This document provides an overview of nanopore sensors and their applications. It discusses biological nanopores formed by pore-forming proteins, solid state nanopores fabricated in materials like silicon nitride, and hybrid nanopores combining biological and solid state elements. The document outlines several applications of nanopore sensors including DNA sequencing, detection of DNA damage, analysis of circulating microRNAs for cancer detection, and single-molecule protein studies. It also discusses the potential of solid state nanopores integrated with nanowire field-effect transistors and hybrid nanopores for improved specificity.

1. NANOPORE SENSORS
AND THEIR APPLICATIONS
NEHA PANT
MTECH 1st YEAR
ROLL NO: 31501107
DEPARTMENT OF CHEMISTRY

2. CONTENTS
 INTRODUCTION
 BIOLOGICAL NANOPORES
 SOLID STATE NANOPORES
 APPLICATIONS OF NANOPORE SENSORS
 NANOPORE SENSORS IN DNA SEQUENCING
 DETECTION OF DNA DAMAGE
 DETECTION OF CIRCULATING MICRO RNAs IN LUNG
CANCER PATIENTS
 SINGLE-MOLECULE STUDY OF PROTEINS
 FUTURE OF NANOPORE SENSORS
 CONCLUSION

3. INTRODUCTION TO
NANOPORE SENSORS
 A nanopore is, essentially, a nano-scale hole. This hole
may be:
• biological: formed by a pore-forming protein in a
membrane such as a lipid bilayer;
• solid-state: formed in synthetic materials such as
silicon nitride or graphene; or
• hybrid: formed by a pore-forming protein set in
synthetic material.
 The concept of using a nanopore as a biosensor was
first proposed in the mid 1990s in academic institutions
such as Oxford, Harvard and UCSC.

4.  Nanopore analysis:
Introduce analyte of interest into the hole  identify “analyte” by
the disruption of electrical current
It uses a voltage to drive molecules through a nanoscale pore
in a membrane between two electrolytes, and monitors how
the ionic current through the nanopore changes as single
molecules pass through it.
 Allows analysis of charged polymers (single-stranded DNA,
double-stranded DNA and RNA) with subnm resolution and
without the need for labels or amplification.
 Structure and dynamic motion of the molecule identified.

5. Nanopore Sensing

6. BIOLOGICAL NANOPORES
PROTEIN NANOPORES:
 NanoPores formed in a membrane(lipid bilayer) by a pore-
forming protein i.e Alpha- hemolysin.
 ADVANTAGES OF BIOLOGICAL NANOPORES:
 Easily reproducible with atomic level precision
 X-Ray crystallography provides information about nanopore
structure at angstrom level
 Heterogeneity observed in terms of size and composition
 Robust, easily reproducible at low cost, and easy to modify
Bala Murali ,Venkatesan ,Rashid Bashir: Nanopore sensors for nucleic acid
analysis, Nature Nanotechnology 6, 615–624 (2011)

7. α-Hemolysin Nanopores:
 Arises from Staphylococcus aureus
 It is an asymmetric, mushroom shape protein nanopore
 Ability to self-assemble into planar lipid membrane
 It is a heptameric protein pore with an inner diameter of 1 nm
 3.6 nm diameter vestibule connected to a transmembrane Beta –
barrel (1.4 nm wide and 5nm long).
 ADVANTAGES:
Excellent stability
Reproducibility
Precise tuning properties via site-directed mutagenesis
Minimal requirements of expensive reagents
 A single-stranded DNA can pass through the nanopore but
double stranded DNA cannot
Bala Murali ,Venkatesan ,Rashid Bashir: Nanopore sensors for nucleic acid analysis,
Nature Nanotechnology 6, 615–624 (2011)

8. Sensors 2014, 14, 18211-18222; doi:10.3390/s141018211

9. •Menestrina, G. Ionic channels formed by Staphylococcus aureus alpha-toxin: Voltage-dependent
inhibition by divalent and trivalent cations. J. Membr. Biol. 1986, 90, 177–190.
•Wang, H.-Y.; Gu, Analysis of a single α-synuclein fibrillation by the interaction with a protein nanopore. Anal.
Chem. 2013, 85, 8254–8261.

10. SOLID STATE NANOPORES
 A nanometer-sized hole formed in a synthetic membrane (usually
SiNx or SiO2).
 The pore is usually fabricated by focused ion or electron beams,
so the size of the pore can be tuned freely.
 Nanopore membrane material of choice: SiN (high chemical
resistance ,low mechanical stress)
 ADVANTAGES:
 Ability to tune the size and shape of the nanopore with subnm
precision
 Superior mechanical, chemical and thermal characteristics
compared with lipid-based systems
 Possibility of integrating with electronic or optical readout
techniques
 Detection of larger biomolecules
o DISADVANTAGE: Intensive electrical noise
Bala Murali ,Venkatesan ,Rashid Bashir: Nanopore sensors for nucleic acid analysis,
Nature Nanotechnology 6, 615–624 (2011)

11. GRAPHENE:
 Robust, 2D single-atom-thick ‘HONEYCOMB’ lattice of carbon
with high electrical conductivity
 It can be used as a base material for nanopore-based DNA
sequencing applications in future
 Extremely strong and chemically inert and also a good
electronic sensor material
 Thinnest membrane able to separate two liquid compartments
from each other.
 Its surface is modified with the appropriate agent in order to
reduce undesirable DNA sticking and ion current signal
fluctuations
 Graphene is ideal for making nanogaps and nanoribbons for
DNA sequencing applications

13. APPLICATIONS
NANOPORE SENSOR FOR DNA SEQUENCING:
 The application of nanopores to DNA sequencing was first
proposed by Church, Deamer, Branton in 1995 (Church, G
et al 1995).
 When a single strand of DNA passes through the nanopore,
the residual ionic current will depend on which nucleotide or
base (adenine (A), cytosine (C), guanine (G) or thymine (T))
is in the nanopore at the time.
 By recording how the ionic current through the nanopore
changes with time, it should be possible to determine the
sequence of bases in the DNA molecule.
 Major Challenge : To reduce the speed at which the DNA
passes or translocates through the nanopore and to improve
the sensitivity of the approach.
Church, G., Deamer, D. W., Branton, D., Baldarelli, R. & Kasianowicz, J. Characterization of
individual polymer molecules based on monomer-interface interactions. US patent 5,795,782
(1995).

14.  Nanopore-based sensing is attractive for DNA sequencing :
 It is a label-free, amplification-free approach
 Single molecule approach
 It typically requires low reagent volumes
 Cost effective
 Can potentially enable de novo sequencing and long-range haplotype
mapping
 Active approaches incorporate enzymes to regulate DNA transport
through the pore.
 An enzyme motor coupled to a nanopore is attractive for 2 reasons:
 The enzyme-DNA complex forms in the bulk solution enabling it to be
electrophoretically captured in the nanopore
 Relatively slow and controlled motion is observed as the enzyme
processively steps the DNA molecule through the nanopore

15. Deamer, D. W. Nanopore analysis of nucleic acids bound to exonucleases and polymerases.
Annu. Rev. Biophys. 39, 79 90 (2010).
Clarke, J. et al. Continuous base identification for single-molecule nanopore DNA sequencing.
Nature Nanotech. 4, 265–270 (2009).

16. DETECTION OF DNA DAMAGE
 This method looks for places where a base is
missing, known as an "ABASIC SITE“.
 DNA abasic (AP) sites are one of the most
frequent lesions in the genome and have a high
mutagenic potential if unrepaired.
 Damage to the bases of DNA contributes to
many age-related diseases, including melanoma;
lung, colon and breast cancers.
 The researchers created damage on some DNA
by removing some bases thus exposing the
sugar in the DNA backbone.
 The backbone is attached to a ring- or crown-
shaped chemical known as an "18-crown-6
ether {2-aminomethyl-18-crown-6 (18c6)} " to
the sugar.
18-crown-
6 ether

18.  The DNA with the crown ether attached pass
through the nanopore slowly enough so missing
bases can be detected.
 The speed of translocation through the tiny pore
depends on the stiffness and size of the crown
ether loop that marks the site of DNA damage (Na
An, Aaron et al 2012).
 Electrolyte: Sodium salt as the DNA and crown
ether marking DNA damage sites both slid
through the nanopore at just the right speed to be
detected.
 It takes about one-millionth of a second for an
undamaged DNA base and about one-
thousandth of a second for a crown ether loop
marking with a missing base.
Na An, Aaron M. Fleming, Henry S. White1 :Crown ether–electrolyte interactions permit
nanopore detection of individual DNA abasic sites in single molecules111504–11509 ∣
PNAS ∣ July 17, 2012 ∣ vol. 109 ∣ no. 29

19. oInteractions between 18c6 and Na ion
produce characteristic pulse-like current
amplitude signatures .
oThis allow the identification of individual
AP sites in single molecules of
homopolymeric or heteropolymeric
DNA sequences.
oAdvantages of 2-aminomethyl-18c6 :
 Water soluble
Ability to interconvert into a large, rigid,
disc-like structure when bound to alkali
metal ions
 And a flexible, collapsed one when
the metal ion dissociates. Fig. 4. Individual i-t traces of AP-18c6 in
homopolymeric strands. (A) Sample
i-t traces of 3′ entry for mono adduct (120 mV
trans vs. cis). (B) Sample i-t traces of 5′ entry for
mono adduct (120 mV trans
vs. cis).

20. SINGLE MOLECULE DETECTION OF CIRCULATING
MICRO RNAS IN LUNG CANCER PATIENTS
miRNAs:
o Are short noncoding RNA molecules (~18-24-nt) , important in
regulating gene expression.
o Involved in Regulation of :Cell proliferation, Differentiation,
Metabolism and Apoptosis
o In cancer cells, dysregulated miRNAs disrupt the homeostasis of the
normal biological processes. Aberrant expression of miRNAs has
been found in all types of tumors.
o Recognized as potential Cancer Biomarkers.
o Potential for noninvasive and cost-effective early diagnosis of lung
cancer (Li-Qun Gu et al 2012).
o It is a simple, sensitive, label-free technique requiring no
amplification for miRNA
Li-Qun Gu, Meni Wanunu, Michael X. Wang: Detection of miRNAs with a nanopore single-
molecule counter, 2012 Jul; 12(6): 573–584.

21.  Powerful tool for noninvasive cancer detection, diagnosis,
staging, and monitoring.
 Single miRNA molecules captured in the nanopore produce a
signature current signal that function fingerprints, enabling us to
identify a specific miRNA and quantify its concentration.
 In clinical tests, the nanopore has shown the power to
differentiate miRNA levels in blood from lung cancer patients and
healthy people.
 miR-155, miR-197 and miR-182, are significantly elevated in
lung cancer patients compared with cancer-free controls. The
combination of the three miRNAs yielded 81% sensitivity and
87% specificity.
 One only needs to count the occurrence frequency of the
current pulse to calculate the concentration of the target
miRNA.

22. Circulating miRNA detection using protein nanopores
Li-Qun Gu, Meni Wanunu, Michael X. Wang: Detection of miRNAs with a nanopore
single-molecule counter, 2012 Jul; 12(6): 573–584.

23. SINGLE-MOLECULE STUDY OF PROTEINS BY
BIOLOGICAL NANOPORE SENSORS:
 Rapid and label-free single molecule analysis in the field of
analytical chemistry.
 Proteins subjected to an electric field pass through a
nanopore induce blockades of ionic current that depend on
the protein and nanopore characteristics and interactions
between them (Dongmei Wu et al 2014).
 Can be used to study protein translocation and protein folding,
proteins with DNA and RNA aptamers, and protein−pore
interactions.
 Different types of membranes can be used, such as silica
membranes (Si3N4, SiO2, Al2O3), polymer membranes, and
recently, graphene membranes
Dongmei Wu 1,*, Sheng Bi 2,*, Liyu Zhang 1 and Jun Yang
:Single-Molecule Study of Proteins by Biological Nanopore Sensors , 2014, 14, 18211-18222

24.  Biological membrane such as Aerolysin secreted by Aeromonas
hydrophila ( Beta forming protein with diameter of 1- 1.7 nm) ,
found to be most suitable for studying the conformational changes
of positive charged proteins and peptides.
 The data of current blockades under different cleavage times can
reveal the composition of polypeptides and the process of peptide
cleavage. This approach may be a basis for research on
polypeptides in the future.
 Trypsin is widely used in the cleavage of the polypeptides in vivo
and in vitro to detect the structure and the enzymatic cleavage
sites of polypeptides.
 Trypsin can specifically identify the polypeptides containing Arg
or Lys residues under certain conditions

25. FUTURE OF NANOPORE SENSORS
SOLID STATE NANOPORE SENSORS:
 Biological Nanopores show very exciting experimental results for
ssDNA sequencing, but they have a constant pore size, profile and
lack of stability (Dario Anselmetti et al 2012).
 SOLID-STATE NANOPORES ADVANTAGES :
Chemical, thermal, and mechanical stability
Size adjustability, and integration
 Solid state nanopores proves to be a versatile new single molecule
tool for biophysics and biotechnology.
 Scientists have combined solid-state nanopores with Silicon-
Nanowire Field-effect Transistors (FETs) to create devices ,capable
of sensing single molecule DNA translocation events with a
sensitivity similar to that of ionic-current sensing (B.M. Venkatesan
et al 2009).
•Dario Anselmetti :Tiny holes with great promise, Nature Nanotechnology, Vol 7, February 2012
•B.M. Venkatesan, B. Dorvel, S. Yemenicioglu, N. Watkins, I. Petrov, R. Bashir Highly sensitive, mechanically
stable nanopore sensors for DNA analysis Adv Mater, 21 (2009), p. 2771

26.  Sensors with combination of Solid-state Nanopores and
Nanowire Field-effect Transistors can be used to detect single
DNA molecules quickly and with high sensitivity in near future.
 The nanowire–nanopore devices are composed of a thin
Silicon Nitride membrane separating two fluid compartments.
 The variations in the current of the FET are ten times larger
than those in the ionic current.
 FET signal can provide higher bandwidth recordings, which
could potentially be increased by orders of magnitude
 The technique provides exciting possibilities for future
biosensing and diagnostic applications.

27. Dario Anselmetti :Tiny holes with great promise, Nature Nanotechnology, Vol 7, February
2012

28. HYBRID NANOPORE SENSORS:
 A major drawback of solid-state nanopores is the lack of chemical
differentiation from the target molecules of approximately the
same size (B.M. Venkatesan et al 2009).
 This chemical specificity can be improved by functionalizing
surfaces or attaching specific recognition sequences and
receptors to the nanopores.
 The synthetic nanopores can be coated with a fluid lipid bilayer to
control protein translocations
 The thickness and surface chemistry of the coating surface can
be accurately controlled by various lipids.
 Excellent electrical properties and enhanced mechanical stability
and, therefore, may find broader applications in nano-
biotechnology (Yanxiao Feng et al).
•Yanxiao Feng, Yuechuan Zhang1:Nanopore-based Fourth-generation DNA Sequencing
Technology
•B.M. Venkatesan, B. Dorvel, S. Yemenicioglu, N. Watkins, I. Petrov, R. Bashir Highly sensitive,
mechanically stable nanopore sensors for DNA analysis Adv Mater, 21 (2009), p. 2771

29. Iqbal, S. M. Akin, D & Bhaskar : Solid state nanopore channels with DNA selectivity.
Nature Nanotech. 2, 243-248 (2007)

30. CONCLUSION
Detection of single molecules using nanopore-based technology has
been used for the identification and quantification of a wide variety of
analytes. As the fourth-generation sequencing technique, nanopores
have the potential to become a label-free, rapid, and low-cost DNA
sequencing technology. At the same time, nanopores provide several
advantages, including minimal sample preparation, elimination of the
need for amplification or modification (nucleotides, polymerases or
ligases), and long read lengths (10,000–50,000 bases). Although
nanopore DNA sequencing has good maneuverability, there are still
significant challenges remaining to be overcome. Among them, a key
limitation of nanopore-based DNA sequencing at the single-molecule
level is the requirement of ultra-precise, high-speed DNA detection
beyond the spatial and temporal resolutions of existing optical and
electrical technologies. Therefore, single base recognition and slowing
down the rate of DNA velocity are still the principal challenges.
Nevertheless, nanopore technology will have a tremendous impact on
DNA sequencing and the future of personal health and disease
diagnosis.