Block 4

UNIT 14 NANOBIOANALYTICAL
TECHNIQUES
Structure

14.0 Introduction

14.1 Objectives
14.2 Nanopore Sequencing

14.2.1 DNA Sequencing

14.2.2 Nanopore sequencing

14.2.3 Types of Nanopore sequencing

14.2.4 Pros and cons of Nanopore sequencing

14.3 Nanowires

14.3.1 Introduction

14.3.2 Synthesis of Nanowires

14.4 Nanogold

14.4.1 Properties of gold nanoparticle

14.4.2 Surface Plasmon Resonance

14.5 Nanoscale Optofluidic Sensor Array

14.5.1 Introduction

14.5.2 Nanofluidics

14.5.3 Assembly and Working

14.5.4 Advantages and disadvantages

14.6 Application of Bio-analytical Techniques in Environmental Monitoring

14.6.1 Introduction

14.6.2 Nanocontacts

14.6.3 Cantilever Sensor or nanoarm

14.6.4 Nanowires

14.6.5 Conducting polymer

14.6.6 Peptide nanoelectrode

14.7 Let Us Sum Up

14.8 References and Suggested Readings

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 Nanobioanalytical
14.0 INTRODUCTION Techniques

Nanotechnology is a new and exciting interdisciplinary branch of science which

has opened up unexplored avenues in analytical, diagnostic and synthetic fields.
This new field demands synergy of all branches of sciences and has also
benefitted all branches of science like never before. As this field is still evolving
new and exciting developments are taking place every day. We will learn about
some of the basic principles of nanotechnologies which have fine-tuned our
foresaid scientific abilities. We will learn how it has benefitted the biological
field by taking an example of Nanopore sequencing where we can now read
the sequence of monomers in a polymer, for example, base pairs in DNA and
RNA. We will learn how nanostructures are engineered by studying about
nanowires. We will also learn how properties of nanoparticles can be exploited
to develop analytical techniquesby taking the example of nanogold. In the last
we will learn how all the above expertise can be put together to develop sensory
array.

We will also learn how all of these are helping us in monitoring various
parameters of environment.

14.1 OBJECTIVES
After studying this unit, you should be able to

z Understand nanopore sequencing

z Describe nanowires and their synthesis
z Describe various properties of nanogold
z Explain nanoscale optofluidic sensor array and its working method
z Explain applications of Bio-analytical techniques in Environmental
monitoring

14.2 NANOPORE SEQUENCING

14.2.1 DNA Sequencing
The DNA contains four base pairs viz adenine, cytosine, guanine and cytosine.
The sequence of these base pairs in DNA determines its function which in turn
regulates the entire growth and development of the organism. Scientists have
always attempted to unravel this mystery in order to get a better understanding
of the functioning of genes, biochemical pathways, diseases which can be
prevented by intervening these sequence, history and evolution of species and
so on.

Sanger introduced chain termination method for DNA Sequencing. This process
required multiple copies of the DNA to be sequenced in addition to fluorescent
dye to help identify the four bases separately. Maxim and Gilbert Method
required radioactive labeling of one end of the DNA to be sequenced and
hazardous chemicals for chain termination reaction. In addition their method
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Block 4 could not be scaled up. DNA sequencing in Human Genome Project was done
using Sangers method. It took 13 years to sequence entire human genome at a
cost of approximately 2.5 billion dollars. Now new technologies are available
which have drastically reduced the time and money involved in DNA
sequencing.

14.2.2 Nanopore sequencing

This method is used for sequencing any polymer e.g. base pairs of RNA, DNA
and amino acid sequence of proteins.

This method employs a nanopore through which the polymer to be sequenced

is passed through. The nanopore is present on a matrix. This matrix can be a
natural or synthetic biological membrane or some solid surface. A constant
DC electric current is passed through the matrix. The intensity of the electric
current changes when anymolecule is present in the nanopores.This changed
electric current is related to the structure of the molecule occupying the pore
at that time.The units of the polymers are read one by one in sequence as they
pass through the pore. Any change in the structure of the molecule or mutation
is immediately known.

14.2.3 Types of Nanopore sequencing

Nanopore sequencing can be of 3 types

A. Membrane/Protein based nanopore sequencing

B. Solid state nanopore sequencing

C. Hybrid nanopores

A. Membrane/Protein based nanopore sequencing:

Many bacteria secrete haemolytic proteins to kill other bacterial species.

Hemolytic proteins cause lysis of cell membranes. It creates nanopores in
the membrane through which solute, water and other vital molecules leak
out and result in death of susceptible bacteria. Staphylococcus aureus
secretesalpha-heamolysin protein. It is used to synthesize nanopore. Alpha-
heamolysin protein is able to spontaneously bind to lipid and protein
layers.It is mushroom shaped protein.The stem protrudes into the
membrane of the lipid. The cap encloses a pore which continues in stem..
The diameter of the pore is 1-5nm.The diameter of the pore is minimum in
the stem.Size of the pore is large enough to allow just a single stranded
DNA to pass through, one nucleotide base at a time. A constant electric
current is passed across the membrane. When a DNA base enters into the
pore (which has a negative charge as no histones are attached to it) the
electric current is interrupted or changed. The intensity and time for which
the electric current was stopped or altered can be correlated to the structure
and properties of the monomer base or unit.

Mycobacterium smegmatis porin A (MspA): It is a porin present on the

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 outer cell wall and membrane. It helps the bacterium to absorb water soluble Nanobioanalytical
Techniques
nutrient from surrounding. It is a tunnel likeprotein structure with rotational
and octomer symmetry. It has a goblet like structure with broader side
outwards. It is narrower than alpha-heamolysin protein. Its narrowest
diameter is approximately 1nm.

B. Solid state nanopore sequencing:

Although membrane based nanopores are very stable, reproducible, cost

effective and widely used also but they have their own set of problems.
One of them is their stability. The stability is affected by pH, temperature
and other external factors. The other problem with them is their diameter
which cannot be increased or decreased as per need. Solid state nanopores
are engineered nanopores of desired diameter and structure. These are
most commonly made of silicon compound or grapheme.

C. Hybrid nanopores:

This combines best features of both the above types. For example when
membrane nanopore can be embedded in the solid matrix it gets a firmer
support which increases its stability. The diameter of the pore can be
reduced below 5 nm which is not possible in solid state nanopores.The
conductivity of the matrix can be according to the requirement.

14.2.4 Pros and cons of Nanopore sequencing:

Pros

1. Very large fragments of genome can be read at once which helps in

detection of structural variations in DNA.

2. Does not require multiple copies of genome to be made.

3. Very cost effective - the entire human genome can be sequenced under
1000 US Dollars.

4. Real time analysis.

5. Commercial hand held sequencers are available. egMinION by Oxford

Nano Pore Technology

Cons:

1. The results may not be very accurate.

2. It produces lots of background noise.

3. The speed of the DNA molecule travelling through the pore need to be
better controlled otherwise some molecules may go undetected.

4. Cannot read very large sequence of DNA molecules at a time as the pore
may rupture.

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Block 4 Check your progress 1

Note: a) Use the space below for your answer.

b) Compare your answers with those given at the end of the unit.

Q.1 How are the hemolytic proteins used in the membrane/Protein based
nanopore sequencing?
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Q 2 What are hybrid nanopores? What advantages they have over other
nanopore sequencing methods?
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14.3 NANOWIRES
14.3.1 Introduction
Fibres or wires are the structure with its length larger than its diameter.
Nanowires have their diameter in nanoscale. Their length can be in microscale.
Nanowires are also known as nanofibres, nanorods or nanotubes (carbon
nanotube being most widely used among the nanotubes) etc. As the diameter
of the fibre decreases its strength increases. These have immense application
in electrical conduction, signal transmission, material delivery and deposition.
As with other nanoforms, nanowires also show different properties from bulk
material which affects its conductivity- electrical as well optical and magnetic
properties. Nanowires can be used as a Lego piece to assemble a larger structure.

14.3.2 Synthesis of nanowires

Synthesis of nanowires can be either top-down or down-top.

A. Template Assisted: A solid template with pores is used to synthesize

nanowires. By this method nanowires with more uniform diameters are
produced. The material of which nanowire is to be made is filled in the
pores. Afterwards the template is removed and nanowires are obtained. A
template having compatible physical and chemical properties with regards
to the material to be filled in is chosen. It can be mica wafer, metal plates,
glass surface or any other material.

B. Use of pressure injection: This method is used to get a metallic nanowire

of uniform thickness. The substrate used for synthesizing nanowire has
pores of required diameter. The molten metal has to be filled in the pores.
The surface tension of the molten metal will pose a hurdle in this process
and the metal will not be able to get inside the pore. To overcome the
surface tension external pressure has to be applied. Smaller the diameter
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 more is the pressure required. For this hydraulic pressure injection or gas Nanobioanalytical
Techniques
pressure injection technique is used.

C. Electrochemical deposition: Nanomaterials are deposited with the help

of electrochemical reaction. The disadvantage is that the dimension of the
wire cannot be controlled.

D. VLS Method (Vapor Liquid Solid method): In this method super saturated
vapor of synthesizing material is directed on an inertsupport made of
nanoparticles.As the vapor cools down, the material is deposited on the
support in crystalline form. The length of the wire will be proportional to
the time of deposition of vapor.

E. Electro deposition/ sharp edge deposition: A layer of nanomaterial can

be deposited on an edge of the substrate. But it is difficult to separate the
deposited material from the substrate.

F. Arc Method: In this method two pure graphite rods are used in an inert
environment as cathode as well as anode. Graphite used is 99.9% pure.
The type of impurity affects the structure and properties of nanotube.
Helium gas is used to maintain inert condition. A direct current of 50-100
A and a voltage of 20-50 V is maintained between the electrodes which
are 1mm apart. It creates an arc with very high temperature which results
in deposition of carbon (not soot) in nanotube form on the cathode.

G. Laser vaporization: Laser beam is used to vaporize the material of which

nanowire has to be made. It is generally a metal. The vaporized metal
particles then react with the gas present in the medium and condense as
nanoparticle.The particles are deposited over each other and result in a
rope or web like structure.

Check your progress 2

Note: a) Use the space below for your answer.

b) Compare your answers with those given at the end of the unit.

Q. 1 How the nanowires are synthesized using Arc method?

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14.4 NANOGOLD
14.4.1 Properties of Gold Nanoparticles
You have already learned before that nanoparticleof material exhibit different
properties from its bulk material. These properties are a function of the size,
shape and aggregation of the nanoparticles.As with other nanoparticle, the
surface area of gold nanoparticle is large as compared to its volume, quantum
size effect and electrodynamic interaction. This large surface area affects
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Block 4 physical and chemical properties and surface charge of Au Nano. By controlling
shape and size of the nanoparticle it is possible to create nanoparticle of specific
properties. These specific properties are governed by the application we want
to put them to.

Shape: Nano Au particles can be spherical, hexagon, octagon,ring, rod,

star,diamond, flower shaped.

Size:Nanoparticle are sized between molecules and bulk material. On the basis
of size gold nanoparticles are of 3 types viz colloids,clusters and quantum
dots. Colloids range from 1-100nm in diameter. They are prone to aggregation.
Aggregation can be prevented by a using a substance that can adsorb over its
surface and hence prevent aggregation of nano particle. Clusters are less than
10nm in diameter and are covered by chemical ligands.

When gold colloids occur in cluster form, these are called colloidal cluster.
These are covered with alkenethiols or proteins.

Quantum dots: these are a cluster of definite number of Au nanoparticles

which are stabilized by a chemical monolayer. These exhibit fluorescent
luminance hence are used for lightening and color display.

Optical properties:One of the interesting opticalproperties exhibited by gold

nano particle is Surface Plasmon resonance.

14.4.2 Surface Plasmon Resonance

When the electromagnetic waves(e.g. light) strike a solid surface, it causes
polarization of its electron cloud. The electrons present at the surface of solid
material are collectively called Plasmon. Nanoparticles are much smaller than
wavelength of light. When light strikes nanoparticles its electron cloud is
delocalized. The energy of light is transferred to plasmons at a particular
wavelength. This causes all the free electrons on the surface of metal to
undergophased oscillation. This phenomenon is called Surface Plasmon
Resonance or SPR. SPR can be used in analytical techniques, as a probe.

Surface Plasmon results in different color of gold nanoparticle depending upon

its shape and size like spherical gold nanoparticles of 100nm or less are deep
red in color whereas for particles above 100nm it is blue in color.

Colloidal gold was used in ancient times to color glass. These glass articles
changed their color depending upon the light source.

Plasmonics: scientific and technological application of optical properties of

metal nanoparticles and nanostructures.

Magnetism: Bulk gold is dimagnetic whereas nanogold exhibits magnetic

properties. Its magnetism is maintained at high temperature also(400k). Many
theories have been put forward to explain magnetism in gold nanoparticle but
still there are gaps in our understanding. Though what we know is the
magnetism is due to change in spin of electron of outermost shell.

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Solubility: A material is soluble in polar or non –polar solvent. Bulk metals Nanobioanalytical
Techniques
are insoluble in water. Gold nanoparticle when coated with thiols develops
hydrophilic property and disperses uniformly in solvents. If the structure of
thiol is altered the Au nanoparticle can be made to dissolve in polar solvent. If
its surface can become amphipholic hence it can dissolve in polar and non-
polar solvents. Gold is an inert material and is not rejected by the body. If it
can be dissolved in aqueous and non-aqueous solvents, it can be used for
diagnosis, as a carrier molecule for drugs and other medical application. The
solubility of Au nanoparticle is size and temperature dependent. The particles
below 5 nm are preferred for make gold solution.

Check your progress 3

Note: a) Use the space below for your answer.

b) Compare your answers with those given at the end of the unit.

Q. 1 How the solubility of gold is changed for its specific roles?

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14.5 NANOSCALE OPTOFLUIDIC SENSOR

ARRAY
14.5.1 Introduction
Optofluidics combines the principles of two fields of science i.e optics and
hydraulics. It has been used to form fluid lens with the help of one or more
liquids when limitations of glass lens became evident (e.g. mirror lens of Hubble
telescope). It manipulates the meniscus of the same liquid to form concave or
convex lens. This technology is used to form lens of telescope (using mercury),
camera of smart phones (using one conductive and one non-conductive liquid).
The meniscus of the liquid working as lens can be controlled byapplying electric
current across it.

14.5.2 Nanofluidics
Nanoscale optofluidic technology is one of the most recent fields of research
in nanotechnology. The resulted technology has made bio-imaging, sensors,
and lab- on -chip more precise. This is very useful in quality control as single
molecule detection of pathogen or pollutant without any mounting media is
possible and real time results are generated.
When a fluid is constrained in a structure of nanoscale dimension it is called
nanofluidics. As with other nanomaterials, the property of nanofluids is different
from those confined in larger structure. The thermodynamic properties,
viscosity and reactivity of the liquid is changed at the nanoscale.
At nanoscale fluids do not follow Newton’s law for fluids but follow Reynolds
Equation.
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Block 4 14.5.3 Assembly and Working
Assembly: Nanoscale optofluidic sensory array has following parts. A chip,
nanofluidic channels,nanovalves, nanopump, and source of light

Chip for fabrication of Nano channels: The chip can be of glass, silicone,
borosilicate etc. The choice of economic material like plastic can bring down
the cost of the set up.

The nanofluidchannels: The fluid channels are equivalent to wires in the

circuit. Their depth is in nanodimention. Some of the methods by which
nanochannels are madeis by engraving it on a base material by dry or wet
engraving, different types of lithography (e.g. photolithography, interference
lithography),mouldingpolydimethylsiloxane (PDMS) processing,deposition
and bonding, peeling, molecule deposition or 3D printing. New methods are
added on a frequent basis.A laminar of nanochannels is fabricated on a single
chip to perform many reactions or detections simultaneously. So this is known
as Lab-on- chip technology.

Nanovalve: These can be the molecules sensitive to astimuli like pH,

temperature or a substrate that can precipitate or dissolve in response to these
stimuli. The difference in the densities of the liquids can also act as nanovalve.

Nanopump: The charge difference is maintained at the two terminals which

provides electromagnetic gradient for molecules to move in the desired
direction. This electromagnetic gradient acts as a nanopump.

Light Source: A stationary source of light is used.Usually it is a laser. It can

be a dye induced laser,fluorescent laser etc. Optical crystals can also be used
to provide illumination.

Working: The sample containing particle of interest is introduced in the

channel. With the help of nanovalves andnanopump it is propelled towards
that part of channel where light photons are incident. The light is incident
across the channel from opposite side on the sample. The photons from the
opposite side transfer their momentum to the particle under consideration.
The momentum from two opposite side controls the speed of the particle in
the channel. The interaction between the light and particle results in difference
in wavelength of incident and reflected ray. This data is used to characterize
the particle of interest.

Nanotweezers: Many a time the biological molecule in the sample may fold
on itself and do not show 3D configuration. To study such
moleculesnanotweezers are used. Nano tweezers stretch the molecule so that
it can be studied.For it the sample is attached on one end on nanotube or wire
and is manipulated by laser beam. The beam is so designed that it adapts to the
structure of the sample molecule. It does not increase the temperature of the
sample so is safe for thermo labile molecules.

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14.5.4 Advantages and disadvantages Nanobioanalytical
Techniques
Advantages:

1. Extremely small amount of sample is required.

2. Mounting medium andmarkers are not required hence the original structure
of sample is maintained.

3. Results are obtained in real time

4. Many parameters can be measured simultaneously

5. High precision

6. A single molecule can be detected

7. Number of molecules can be counted

8. Can be used for quality control, biological and chemical sensing

Disadvantages:

1. Fabrication of channel and machinery set up requires a very high level of

expertise and precision.

2. Can produce high amount of background noise

3. Blockage of nanochannel by macromolecules.

Check your progress 4

Note: a) Use the space below for your answer.

b) Compare your answers with those given at the end of the unit.

Q. 1 What are nanotweezers? What is their application?

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14.6 APPLICATION OF BIO-ANALYTICAL

TECHNIQUES IN ENVIRONMENTAL
MONITORING
There are several applications of Bio-analytical Techniques in Environmental
Monitoring:

14.6.1 Introduction
Environmental monitoring is the first step in controlling environmental
pollution. In today’s fast paced and data driven world, monitoring devices
should be portable, inexpensive, highly accurate, extremely sensitive, use
minimum sample quantity, be uncomplicated to use and give results in real
time. We should be able to use it in field, our homes and in labs. Nanotechnology
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Block 4 is fulfilling the demand for development of future miniaturized electronic and
optical devices and systems which are calledLab-on-chip. Various
nanotechnology driven analytical sensors have been developed and are being
used on commercial scale today and many more are in different states of
standardization for release in market. These sensors are able to detect very
small concentration of pollutant in the range of ppm and give the result in the
form of readable electrical signal.

14.6.2 Nanocontacts:
These are made up of two nanoelectrodes separated by a gap of molecular
width of target pollutant. This circuit is assembled on a silicon chip. When the
sample is loaded on the chip, molecules of pollutants settle between the nano-
electrodes. So these molecules fill in the gap between the nanoelectrode and
hence bring them in contact. This contact results in jump in conductance. Hence
presence of pollutant at molecular level can be detected. This method can be
used to detect heavy metal in water.

14.6.3 Cantilever Sensor or nanoarm:

This sensor is made up of silicon cantilever array which has a nanocoating of
material that can attract the specific pollutant.These cantilevers are 10-500
nanometers long and thickness is less than few micrometers. When these sensors
are exposed to samples then the pollutant settle on the nanocoating of the
cantilever sensor. This causes cantilever sensor to bend. The angle of the bend
is detected by a laser beam. The degree of bend can be correlated with the
amount of pollutant deposited. This is used to detect the presence of heavy
metals, pesticides, bacteria etc.

14.6.4 Nanowires:
Single Walled Nano Tubes (SWNT)is used to detect gases like NO2 and NH3
or any biological entity. These SWNT are coated with material to detect specific
gaseous or biological entity.The gas molecule or the biological entity directly
binds to the coating over the SWNT. As a result of this the electrical conductivity
of the sensor either increases or decreases from the normal value. SWNT
nanosensors work at room temperature whereas to detect these gases
conventional sensors require a temperature of 200-6000C. These can be used
to detect pathogen in environment.

14.6.5 Conducting polymer:

Conducting polymers were discovered in 1977 by Heideki Shirakawa,Alan
Heeger and Alan MacDiarmid. They were awarded Nobel Prize,”for the
Discovery and Development of Conductive Polymers”. Before this discovery
polymers were used as insulators. Polymers with conjugated double bond can
function as conducting polymers because they have sigma bond (a strong bond)
and a pi bond [weak bond] localized over conjugated double bond. In addition
the structure of the polymer is disturbed by doping which is either adding
electrons or extracting electrons in the polymer. The doping produces empty
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holes in which charge can move and thus they become conducting. The most Nanobioanalytical
Techniques
common conducting polymer is polyaniline. In the sensors using conducting
polymers, a conducting polymer layer is deposited between the electrodes. On
this conducting polymer layer enzyme or binding agent for target molecule is
immobilized. When the sample is loaded on the sensor there is change in
conductance and the signal can be read. These sensors can detect organic
pollutant, pathogens, and allergy causing chemicals in environment.

14.6.6 Peptide nanoelectrode:

It works on the principle of thermocouple. In thermocouple, two dissimilar
electrical conductors form electrical junction at different temperature. In peptide
nanoelectrode, a peptide molecule is placed at ‘nano-distance’ separation gap,
to form a molecular junction. When a specific metal ion is bound to the gap,
the electrical current will result conductance in a unique value. Thus, the metal
ion will be identified easily.

Check your progress 5

Note: a) Use the space below for your answer.

b) Compare your answers with those given at the end of the unit.

Q. 1 What are peptide nanoelectrodes? What is their principle of working?

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14.7 LET US SUM UP

Nanoparticles have found their applications in various fields like agriculture,
health, science and technology. In recent times they have also become very
useful in the environmental technology. Nanoparticles have been synthesized
by different methods depending on their applications and cost factor. Different
types of nanostructures have been constructed for different type of functions.
Nanowires (or nanotubes) have found greater applications in science and
technology. Nanoparticles and nanostructures are also being used as
bioanalytical techniques for environmental monitoring.

ANSWERS TO CHECK YOUR PROGRESS

Check your progress Exercise 1

Q1 Your answer must include the following points:

z Membrane/Protein based nanopore sequencing,

z Hemolytic proteins

z mechanism of creation of pores by hemolytic proteins

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Block 4 Q2 Your answer must include the following points:

z Nanopores sequencing,

z Hybrid nanopores and their advantages

Check your progress Exercise 2

Q.1 Your answer must include the following points:

z Nanowires

z Methods used to synthesized nanowires

z Arc method

Check your progress Exercise 3

Q.1 Your answer must include the following points:

z Nanogold

z Solubility of nanogold

Check your progress Exercise 4

Q.1 Your answer must include the following points:

z Nanotweezers

z Applications of Nanotweezers

Check your progress Exercise 5

Q.1 Your answer must include the following points:

z peptide nanoelectrodes

z working principle of peptide nanoelectrodes

14.8 REFERENCES AND SUGGESTED READINGS

1. Introduction to Nano: basics to nanoscience and nanotechnology by
Amretashis Sengupta (Editor); Chandan Kumar Sarkar (Editor)

2. Microfluidic Devices in Nanotechnology by Challa S. S. R. Kumar (Editor)

3. Soft Matter Nanotechnology by Xiaodong Chen; Harald Fuchs

4. Nanotechnology: Principles and Practices by Sulabha K. Kulkarni

5. Sudeep Mandal and David Erickson, “Nanoscale optofluidic sensor arrays,”

Opt. Express 16, 1623-1631 (2008)

6. Gold Nanoparticles for Physics, Chemistry and Biology by Catherine Louis

and Olivier Pluchery

7. Nanowires Building Blocks for Nanoscience and Nanotechnology Authors:

Zhang,Anqi, Zheng,Gengfeng, Lieber, Charles
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8. Environmental Monitoring By G. Bruce Wiersma Nanobioanalytical
Techniques
9. Bioanalytical Techniques by Abhilasha Shourie and Shilpa S
Chapadgaonkar

10. Nanoelectronics: Materials, Devices, Applications. Editor(s): Dr. Dr. h.c.

Marcel Van de Voorde, Robert Puers, Dr. LivioBaldi, Dr. Sebastiaan E
van Nooten

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