNA of-

fers better identiﬁcation by changing the electronic environment

of the graphene surface.

Apart from graphene, other solid-state 2D materials have

also been explored, such as silicene, hexagonal boron nitride

(h-BN), phosphorene, and molybdenum disulﬁde (MoS2)for

nanochannel-based DNA sequencing. The potential of armchair

nanoribbons of diﬀerent materials, namely, silicene, hexago-

nal boron nitride (h-BN), and molybdenum disulﬁde (MoS2),

has been explored in ultrafast DNA sequencing, and it is no-

ticed that edge-modiﬁed h-BN nanoribbon is a promising alter-

native for graphene to achieve single molecule DNA sequenc-

ing (Figure 20a).[221,144]In h-BN nanoribbons, the shift of dip

in transmission as a function of the bias voltage is noticed

to be relatively high, which may be a reason behind the in-

creased sensitivity. Along with nucleobase identiﬁcation, the

silicene nanoribbon has also been found to be promising for

methylation detection based on electric current signals.[222,223]

The epigenetic modiﬁcation of DNA nucleobases is associ-

ated with various forms of cancer. Thereby, methylation de-

tection is crucial for developing a universal cancer screen-

ing test. The current–voltage (I–V) characteristics of silicene

nanoribbon with adsorbed nucleobases reveal that silicene can

identify all eight nucleobases (DNA and methylated DNA) at

two distinct 0.5 and 1.0 V bias voltages, making it a promis-

ing material for DNA sequencing and cancerous methylation

detection.

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Figure 21. Solid-state 2D materials-based nanochannels for DNA sequencing utilizing transverse tunneling current approach. a) Schematic for DNA

sequencing through extended topological line defected graphene nanoribbons and related zero bias transmission function;[225]Reproduced with

Pathak and co-workers further explored the potential of phos-

phorene armchair nanoribbons for individual identiﬁcation of

nucleobases (Figure 20b).[224]Phosphorene has various advanta-

geous properties over graphene, such as hydrophilic surface, tun-

able band gap, large surface-to-volume ratio, and biocompatibil-

ity. On account of molecular orbital coupling between phospho-

rene nanoribbon and nucleobases, a dip in the transmission is

observed, allowing the identiﬁcation of DNA nucleobases at sin-

gle molecule resolution. The authors also studied the extended

line defects-based graphene nanodevice for the identiﬁcation of

DNA nucleobases through conductance measurements, as given

in Figure 21a.[225]Here, the advantage is that extended line de-

fects embedded in a graphene sheet help in precisely controlled

translocation of DNA nucleobase over the nanochannel surface,

promising spatial resolution. The results show strong Fano res-

onance in the transmission function, enabling recognition of all

four DNA nucleobases with good sensitivity and selectivity. In a

very recent study, armchair germanene nanoribbons have been

theoretically explored for sequencing both DNA and RNA nucle-

obases via two-dimensional molecular electronic spectroscopy, as

shown in Figure 21b.[226]Additionally, the germanene nanorib-

bons are also reported to be capable of recognizing cancerous

methylated DNA nucleobases. Here, the advantage is that, com-

pared to gold standard graphene, germanene nanoribbon ex-

hibits more distinct adsorption energy values for diﬀerent nu-

cleobases.

Despite signiﬁcant progress in the ﬁeld, very few studies have

been reported for nanoribbon/nanochannel sequencing. In the

absence of experimental demonstration, the discussion so far in-

cludes only theoretical results. Nanoribbons are potential alterna-

tives to the nanopore/nanogap sequencing technique. However,

to provide more insight into real DNA sequencing, more detailed

theoretical (DFT and MD) and experimental studies are needed.

We believe that in the near future, more studies will be reported

on nanochannel/nanoribbon-based DNA sequencing to ﬁll the

gap between nanopore/nanogap and nanochannel sequencing.

To provide a brief overview of nanochannels/nanoribbons se-

quencing, studies utilizing transverse tunneling current for DNA

sequencing are summarized in Table 5.

3.2.4. Heterostructures

So far, we have discussed nanopore, nanogap, and nanochan-

nel/nanoribbons for DNA sequencing based on transverse cur-

rent measurements. Apart from this, solid-state heterostructures

have also emerged as promising platforms for sequencing DNA.

As previously discussed, graphene has some disadvantages. To

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Tabl e 5 . Year-wise documentation of transverse current studies reported on nanochannels/nanoribbons for DNA sequencing.

Materials Nanodevice Molecular carrier Study Comments Year Refs.

Graphene Armchair ssDNA DFT +NEGF Ultrasensitive characteristic Fano

resonance-driven conductance

2011 [212]

Graphene Armchair DNA nucleobases and

methylated DNA

DFT +NEGF Two-dimensional molecular electronics

spectroscopy is demonstrated

2014 [213]

h-BN, MoS2, Silicene,

Graphene

Armchair DNA nucleobases DFT +NEGF Distinct transmission dips for graphene

and h-BN

2014 [221]

Silicene Zigzag DNA nucleobases DFT +NEGF Guanine is distinguishable from the other

three nucleobases

2018 [223]

Doped graphene nanoribbon Armchair DNA nucleobases DFT +NEGF Doping leads to enhanced features in the

transmission and modiﬁcation of

transport properties

2019 [219]

Graphene Armchair nanoribbon ssDNA and dsDNA QM/MM +NEGF The presence of water decreased

transmission function intensity.

2019 [220]

Phosphorene Armchair DNA nucleobases DFT +NEGF G >A>C≥T 2019 [224]

Extended line defected

graphene nanoribbon

Zigzag DNA nucleobases DFT +NEGF Precisely controlled translocation of DNA

nucleobases

2020 [225]

Silicene Zigzag Methylated nucleobases DFT +NEGF Diﬀerentiation possible of DNA and

methylated DNA nucleobases at the bias

of 0.5 and 1.0 V

2020 [222]

Germanene Armchair DNA/RNA and methylated

nucleobases

DFT+NEGF 2D and 3D conductance maps exhibit

explicitly distinct features

2022 [226]

overcome these drawbacks, several eﬀorts are being made by re-

searchers. It has been shown that the graphene heterostructure

with other solid-state 2D materials can suppress the associated

challenges while preserving the advantages of graphene-based

heterostructures.

Souza et al. proposed a novel single modulation device com-

prising a nanopore formed within a heterostructure composed

of graphene and h-BN for DNA sequencing, as shown in

Figure 22a.[163]The results show that local current is concentrated

in the carbon chain binding nanopore and h-BN part, facilitating

unique current modulation due to the unique dipole moment

of each nucleotide. The study emphasizes that dipole-induced

conduction modulation is a promising approach for sequencing

biomolecules. Later, the device is also found to be capable of de-

tecting hachimoji or unnatural DNA nucleotides.[227]The hachi-

moji nucleobases have low binding aﬃnity to the nanopore, re-

sulting in short resident time during translocation. Moreover,

these diﬀerent artiﬁcial nucleobases in the nanopore cause sig-

niﬁcant variations in the electron transmission properties, lead-

ingtosensitivityupto80%.

Besides this nanopore technology, heterostructures based

on nanogap have also been reported. Shukla et al. studied

the graphene/h-BN heterostructure nanogap architecture for

DNA sequencing based on transverse current measurements

(Figure 22b).[228]The transverse transmission and I–Vcharacter-

istics of the proposed setup are prominent for single nucleotide

sensing. Moreover, graphene edge passivation with h-BN is re-

ported to solve the major bottlenecks of graphene, such as dif-

ﬁculty in controlled engineering, hydrophobic interactions, and

higher noise levels. Ralph and co-workers proposed a 2D hybrid

graphene/h-BN double modulation device with a nanopore em-

bedded in a graphene part and found characteristic ﬁngerprints

for each DNA nucleotide (Figure 22c).[229]In the proposed archi-

tecture, by applying a speciﬁc gate voltage, local current pathways

can be controlled.

Recently, a nanopore structure has been explored embedded in

graphene/h-BN/graphene heterostructure with 3, 5, and 7 h-BN

layers in the central part. The DFT calculations reveal that nu-

cleotides show better interactions with nanopores in h-BN rather

than nanopores in pristine graphene (Figure 22d).[230]In the case

of graphene/h-BN/graphene heterostructure, higher sensitivity

is observed compared to a similar structure consisting of pris-

tine graphene. Though these theoretical reports are promising

for sequencing, no experimental studies have been reported so

far on solid-state heterostructures utilizing the transverse tunnel-

ing current approach.

Solid-state hybrid nanopores resolved the resolution and

single-molecule sensing issues to a great extent. However, there

still exist some daunting challenges. These consist of high leak-

iness of the nanopore structures, uniform fabrication of het-

erostructure nanopore/nanogap, and the controlled orientation

of nucleotides. Besides, the smooth aggregation of functionalized

pores is also very diﬃcult. These issues require further advance-

ment in the ﬁeld to improve the performance and reliability of

solid-state heterostructures. To provide a brief overview of DNA

sequencing with solid-state heterostructures, transverse cur-

rent studies reported with heterostructures are summarized in

Table 6.

So far, we have provided a detailed discussion of ionic and

transverse current methods for DNA sequencing across a broad

range of next-generation architectures, including nanopore,

nanogap, nanochannel/nanoribbon, hybrid nanopores, and het-

erostructures. The merits and demerits of each electrical tech-

nique are thoroughly investigated with both experimental and

theoretical studies. For a better understanding, a summary com-

paring the advantages and disadvantages of the ionic current

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Figure 22. Schematic representation of solid-state heterostructure DNA sequencing devices utilizing transverse tunneling current approach. a) Sin-

blockade and the transverse tunneling current method is shown

in Table 7.

4. Challenges of DNA Sequencing

Though biological nanopores have certain advantages, such as

reproducibility at the atomic level crucial for consistent and re-

liable sequencing, wide pH stability range (pH 2–12) desired

for various experimental conditions, suitable pore sizes facilitat-

ing eﬃcient molecule translocation and amenability to surface

functionalization. However, despite signiﬁcant progress in se-

quencing techniques and the emergence of biological nanopores

in commercial applications, achieving high-throughput and ac-

curate DNA sequencing remains a challenge that needs to

be addressed.[231,232]This includes mechanical fragility of the

lipid bilayer, complex fabrication, breakdown under thermal and

Tabl e 6 . Year-wise documentation of ionic and transverse current studies reported on hybrid/heterostructures for DNA sequencing.

Materials Nanodevice Molecular carrier Study Comments Year Refs.

Graphene/h-BN Nanopore (single

modulation

device)

DNA nucleotides DFT +NEGF Detection of DNA nucleotides based on

current-modulation signature

2017 [163]

Graphene/h-BN Nanogap DNA nucleotides DFT +NEGF Unique identiﬁcation of DNA nucleotides

from current traces within two bias

windows

2017 [228]

Graphene/h-BN Nanopore (double

modulation

device)

DNA nucleotides DFT +NEGF Current modulation control and electrical

readout allow single nucleotides to be

identiﬁed

2019 [229]

Graphene/h-BN Nanopore Hachimoji DNA

nucleobases

DFT +NEGF Signiﬁcant changes in the transmission

function leading to sensitivity in

distinction of up to 80%

2020 [227]

Graphene/h-BN/Graphene Nanopore DNA nucleotides DFT+NEGF Improved sensitivity compared to pure

graphene nanopore

2021 [230]

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Tabl e 7 . Comparison of ionic and transverse current methods for next-generation DNA sequencing.

Aspect Ionic Current Blockade Method (Nanopore Sequencing) Transverse Tunneling Current Method

Principle Detection of changes in ionic current as DNA passes through a

nanopore

Detection of changes in tunneling current as DNA interacts with

electrodes

Detection Method Electrolyte solution with nanopore; electrical signal changes due to

DNA translocation

Solid-state device with electrodes; electrical signal changes due to

DNA-electrode interaction

Read Length Potentially long reads, depending on nanopore size and technology Not typically speciﬁed, but potential for high resolution down to single

nucleotide level

Resolution limited resolution, may struggle with homopolymer regions and base

calling accuracy

High resolution, potentially down to single nucleotide level

Throughput Moderate to high throughput, depending on the number of

nanopores and sequencing speed

Not typically speciﬁed, may be limited by the number of electrodes and

measurement speed

Sample Preparation Relatively simple sample preparation typically involves DNA library

preparation

Sample preparation may involve surface functionalization and

immobilization of DNA

Labeling Requirement Label-free detection; DNA molecules are detected as they pass

through the nanopore

Label-free detection; DNA molecules interact directly with electrodes

Error Rates Prone to errors, including insertions, deletions, and base

misassignments

Error rates may vary but potentially lower due to high-resolution

Commercial Platforms Multiple commercial platforms are available, including Oxford

Nanopore and others

primarily experimental at this stage

Scalability Scalable with multiple nanopores, enabling parallelization for

high-throughput sequencing

Scalable but may require optimization for parallelization

Complexity Relatively straightforward instrumentation and experimental setup More complex instrumentation and experimental setup may be required

Environmental

Sensitivity

Sensitive to environmental factors such as temperature and ionic

strength.

Sensitive to environmental factors such as temperature and humidity.

Potential Applications Whole-genome sequencing, Epigenetics studies, RNA sequencing,

real-time monitoring

High-resolution DNA sequencing, detection of base modiﬁcations,

single-molecule analysis

electrical stress, poor fabrication yield, high translocation rate,

and low sensitivity that need to be solved.

In this direction, enzyme-integrated bionanopores have been

successfully raised as a promising solution.[26,233–236]The key ad-

vantages of enzyme-integrated bionanopore lie in improving the

signal-to-noise ratio, reduction in the translocation kinetics varia-

tion, controlled translocation, and helicase activity helpful in un-

winding dsDNA or DNA-RNA duplexes. Apart from the devel-

opment of enzyme-integrated bionanopores, the above-discussed

NGS architectures: solid-state nanopore, nanogap, nanochannel,

hybrid nanopores, and heterostructures have also been realized

in experimental and theoretical arenas. However, the existing

DNA sequencing methods are still gaining their potential from

the perspective of cost and base-by-base read eﬃciency. Herein,

we have summarized the major challenges of DNA sequencing,

followed by the relevant discussion on how to resolve them.

4.1. Limitations of the Ionic Current Blockade Method

Although several experimental and theoretical studies have been

done on ionic current-based DNA sequencing, none of the stud-

ies demonstrate a device that can detect only one nucleotide

at a particular time. Traditional biological nanopore sequenc-

ing still needs advancement as it cannot detect the single bases

separated by ≈0.4 nm.[25]Biological nanopores having channels

shorter than ≈5 nm yet need to be realized to identify the sin-

gle nucleotide.[25]Currently, the major challenge associated with

nanopore sequencing is controlling the high translocation speed

of DNA.[99,237,238]The high velocity of DNA issues aﬀects DNA

translocation dynamics and widens the statistical data measured

in the sequencing results.[49]As DNA translocates through a

nanopore in an ionic solution under an applied electric ﬁeld,

its dwell time within the nanopore is typically less than one nu-

cleotide per microsecond (1 base μs−1).[118,239]This short dwell

time presents diﬃculties in accurately detecting the ionic-current

signal associated with each single nucleotide using commercially

available ampliﬁers. To ensure satisfactory electric signal record-

ing, the optimal DNA translocation speed within a nanopore

should fall within the range of 1–100 base pairs per millisecond

(1–100 bp ms−1).[240]

Various methods have been utilized in recent years to decrease

the translocation rate of DNA, including the immobilization

of DNA polynucleotides using streptavidin, the creation of a

rotaxane, and the use of enzymes as binding agents.[234–236,241]

The translocation speed of ssDNA in biological nanopores

is slower (≈1ntμS−1) compared to solid-state nanopores

(≈10 nt μS−1). Speciﬁcally, phi29 DNA polymerase is reported

to have decreased translocation velocity by four orders of mag-

nitude with ratcheting DNA during translocation through the

biological nanopore.[242,243]Atomic force microscopy has also

been developed to control the translocation rate.[244,245]Along

with that, various strategies have also been reported to slow

down DNA translocation and improve the temporal resolution of

ionic current-based solid-state nanopores. The optimization of

experimental physical conditions can be achieved by modulating

inﬂuencing factors: electrolyte viscosity,[246–248]temperature,[239]

electrolyte concentration,[249,250]lowering applied bias

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voltage,[246,251]small size/double nanopore,[77,252 ]and surface

charge density of nanopore.[99]

Kowalczyk et al. reported that diﬀerent ions exhibit diﬀerent

binding strengths with the nucleotides.[249]When the counte-

rion size is decreased, the translocation time of DNA molecules

through a solid-state nanopore can increase dramatically. With

LiCl ion solution, the translocation speed of both ssDNA and

dsDNA has been found to be signiﬁcantly reduced. This is be-

cause the smaller size of Li+ions results in stronger interactions

with the negatively charged DNA molecules, slowing down their

movement through the nanopore. This eﬀect can be useful in

controlling the translocation rate of DNA, as it provides a way to

regulate the speed and orientation of the DNA molecule. How-

ever, it can also pose challenges in DNA sequencing applications,

where it is important to maintain a consistent and predictable

translocation rate. Therefore, careful selection of the electrolyte

solution and optimization of the experimental conditions are re-

quired to ensure reliable and accurate DNA sequencing results.

Moreover, Feng et al. reported that a viscosity gradient system

based on room-temperature ionic liquids (RTILs) can control the

dynamics of DNA and slow down DNA translocation through

MoS2nanopore.[247 ]With the introduction of high viscosity, the

nucleotide translocation velocity falls within an optimal speed

(1–50 nt ms−1) while maintaining a high signal-to-noise ratio

(SNR >10).

Another major challenge is the low level of signals in the or-

der of pico-ampere. Low ionic blockage current decreases the ef-

ﬁciency of accurate nucleobase identiﬁcation.[253,254,52,255–257]The

high-quality resolution with a high signal-to-noise ratio can be

achieved by considering the size of solid-state nanopores compa-

rable with DNA nucleotide diameter, and the nanopore thickness

should be within the range of 0.4–0.6 nm. In biological pores, the

major issue is the pore thickness, which is around ≈5 nm, consid-

erably longer, and can easily accommodate more than ≈10–15 nu-

cleotides, resulting in a low signal-to-noise ratio and a high error

rate of base identiﬁcation.[258]In this regard, it is nearly impos-

sible to achieve single-nucleobase resolution with the biological

nanopore. A comparative study of current noise between biologi-

cal and solid-state nanopores suggests that solid-state nanopores

are more trustworthy for a large signal-to-noise ratio compared

to their biological counterparts.[259]

4.2. Limitations of Transverse Current-Based DNA Sequencing

The challenges of the transverse current-based approach can be

broadly classiﬁed into four categories[260]: i) optimization of a

voltage bias and solution conditions, as it is really diﬃcult to pre-

dict exactly the electronic response of the detector with diﬀerent

nucleotides in the solvent conditions, ii) fabrication of the device

enabling each base to assume a reproducible orientation and dis-

tances on the collector probe as minor variations in the orienta-

tions and positions of atoms can have a signiﬁcant impact on tun-

neling current, iii) translocation of DNA must be controlled for

minimum read-out error, and iv) it still remains unclear whether

the tunneling current calculations can provide suﬃcient contrast

to base discrimination and signal characteristics of the gap be-

tween the nucleobases.

In addition to the aforementioned challenges, there are

other signiﬁcant obstacles associated with the transverse current

method. Two of these major challenges are controlling the ve-

locity and orientation of DNA during translocation and achiev-

ing a high signal-to-noise ratio in the current signals. The sen-

sitivity of solid-state materials needs to be improved to discrim-

inate biomolecules of similar sizes. Electronic transport proper-

ties of probe-DNA interactions need to be optimized. Eﬀorts are

still under consideration to fabricate more eﬃcient nanopore and

nanogap-based transverse tunneling current detectors.

Single-walled carbon nanotubes (SWCNTs) have been pro-

posed as a promising method to address the challenges asso-

ciated with DNA translocation-based detection.[25,261]One ad-

vantage of SWCNTs is their ability to interact with DNA in

a nucleobase-speciﬁc manner. This means that diﬀerent DNA

bases can be identiﬁed based on their distinct interaction with

the SWCNT surface, allowing for more accurate and reliable se-

quencing. In addition, the translocation rate of DNA through a

SWCNT nanopore can be controlled by modulating the tempera-

ture, ionic strength, or voltage bias. This provides a way to regu-

late the speed and orientation of the DNA molecule, enabling bet-

ter control over the sequencing process. Base-speciﬁc hydrogen

bonding interactions between chemically modiﬁed metal elec-

trodes and nucleobases may also be helpful in resolving the above

issues.

4.3. Low Signal-To-Noise Ratio

Noise is an unwanted signal that shadows the actual signals,

decreasing the eﬃciency of the device and signal-to-noise ratio

(SNR) during detection. Hence, there is a substantial demand

for strategies to reduce noise. It is reported that even though

biological pores generally exhibit lower noise, solid-state pores

have substantially higher signal-to-noise (SNR) than biological

nanopores.[259]It can be attributed to the higher currents that

solid-state systems oﬀer, along with relatively low high-frequency

noise.

In solid-state nanopores, noise originates from the charge ﬂuc-

tuations at the nanopore surface, ﬂuctuations of the total num-

ber of charge carriers, and nanometer-sized gaseous bubbles in-

side the pore during the translocation of DNA molecules.[259,262]

Based on its frequency range, noise can be divided into four cate-

gories: a) low-frequency noise, b) thermal current noise, c) high-

frequency dielectric noise, and d) capacitive noise.[263–265]Con-

siderable research has been devoted to unraveling the origins

of noise in both biological and solid-state nanopores.[263,266,267]

To reduce the low-frequency noise, researchers are using more

mechanically stable nanopores and thicker membranes for the

studies. Graphene membranes coated with a TiO2layer or a

multi-layered graphene-Al2O3stack structure with a thickness

of approximately 20 nm are reported to be more eﬀective in re-

ducing electrical noise than pure graphene nanopores in single

molecule-level DNA sequencing.[268]High-frequency noise is re-

ported to be reduced by integrating complementary metal-oxide

semiconductor (CMOS) preampliﬁers with solid-state nanopores

in thin silicon nitride membranes.[269]Further, signal-to-noise

ratios (SNRs) in nanopore and ion channel recordings can be

improved with wavelet-denoising techniques.[270]With the ad-

vancement of ML techniques, deep learning methods are also

being used for denoising electric signals with convolutional

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auto-encoding neural networks.[271]This network is speciﬁcally

engineered to iteratively analyze and minimize disparities be-

tween two waveforms by employing gradient descent optimiza-

tion. Utilizing a permittivity gradient approach, Tsutsui et al.

have reported an enhancement in the single-molecule sensitiv-

ity of nanopores by amplifying ionic current signals while mini-

mizing interference with other features.[272]Drawing inspiration

from concepts in both electronics and biology, an abiotic ionic

circuit demonstrates signal ampliﬁcation by transforming small

ionic signals into ampliﬁed ionic outputs.[273]In the case of trans-

verse tunneling methods, the insulator-protected mechanically-

controllable break junctions (MCBJs) demonstrated fast tunnel-

ing current measurements and improved the signal-to-noise ratio

in the electrical single-molecule detection.[274]

The sensing performance of 2D materials can also be hindered

by noise resulting from tunneling current across the transmem-

brane sheet. However, till now, it is a less explored area. Accord-

ing to a recent experimental study by Graf et al., the transverse

current demonstrated a 40% improvement in signal-to-noise ra-

tio compared to the ionic current in simultaneous measurements

using a MoS2nanopore.[170]This suggests that the transverse cur-

rent approach has potential advantages over the ionic current ap-

proach for DNA sequencing.

4.4. To Achieve Long Readability

One of the potential challenges of DNA sequencing is achiev-

ing long reads for signiﬁcant nucleotide identiﬁcation. There

are some ﬂuctuations in translocation kinetics owing to device-

surface interaction, which is the key challenge to achieve long

reads. In addition, the translocation of DNA in an unfolded state

is also a key requirement for more reliable DNA sequencing.

The nanopore sequencing turned out to be the most promising

in achieving long readability, as the nanopore sensor reads the

DNA molecule base by base, and the accuracy of nucleobase at

one instance of time is independent of the prior history of the

system. The MinION device of Oxford Nanopore indeed repre-

sented a signiﬁcant advancement in portable DNA sequencing

technology. The MinION device, resembling a USB drive in ap-

pearance, oﬀered real-time DNA sequencing capabilities by uti-

lizing nanopore technology.[24,275]

Nevertheless, some limitations still need to be explored in the

experimental studies, such as avoiding shearing during sample

preparation and threading exceptionally long molecules through

the pore. The experimental DNA sequencing analysis of nanogap

and nanochannel devices with the transverse tunneling current

method is still in the infant stage. A more detailed exploration

of these speciﬁc devices can shed light on their long-strand DNA

sequencing capabilities.

4.5. Challenges in the Solid-State Device Fabrication

Several strategies have been employed for solid-state nanopore

fabrication, such as ion-beam sculpting, electron-beam drilling,

and atomic layer deposition, but a potential method capable of

generating a large number of uniform solid-state nanopores with

suitable diameter ranges from 1.5 to 2.0 nm remains a daunting

challenge. The problem with focused ion beam drilling is that the

resolution is insuﬃcient to fabricate nanopores with a diameter

smaller than 10 nm.[276]Similarly, achieving precise control over

the geometry and size of nanopores is a signiﬁcant challenge, as

it is essential for ensuring the reproducibility of nanopore sens-

ing experiments. Solid-state nanopore fabrication with thin-ﬁlm

growth is unscalable, costly, and time-consuming. There are ma-

jor diﬃculties as well in the fabrication of hybrid nanopores, such

as the condition of insertion with controlled precision and prepa-

ration of an inner volume of the nanopore.

Moreover, the reproducible and controlled fabrication of

nanogap electrodes with a sub-5nm gap remains a formidable

challenge. The potential techniques for the fabrication of

nanogap are reported to be focused ion beam (FIB) milling, pho-

tolithography, and electron-beam lithography (EBL).[277]In the

case of nanoribbons, one major challenge is the fabrication of

a device with a width of ≈1 nm, which is important for single-

base resolution.[278]An additional challenge is the controlled

alignment of nanoribbons within a nanoﬂuidic channel. This

issue may be resolved by nanowire lithography[279]and the un-

zipping of carbon nanotubes.[280]Nanopore fabrication utilizing

dielectric-controlled breakdown (CBD) of thin membranes di-

rectly in an aqueous solution is currently undergoing rapid ad-

vancement and active investigation to enhance the scalability of

solid-state nanopores.[281]The CBD method presents three signif-

icant advantages: a) cost-eﬀective in situ fabrication, b) the abil-

ity to fabricate planar nanopores within existing nanostructures,

and c) high accessibility and ease of handling.[282,283]Nonetheless,

signiﬁcant hurdles persist, particularly in controlling the precise

location of pore formation and establishing scalable manufactur-

ing processes. Addressing these challenges is pivotal in the seam-

less integration of solid-state pore-based platforms into DNA se-

quencing applications.

Lastly, a strong collaboration work in both experimental and

computational research can reduce lots of eﬀort, time, and cost

by providing simulated information for a more precise selec-

tion of devices and their applications for DNA sequencing. For

a more eﬀective interpretation of experimental results with com-

putational reports, transmembrane-applied bias voltages should

not be higher than the experimental voltage.

4.6. Orientational Fluctuations of the DNA Molecule

Environmental factors mostly lead to conformational, structural,

and orientational ﬂuctuations in DNA nucleotides inside the

nanopore. DNA translocation is stochastic, and the ﬂuctuation

occurs due to translocation kinetics, which gives rise to a broad

distribution of dwell times. These variations due to translocation

kinetics cause uncertainties in the number of DNA nucleotides

translocating through nanopores. As a result of these ﬂuctua-

tions, there can be signiﬁcant variations in the measured electric

current signals, leading to a higher potential for read-out errors.

In biological nanopores, the problem of stochastic translocation

kinetics can be reduced using a processive enzyme,[74]while in

solid-state nanopores, a possible way to control the nucleobase

motions is the use of a multilayered membrane transistor with

a motion-control electrode layer that can facilitate decrease in

stochastic ﬂuctuations of trapped DNA molecule.[158]

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In nanopores, any ﬂuctuation in the DNA molecule is critical

to the transverse tunneling current approach as it can consider-

ably inﬂuence the coupling strength by aﬀecting the eigenstates

of DNA nucleotides with the nanopore/nanogap edges and af-

fecting the distribution of the recorded current values, resulting

in overlap in the signature signal of DNA nucleotides. Most of the

previous computational studies either excluded the environmen-

tal eﬀects or simulated homogenous DNA strands by considering

only arbitrary structural and conformational orientations.[148,284]

In a realistic simulation, the eﬀects of embedded material, tem-

perature, solvent, and salt concentration come into the picture,

which triggers thermal ﬂuctuation. The most promising architec-

ture for reducing the orientational ﬂuctuation is the nanochan-

nel. There are several tactical methods that have been pro-

posed to reduce conformational instabilities, and stochastic nu-

cleobase motions and resolve the signal overlap between diﬀer-

ent DNA nucleobases. Some important methods are the func-

tionalization of nanogap electrodes,[180,285,204]labeling of DNA

nucleotides,[189,286]and double nanopore,[287,288 ]among others.

5. Conclusions

Solid-state material-based DNA sequencing has emerged as a

powerful paradigm with the potential to transform comprehen-

sive genomic and medical research. Signiﬁcant eﬀorts are be-

ing made to achieve an ultrafast, cost-eﬀective, and reproducible

DNA sequencing technique that recognizes a single base within

a DNA strand. Though biological nanopores with ionic cur-

rent methods have already been commercialized, solid-state 2D

materials-based electrical detection techniques will be the key to

achieve single nucleotide resolution. In the present review, we

have provided a comprehensive discussion of all the prominent

aspects of various DNA sequencing techniques proposed so far,

including their challenges, opportunities, and future directions.

The review highlights the ionic and transverse tunneling current

methods involving nanopores, nanogaps, nanochannels, and hy-

brid/heterostructures for DNA sequencing.

Despite currently being the advanced method, the ionic cur-

rent approach suﬀers from major bottlenecks that need to be

resolved, such as scalability, slowing down DNA translocation

speed, resolving nucleotides signal overlap, reducing nucle-

obases stochastic motion, and low signal-to-noise ratio. Recently,

transverse tunneling current detection technique is also moving

fast in the direction of sequencing biomolecules. The technique

has several advantages, such as the potential to sequence many

times faster than the ionic current method, single-base resolu-

tion, high readability, and ﬁdelity, along with lower noise levels.

Moreover, in tunneling current measurements, the recognition

can be achieved without complex enzymatic processing. How-

ever, the technique is extremely sensitive to molecule sizes, com-

position, and thermal ﬂuctuations in an aqueous solution. Cur-

rently, as the promising frontier, more theoretical and experimen-

tal reports are needed to comment more on the potential of the

transverse current method for ultrafast DNA sequencing.

We believe that solid-state 2D materials are more promis-

ing for single-molecule DNA detection. Solid-state 2D materi-

als have the advantages of long-term stability, high surface-to-

volume ratio, atomically thin membranes, higher durability, and

superior mechanical and electrical properties. Till now, graphene

is reported to be the most promising material to achieve single

nucleotide recognition. Nevertheless, graphene nanostructures

have certain demerits, such as hydrophobic interactions, pore-

clogging, and high noise levels, which need to be resolved. We

anticipate that the commercialization of solid-state 2D materials

based on nanopore/nanogap sequencing techniques may appear

in the near future for decoding DNA with long readability in a

very short time, reasonable cost, and high accuracy.

A remaining question is which nanostructure (nanopore,

nanogap, and nanochannel) will provide suﬃcient distinguisha-

bility among the DNA nucleotides. At present, the technique that

is commercialized is the nanopore technique utilizing an ionic

current approach. However, the technique has certain shortcom-

ings, including irreversible pore clogging, low signal-to-noise ra-

tio, high translocation speed, and conformational ﬂuctuations,

which need to be overcome so that more accurate sequencing

can be achieved. The nanogap sequencing technique is based

on the quantum tunneling current method. Interestingly, in this

method, each targeted nucleotide exhibits a current signal inde-

pendent of the neighbor nucleotide. Nevertheless, the drawback

is the possibility of signiﬁcant ﬂuctuations in the tunneling cur-

rent. In the nanochannel technique, the merit is controlled orien-

tational ﬂuctuation, which may provide better signal readability.

The demerit of the nanochannel is its ultra-thin fabrication, lim-

iting its capacity to adsorb and detect only a single molecule at a

time. After thoroughly analyzing all the prominent aspects of cur-

rently available architectures, we believe that both nanopore and

nanogap architectures are promising for single nucleotide iden-

tiﬁcation. Moreover, machine learning integrated nanopore de-

vices with transverse current method has shown potential for pre-

diction of nucleotide ﬁngerprint conductance signals from train-

ing with single nucleotide datasets and helps in the classiﬁcation

of overlapped complex, noisy nucleotide signals with high accu-

racy.

Despite all the achievements, there is an urgent need to dis-

cover new sensing methods, such as alternative read-out schemes

and other 2D solid-state materials, which can have the potential

to suppress stochastic nucleotide motions, control DNA translo-

cation rate, conformational ﬂuctuations, and resolve the signal

overlap. Considering the incredible progress in nanopore DNA

sequencing, there is no doubt that it is currently the most promis-

ing sequencing technique, even capable of sequencing the hu-

man genome in a very fast and cost-eﬀective manner. There

are still lots of things that need to be explored further in this

ﬁeld. Furthermore, fabrication techniques need to be improved

to achieve uniform nanostructures and enable high-throughput

DNA sequencing.

6. Future Perspectives

The future of DNA sequencing research hinges on the pursuit

of their aﬀordability, rapidity, and precision. Remarkable strides

have been made in the realm of solid-state nanopores. However,

it is worth noting that commercialization of DNA sequencing

is only achieved by biological nanopores with the ionic block-

ade current approach. For a successful transition from biologi-

cal nanopores to solid-state material-based devices into commer-

cial viability for DNA sequencing, an emphasis on extensive ex-

perimental investigations under realistic conditions is crucial.

2401112 (37 of 43)

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Moreover, the transverse tunneling current method using solid-

state 2D materials has been emerging as a potential strategy for

single molecule DNA sequencing. Several theoretical investiga-

tions emphasize the promise of this approach. However, more ex-

perimental studies can provide better insights into the complex-

ities and accuracy of identiﬁcation with the transverse current

approach, which is still in the nascent stage of technological evo-

lution. The solid-state 2D material-based sequencing paradigm

warrants a patient outlook, drawing parallels to the evolution-

ary trajectory of biological nanopores, which required nearly two

decades of research to achieve commercialization. From the ex-

tensive experimental and computational analysis, it is evident

that amongst the solid-state materials, the commercialization of

graphene nanopores can be achieved in the future. Recently, the

integration of machine learning and artiﬁcial intelligence tech-

nologies into sequencing devices has helped signal processing,

denoising large complex data, and facilitating accurate nucleotide

identiﬁcation and classiﬁcation through the extraction of elec-

tronic signal information. From our understanding, we feel the

future of DNA sequencing will be taken over by the application

of artiﬁcial intelligence and data-driven prediction of nucleotide

sequencing. Additionally, by leveraging the lessons learned from

biological pore commercialization and harnessing the capabili-

ties of machine learning, ionic current-based DNA sequencing

stands poised for substantial advancement.

Acknowledgements

M.K.J. and S.M. contributed equally to this work. This work was sup-

ported by grants DST-SERB (project number: CRG/2022/000836), CSIR

(project number: 01(3046)/21/EMR-II), and BRNS (project number: 2023-

BRNS/12356). R.L.K. and M.K.J. acknowledge MHRD, and S.M. acknowl-

edge UGC for the research fellowship, respectively.

Conﬂict of Interest

The authors declare no conﬂict of interest.

Keywords

DNA sequencing, ionic current, machine learning, nanopore, transverse

current

Received: February 13, 2024

Revised: April 5, 2024

Published online:

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Rameshwar L. Kumawat was born in Jaipur,Rajasthan, India. He obtained his Bachelor and Master

of Technology in Nanotechnology (major) and Information and Communication Technology (minor)

in 2015 and 2016 from the Centre for Converging Technologies-University of Rajasthan, Jaipur, Ra-

jasthan, India. In July 2017, he joined Prof. Biswarup Pathak’s lab as a doctoral student in the Depart-

ment of Metallurgy Engineering and Materials Science, Indian Institute of Technology Indore (IITI),

India. His Ph.D. thesis was titled “Nano-electrodes for ultrafast DNA/protein sequencing: Ab-initio

quantum transport studies.” He extensively used atomistic simulations to shed light on the DNA and

protein sensing and detection physics of solid-state nanopores, nanogaps, and nanochannels. He

also worked on tuning the physical and chemical properties of low-dimensional materials. Soon af-

ter that, he joined Regents’ Prof. C. David Sherrill’s lab as a Postdoctoral Fellow at Georgia Institute

of Technology, Georgia, Atlanta, USA. His current research focuses on developing and applying new

models for intermolecular interactions and their implementation as software and machine learn-

ing for high-throughput screening of materials/molecules and their chemical and physical property

prediction at Georgia Tech.

Milan Kumar Jena obtained his UG degree from Ravenshaw University,Cuttack, Odisha. He com-

pleted his master’s degree from the Indian Institute of Technology Guwahati, Assam. Then, he joined

Prof. Biswarup Pathak’s group as a graduate student in January 2020 at the Department of Chemistry,

Indian Institute of Technology Indore, where his primary research interest is the computational inves-

tigation of solid-state nanopores/nanogaps for next-generation DNA sequencing.

Sneha Mittal received her M.Sc. degree in chemistry from Ramjas College, University of Delhi (DU),

in 2018. Currently,she is carrying out her doctorate studies under the supervision of Prof. Biswarup

Pathak at the Department of Chemistry,Indian Institute of Technology Indore (IITI), India. Her current

research work focuses on the application of molecular electronics and machine learning in single-

molecule DNA and protein sequencing.

Biswarup Pathak was born in Bankura, West Bengal, India. He obtained his B.Sc. (Chemistry, 2000)

from Bankura Christian College and M.Sc. (Physical Chemistry, 2002) from Banaras Hindu University

(BHU). After postgraduation from BHU, he obtained his Ph.D. from Hyderabad Central University

under the supervision of Professor E.D. Jemmis. During his Ph.D., he was involved in computational

studies on the structure, bonding, and reactivity of boranes, carboranes, metallaboranes, and transi-

tion metal complexes. Soon after his Ph.D., he completed his four-year postdoctoral study with Prof.

Jerzy Leszczynski’s group at Jackson State University,USA (January 2008–July 2009) and Prof. Rajeev

Ahuja’s group at Uppsala University,Sweden (September 2009–May 2012). He joined IIT Indore as

an Assistant Professor in 2012 and became a Professor in 2022. Dr.Pathak is currently working on de-

veloping solid-state materials for clean energy (hydrogen storage, photocatalysis, fuel cells, batteries,

and solar cells) and biological (DNA sequencing) applications.

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Towards Graphene Semi/Hybrid-Nanogap: A New Architecture for Ultrafast DNA Sequencing

Article

Full-text available

Dec 2022

The tremendous upsurge in the research of next-generation sequencing (NGS) methods has broadly been driven by the rise of the wonder material graphene and continues to dominate the futuristic approaches for fast and accurate DNA sequencing. The success of graphene has also triggered the search for many new potential NGS methods capable of ultrafast, reliable, and controlled DNA sequencing. The present study delves into the potential of a new NGS architecture utilizing graphene, namely, a 'semi/hybrid-nanogap' for the identification of DNA nucleobases with single-base resolution. In the framework of first-principles density functional theory methods, we have calculated the transmission function and current-voltage (I-V) characteristics which are of particular significance for DNA sequencing applications. It is noted that the interaction energy values are significantly reduced compared to the previously reported graphene nanodevices, which can lead to a controlled translocation during experimental measurements. Based on the transmission function, each nucleobase can be identified with pertinent sensitivity. It is noticed that the use of highly conductive nucleobase analogs can facilitate improved single nucleobase sensing by increasing the transmission sensitivity. Therefore, we believe that the present study opens up promising frontiers for sequencing applications.

Ionic Signal Amplification of DNA in a Nanopore

Article

Full-text available

Oct 2022

Ionic signal amplification is a key challenge for single‐molecule analyses by solid‐state nanopore sensing. Here, a permittivity gradient approach for amplifying ionic blockade characteristics of DNA in a nanofluidic channel is reported. The transmembrane ionic current response is found to change substantially through modifying the liquid permittivity at one side of a pore with an organic solvent. Imposing positive liquid permittivity gradients with respect to the direction of DNA electrophoresis, this study observes the resistive ionic signals to become larger due to the varying contributions of molecular counterions. On the contrary, negative gradients render adverse effects causing conductive ionic current pulses upon polynucleotide translocations. Most importantly, both the positive and negative gradients are demonstrated to be capable of amplifying the ionic signals by an order of magnitude with a 1.3‐fold difference in the transmembrane liquid dielectric constants. This phenomenon allows a novel way to enhance the single‐molecule sensitivity of nanopore sensing that may be useful in analyzing secondary structures and genome sequence of DNA by ionic current measurements.

Identification of DNA Nucleotides by Conductance and Tunnelling Current Variation through Borophene Nanogap

Article

Full-text available

Sep 2022

Rapid and inexpensive DNA sequencing is critical to biomedical research and healthcare for the accomplishment of personalized medicine. Solid-state nanopores and nanogaps have marshalled themselves in the fascinating paradigm of nano-research since the advent of its application in DNA sequencing by analyzing the quantum conductance and electric current signals. In this study, the feasibility of the considered borophene nanogaps for DNA sequencing purposes via the electronic tunnelling current approach was investigated by utilizing combined density functional theory with non-equilibrium Green's function (DFT-NEGF) techniques. The interaction energy (Ei) and the charge density difference (CDD) plots exploit the charge modulation around the nanogap edges due to the presence of each nucleotide. Our results revealed a distinct variation in the tunnelling conductance, as a characteristic fingerprint of each nucleotide at the Fermi level. The calculated tunnelling current variation across the nanogap under an applied bias voltage was also significant due to the effective coupling of nucleotides with the electrode edges. The current was in the picoampere (pA) range, which was fairly higher than the electrical background noise and also experimentally detectable by the canning tunnelling microscopy (STM) technique. Our findings demonstrated that in the borophene nanopore vs. nanogap scenario, the nanogap has several advantages and is a more promising nanobiosensor. Moreover, we also compared our results with various previous experimental and theoretical reports on nanogaps as well as nanopores for gaining better insights.

Single-molecule DNA sequencing using two-dimensional Ti2C(OH)2 MXene nanopores: A first-principles investigation

Article

Full-text available

Jun 2022

Nanopore-based devices have provided exciting opportunities to develop affordable label-free DNA sequencing platforms. Over a decade ago, graphene has been proposed as a two-dimensional (2D) nanopore membrane in order to achieve single-base resolution. However, it was experimentally revealed that clogging of the graphene nanopore can occur due to the hydrophobic nature of graphene, thus hindering the translocation of DNA. To overcome this problem, the exploration of alternative 2D materials has gained considerable interest over the last decade. Here we show that a Ti 2 C-based MXene nanopore functionalized by hydroxyl groups (−OH) exhibits transverse conductance properties that allow for the distinction between all four naturally occurring DNA bases. We have used a combination of density functional theory and non-equilibrium Green’s function method to sample over multiple orientations of the nucleotides in the nanopore, as generated from molecular dynamics simulations. The conductance variation resulting from sweeping an applied gate voltage demonstrates that the Ti 2 C-based MXene nanopore possesses high potential to rapidly and reliably sequence DNA. Our findings open the door to further theoretical and experimental explorations of MXene nanopores as a promising 2D material for nanopore-based DNA sensing.

The complete sequence of a human genome

Article

Full-text available

Apr 2022
SCIENCE

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion-base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.

A Step toward Amino Acid-Labeled DNA Sequencing: Boosting Transmission Sensitivity of Graphene Nanogap

Article

Dec 2022

Existing obstacles in next-generation DNA sequencing techniques, for instance, high noise, high translocation speed, and configurational fluctuations, call for approaches capable of reaching the goal and accelerating the process of personalized medicine development. The labeling nucleotide approach has the potential to overcome these barriers and boost the recognition sensitivity of a solid-state nanodevice. In this theoretical report, the first-principles density functional theory calculations have been employed to study the role of three different labels, tyrosine (Tyr), aspartic acid (Asp), and arginine (Arg), for labeling DNA nucleotides and study their effect in rapid and controlled DNA sequencing at atomic resolution. Remarkable differences in interaction energy values are noticed in all three cases of differently labeled nucleotides. The zero-bias transmission spectra confirm that proposed labels have the ability to detect the individual nucleotide, amplifying the tunneling current sensitivity by several orders of magnitude. The current-voltage characteristics of Arg-labeled nucleotides are found to be promising for single nucleotide recognition even at a very low bias voltage of 0.1 V.

Machine Learning Aided Interpretable Approach for Single Nucleotide-Based DNA Sequencing using a Model Nanopore

Article

Dec 2022

Solid-state nanopore-based electrical detection of DNA nucleotides with the quantum tunneling technique has emerged as a powerful strategy to be the next-generation sequencing technology. However, experimental complexity has been a foremost obstacle in achieving a more accurate high-throughput analysis with industrial scalability. Here, with one of the nucleotide training data sets of a model monolayer gold nanopore, we have predicted the transmission function for all other nucleotides with root-mean-square error scores as low as 0.12 using the optimized eXtreme Gradient Boosting Regression (XGBR) model. Further, the SHapley Additive exPlanations (SHAP) analysis helped in exploring the interpretability of the XGBR model prediction and revealed the complex relationship between the molecular properties of nucleotides and their transmission functions by both global and local interpretable explanations. Hence, experimental integration of our proposed machine-learning-assisted transmission function prediction method can offer a new direction for the realization of cheap, accurate, and ultrafast DNA sequencing.

Which 2D Material is Better for DNA Detection: Graphene, MoS 2 , or MXene?

Article

Sep 2022

Despite much research on characterizing 2D materials for DNA detection with nanopore technology, a thorough comparison between the performance of different 2D materials is currently lacking. In this work, using extensive molecular dynamics simulations, we compare nanoporous graphene, MoS2 and titanium carbide MXene (Ti3C2) for their DNA detection performance and sensitivity. The ionic current and residence time of DNA are characterized in each nanoporous materials by performing hundreds of simulations. We devised two statistical measures including the Kolmogorov-Smirnov test and the absolute pairwise difference to compare the performance of nanopores. We found that graphene nanopore is the most sensitive membrane for distinguishing DNA bases. The MoS2 is capable of distinguishing the A and T bases from the C and G bases better than graphene and MXene. Physisorption and the orientation of DNA in nanopores are further investigated to provide molecular insight into the performance characteristics of different nanopores.

Amplifying Quantum Tunneling Current Sensitivity through Labeling Nucleotides Using Graphene Nanogap Electrodes

Article

Jul 2022

Recent advances in cost-effective, ultra-rapid, and efficient DNA sequencing are in the saddle of the advancement of the personalization of medicines for understanding and early-stage detection of several killer diseases. Paying attention to a timely need for the development of solid-state nanodevices for rapid and controlled identification of DNA nucleotides, in this report, we theoretically explored the potential of labeling techniques in the sequencing of DNA nucleotides through solid-state graphene nanogap electrodes using the quantum tunneling current approach. Our study boasts the idea that labeling of DNA nucleotides can solve major hurdles of DNA sequencing, such as improving the signal-to-noise ratio, slowing down translocation velocity, and controlling orientational variations. Employing the first-principle density functional theory study, we identify unique interaction energy values for each labeled nucleotide having remarkable differences in the range of 0.10-0.74 eV. The zero-bias transmission spectra of the proposed setup suggest that the detection of the nucleotides is possible by applying very low gate voltages. Moreover, the labeling of nucleotides amplifies the conductance sensitivity considerably. I-V characteristics suggest that electrical recognition of each labeled nucleotide can be carried out at both lower (0.3 V) and higher (0.8 V) bias voltages with single-molecule resolution, although the maximum current sensitivity is observed at a higher bias voltage. The proposed sequencing device possesses high sensitivity and selectivity characteristics that are crucial for experimental purposes. We find that our results are rich compared to unlabeled nucleotides-based graphene nanopore/nanogap devices. Hence, the study will certainly motivate the experimentalists toward the application of a labeled DNA nucleotide system for ultra-rapid DNA sequencing by using the tunneling current approach.

Ionic Liquid Decelerates Single-Stranded DNA Transport through Molybdenum Disulfide Nanopores

Article

Jul 2022

Nanopores in two-dimensional (2D) materials have emerged to offer in principle necessary spatial resolution for high-throughput DNA sequencing. However, their fidelity is severely limited by the fast DNA translocation. A recent experiment indicates that introducing ionic liquids could slow down DNA translocation in a MoS2 nanopore. However, the corresponding in-depth molecular mechanism underlying the experimental findings is not fully understood, which is crucial for the future improvement of rational DNA translocation control. Here, we computationally investigate and then experimentally identify the effect of BmimCl ionic liquid on the retardation of ssDNA translocation through a single-layer MoS2 nanopore. Our all-atom molecular dynamics simulations demonstrate that the strong interaction between Bmim+ and ssDNA offers a considerable dragging force to decelerate the electrophoretic motion of ssDNA in the BmimCl solution. Moreover, we show that Bmim+ ions exhibit preferential binding on the sulfur edges of the nanopore. These Bmim+ in the pore region can not only act as a steric blockage but also form π-π stackings with nucleobases, which provide a further restriction on the ssDNA motion. Therefore, our molecular dynamics simulation investigations deepen the understanding of the critical role of ionic liquid in DNA translocation through a nanopore from a molecular landscape, which may benefit practical implementations of ionic liquids in nanopore sequencing.

Advancement of Next-Generation DNA Sequencing through Ionic Blockade and Transverse Tunneling Current Methods

Abstract

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