Advanced Electrochemistry: Nanopore Sequencing Principle and DNA Identification

Lecture 4 Module Contents

Advanced Electrochemistry CM4027/CM6013 Lecture 4Module contents

• 4 lectures covering:
  Overview of electrochemical techniques
  OImpedance Spectroscopy and Electrode surface functionalisation
  Protein/enzyme film electrochemistry
  Point of care electrochemical sensors

Glucose Sensors (PFE)

Glucose sensors (PFE)

• Different generations of electrochemical glucose sensors
  Indirect glucose sensing
  Direct glucose sensing

glucose gluconate enzyme 3 cofactor O2 H2O2 2e electrode 1 st generation

gluconate glucose N enzyme cofactor Medox Medred 2e electrode electrode 3rd gelectrode 2nd generation

glucose gluconate enzyme cofactor 2e

Nanoconfined Enzyme Cascades and the Electrochemical Leaf (e-leaf)

• Advantages:
• Enzyme cascade reactions can be driven and controlled through
  the electrode potential, and their rates or changes in rate are
  observed directly
• Natural entrapment of enzymes (and co-factors) in the porous
  network, because of their large size, makes it possible to channel
  extended cascades with minimal release of intermediates and
  extremely efficient use of exchangeable cofactors such as
  NAD(P)(H) or ATP
• The catalytic wave-shape observed in CV experiments is very
  informative, and it reports on the factors controlling the
  performance of the cascade

FNR NADP+ NADPH + NH4+ + H2O GLDH NH2 0 0 NADP -10 -- 19 Current / mA -20 -38 -30 -57 conversion 50% -40 75% -- 76 90% -50- Current Density / HA cm-2 .95 0 6 12 18 24 30 36 Time / hour O injection 19.8 mmoles 99.0% yield

Application of Enzyme-Film Electrochemistry

1. Biosensing applications
   I. Detecting enzyme substrate
   (direct detection)
   II. Using enzyme as a label for
   indirect detection (e.g.
   Electrochemical ELISA)
2. Electrosynthesis (e.g. e-Leaf)

ip" I -E(V)

Modes of Electron Transfer at Enzyme-Electrode Interface

Direct Electron Transfer

i. Direct Electron Transfer: occurs when the
enzyme's redox-active site (e.g., heme
groups in cytochromes or flavins in
flavoproteins) interacts directly with the
electrode surface without requiring any
external mediators.

. This mechanism relies on:

a. Proximity of the Active Site: The
enzyme's redox centre must be near
the electrode to facilitate tunnelling of
electrons (e.g. 3rd generation glucose
sensors)
e
e7
O
Using surface topology (rough surface) -> proximity of
the enzyme active sites to the electrode surface

Direct Electron Transfer Enhancements

b. Electron-Conducting Interfaces:
Conductive nanomaterials (e.g.,
carbon nanotubes, graphene, or
gold nanoparticles) can bridge the
gap between the enzyme and
electrode, enhancing DET (e.g. 2nd
generation glucose sensors)
Glucose
¢NQ
3
8
Gluconic
AuNPs
acid
FAD
e
5 nm
Using gold nanoparticles (AuNPs)-> facilitate direct
electron transfer between enzyme and electrode
surface

Mediated Electron Transfer

ii. Mediated Electron Transfer
A redox mediator facilitates the electron
exchange between the enzyme's active site and
the electrode surface

• Mediators shuttle electrons back and forth,
  enabling effective communication even when
  the enzyme's redox site is deeply buried
  within the protein structure
• Common Mediators: Ferrocene derivatives,
  quinones, and metal complexes (e.g.,
  ruthenium or osmium complexes).

Substrate
Product
Enzyme
Active
Site
Med
Med
e
Med
Med
(Ox)
(Red)
Electrode

Microfluidic Devices and Systems for Analytical Applications

Integrating chemical/biochemical operations on a chip

• Microfabrication
  >Semiconductor Technology
• Miniaturization
  >High Throughput Processing
• Automation & Parallelization
  >System Integration
• Wide Variety of Applications
  >Medical/Pharmaceutical, etc.

Reservoir#1 Reservoir#2
Microbiochemical System
Heater
Reaction
Microreactor
Analysis
Buffer
Separation
Channel
Product
Mass
Production
Detector
Waste

Miniaturization of Computing and Lab Equipment

ENIAC and Modern Computers

ENIAC (Electronic Numerical Integrator And Computer),
built between 1943 and 1945-the first large-scale
computer
Lap TopPC
< (several) kg
-> Multi-function/purposes
60years
ENIAC, 1940s
30 ton, 13 x 6.5 m2
-> Single Purpose

Mobile Phones and Lab Miniaturization

Mobile Phones
< 1 kg
-> Multi-function/purposes
70years
ENIAC, 1940s
30 ton, 13 x 6.5 m2
-> Single Purpose
*
fE
Y
Desk Top Lab.
< kg
~ ? years -> Multi-Purposes
Chemistry/Biology Lab.
-Room+Operators
+ Analytical Machines

Biosensor Characteristics

Portability vs. Lab-based Assays

Portability Vs lab-based assays
Is this a biosensor ?!!!!
XXXXXXX
H
H
H
H
x
x

WHO "ASSURED" Criteria for POC Testing

Biosensor Characteristics

• WHO "ASSURED" criteria for POC testing
  Affordable by those at risk
  >Sensitive (few false-negatives)
  Specific (few false-positives)
  > User-friendly (simple to perform and requiring
  minimal training)
  Rapid (to enable treatment at first visit) and
  Robust (does not require refrigerated storage)
  Equipment-free ??
  > Delivered to those who need it
  0
  pregnant
  not pregnant
  D

Biosensors for Diagnostic Applications

Diagnostic Workflow

Biosensors for diagnostic applications
Diagnostic workflow
Advanced healthcare system

• Personalized diagnostics
• Personalized treatment
• Acknowledge genetic and epigenetic variations
• Targeted therapy
• Avoid overtreatment (e.g. cancer chemotherapy)
• Quick and effective decisions (e.g. antibiotic
  administration/ self-isolation)

Patient
Sample
O
Biomarker Analysis
Personalized
Treatment

Current Techniques: ELISA

Current techniques
> 90 % of sample analysis is lab-based
Example: Enzyme linked immunosorbent assay (ELISA)

• Mainly for protein analysis and some biomolecules (hormones)
• Mainly optical transduction

Sample
Label (e.g. enzyme)
Label
(e.g. enzyme)
+
Inhibitor
antigen
Label (e.g. enzyme)
Target
Secondary Ab
Secondary Ab
Secondary Ab
Primary Ab
Target
Target
Target
Sandwich ELISA
Direct ELISA
Indirect ELISA
Competitive ELISA
Secondary Ab
Label (e.g. enzyme)

Limitations of Current Techniques

Current techniques limitations

• Operationally complex (labelling
  and washing)
• High cost (ligand and labels)
• Associated with bulky and
  expensive equipment
• Require extensive optimization
• Require highly trained operator
• Limited access specially at
  areas with low resources
• High cost
• Late or missed diagnosis

Electrochemical Biosensors Examples

Electrochemical Biosensors examples

2000 First Ever
Non invasive
Commercial
product

1991 First ever Continuous
Glucose Measuring Device

1987 First ever self glucose
measurement strip
by Medisense

• Glucose sensor:
  refer to lecture 3

1971Ames Reflectance glucometer by
Anton Hubert

1965 First ever glucose measurement strip developed by
Ames

1962 First glucose biosensor based on enzyme electrode
developed by Clarke and Lyons

Nanopore Sequencing Technology

Historical Background of Oxford Nanopore Sequencing

Nanopore sequencing technology
Historical Background of Oxford Nanopore Sequencing

• In 1989, Professor David Deamer first came up with the idea of using a protein channel in
  membranes to detect individual nucleotides.
• In 2001, Professor Hagan Bayley at Oxford University explained a working nanopore sensor.
• The first nanopore sequencing data was presented in 2012 at a conference, introducing the MinION
  and GridION systems.
• MinION was launched for access to early users in 2014 and it was commercially released in 2015.
• In 2016, new devices like the mobile-compatible SmidgION and the automated sample preparation
  device VolTRAX were announced.
• In 2019, the Flongle adapter was launched for low-cost, smaller sequencing tests.

Nanopore Sequencing Principle

Nanopore Sequencing Principle
1
DNA is unwound by the motor protein
and one strand is translocated
through the pore to the +ve side of
membrane
DNA
Motor
Protein
Nanopore
Membrane
+
O
O
O
lon
O
lonic
Current
A
T
C
G
Base
2
Each base gives a characteristic
reduction in the ionic current,
allowing the DNA to be sequenced
Characteristic
drop

• When nucleic acid
  molecules pass
  through a
  nanopore channel
  in a membrane
  that separates two
  electrolyte-filled
  chambers, it
  disrupts the
  current and
  produces a
  characteristic
  electrical signal

Sequencing Principle Details

Sequencing principle

• The sample chamber incorporate motor proteins that control the
  speed of translocation
• These proteins also have helicase activity which unwinds the double-
  stranded DNA into single-stranded molecule
• When voltage is applied across the membrane, it generates an ionic
  current
• As nucleotides pass through the nanopore, their negative charge
  causes them to move toward the anode, which disrupts the ionic
  current and generates a distinct pattern
• Different nucleotides affect the ionic current differently and produce
  unique patterns due to their mass and electrical properties
• This pattern is detected and interpreted to determine the nucleotide
  sequence.

Sequencing principle

Oxford Nanopore Sequencing Steps

Oxford Nanopore sequencing
Oxford Nanopore Sequencing Steps
1
Sample Preparation
Nasopharyngeal swab
Multiplex PCR
5
3
genome
+
primers
RNA extraction
amplified
Library preparation
Rapid barcoding kit
+
Transposome
Complex
3
Sequencing
Sample pooling and library loading
Active
pores
MinION
sequencer
Nanopore
Base calling
AGTCCCTGAATCGA
Assembly and Analysis
Sequenced Genome
Phylodynamics
cDNA synthesis
2
PCR and Barcoding

Evolution of Sequencing Technologies

Evolution of sequencing technologies
First generation
Second generation
(next generation sequencing)
Third generation
OF
Flongle
Flongle is an ada
cells.
Sanger sequencing
Maxam and Gilbert
Sanger chain termination
454, Solexa,
lon Torrent,
Illumina
PacBio
Oxford Nanopore
Why choose Fld
Infer nucleotide identity using dNTPs,
then visualize with electrophoresis
High throughput from the
parallelization of sequencing reactions
Sequence native DNA in real time
with single-molecule resolution
FROM $1,460
le DNA sequencing, or cDNA sequencing on smaller, single-use flow
500-1,000 bp fragments
~50-500 bp fragments
Tens of kb fragments, on average
Short-read sequencing
Long-read sequencing

• Human Genome Project (HGP) was an international scientific research project with the goal of determining
  the base pairs that make up human DNA
• It started in 1990 and was completed in 2003 with a total cost of $2.7 billion (equivalent to about $5 billion
  in 2021)

Blood Cell Counters (Coulter Counter)

BLOOD CELL COUNTERS (Coulter Counter)

• The blood cell counter count the number of
  RBC or WBC per unit of volume of blood using
  electrical method called aperture impedance
  change
  O
  17.5"
  1.0"
  10.4"
  1

Aperture Impedance Principle

Aperture impedance
Internal
Electrode
Aperture
External
Electrode

• When blood is diluted in the proper type of
  solution, the electrical resistivity of blood cells is
  higher than the resistivity of the surrounding fluid
• The sensor consists of a two-chamber vessel in
  which the dilute blood is on one side of barrier,
  and the waste blood to be discarded is on the
  other
• A hole with a small diameter (50 um) is placed in
  the partition between the tow halves of the cell
• measures the change of resistance when blood
  cells pass through the aperture