MEVE-018 Sampling and

Preservation

Instrumentation Techniques
Indira Gandhi National for Environmental
Open University
School of Monitoring

BLOCK

4
Bio Analytical Techniques
UNIT 12
Biosensors 291

UNIT 13
Microarrays 308

UNIT 14
Nanobioanalytical Techniques 324

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Separation Techniques

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 Sampling and
BLOCK INTRODUCTION Preservation

Block 4 BIO ANALYTICAL TECHNIQUES

This block focuses on theBioanalytical Techniques.This block contains three

units.

Unit 12 deals with Biosensors. The unit gives a detailed account of various
types of Biosensors which is a biological sensor. It offers many rewards over
conventional detection and monitoring techniques for broad spectrum of
environmental contaminants. The function of biosensor depends on the
specificity of the biologically active bioreceptor. It also discussed about the
sensitivity and selectivity of the bioreceptor presents a chance for the
development of exceedingly specific devices for real-time analysis in complex
mixtures, without the requirement for extensive sample pre-treatment or large
sample volumes.

Unit 13 deals with Microarrays. In this unit on microarray, we have discussed

about the principle and applications as ananalytical tool. DNA microarrays
have provided a potential and powerful tool to perform molecularbiology and
clinical diagnostic assays. DNA microarray technology immobilizes
micrometer-sized known DNAsequences called probes on a solid surface and
specifically hybridizes a complementary sequence of the analyte DNA or a
target. A fluorescently labeled reporter facilitates fluorescent detection of the
presence or absence of a particular target or gene in the sample.

Unit 14 deals with Nano bioanalytical Techniques.This unit mainly focused

onNanoparticles that 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. This unit explained the
synthesis 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. At the end of this unit, it explained about various applications
as bioanalytical techniques for environmental monitoring.

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Block 3

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 Biosensors
UNIT 12 BIOSENSORS
Structure

12.0 Introduction
12.1 Objectives

12.2 Environmental Pollution and conventional techniques

12.3 Biosensors

12.3.1 Working of Biosensors

12.4 Classification of Biosensors

12.4.1 Electrochemical Biosensors

12.4.2 Amperometric device1

12.4.3 Cyclic Voltammetry

12.4.4 Conductometry Biosensors

12.4.5 Potentiometric Biosensors

12.4.6 Optical Biosensors

12.5 Application of Biosensors

12.6 Let Us Sum Up

12.7 References and Suggested Readings

12.0 INTRODUCTION
The vast presence and increasing number and amount of potentially harmful
and toxic pollutants in the environment call for the need of rapid and
economical analytical techniques that can be used extensively in environment
monitoring programs. In the last few years, a rising number of proposals and
legislative actions to control environmental pollution have been adopted.
Moreover, nowadays there in massive scientific and social concern in this
area. Learner form a metropolitan city will definitely come across the need to
control environmental pollution. Even in remote and environment hazardous
areas, the need to detect, analyze and control for environment pollutants is
gaining ground. The application of most traditional analytical methods for the
detection and analysis of environmental pollutants often constitutes acentral
impediment for their applications, in particular for environmental
monitoring.There is an urgent need for the development of new technologies
and more suitable methodologies. In such prevailing context, biosensors appear
as a fittingchoice as a complementary analytical tool. Biosensors are
considered as a subgroup of chemical sensors in which a biological mechanism
is used for analyte detection.

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Block 3 The main advantages offered by biosensors over traditional analytical
techniques are the prospect of portability, miniaturization, efficient work on-
site, and the capability to measure pollutants in complex matrices with minimal
sample preparation. Biosensors can be used as environmental quality
monitoring tools in the assessment of biological/ecological quality or for the
chemical monitoring of both inorganic and organic priority pollutants.
Biosensors also guarantee highly sensitive, rapid, reproducible and simple-to-
operate analytical tools. In the present unit we will discuss several aspects of
biosensor and its working as well as its applications.

12.1 OBJECTIVES
After reading this unit, you will be able to:

z describe an overview of biosensor systems for environment applications;

z explain the working of biosensor
z classify the biosensor
z determine the applications of biosensor

12.2 ENVIRONMENT POLLUTION AND

CONVENTIONAL TECHNIQUES
Massive industrialization, transportation and extensive usage of chemicals in
agriculture, have contributed to the increase of many hazardous and toxic
elements in air, soil, and water, which cause environmental pollution. The
determination of these lethal compounds in environment depends generally
on two concepts. Firstly, the pollutants are recognized and measuredwith the
help of traditional analytical techniques such as gas chromatography (GC/
MS) or high performance liquid chromatography (HPLC/MS). A major
limitation of these techniques is that they are timeconsuming. Several steps
involved in usage of these techniques such as sample preparation and
requirement for pre-concentration is expensive, and, in case of water samples,
detection and analysis cannot be executed easily outside the laboratory. Another
limiting factor is that the usage of these techniques is restricted toonly fewset
of substances. The selection of end compounds may totally beunsuccessful to
determine the most harmful toxic constituents, coming from, for example,
degradation processes. Secondlywithout identifying the compounds their
measurements may or may not allow the evaluation of toxicity of the tested
samples. In the nutshell these techniques for assessing environmental pollutants
are very useful only under certain set of conditions. A wide variety of detection
and toxicity measurement systems exist, including those based on bacteria
and algae, animal cells, small mammals, fish fly, and zooplankton. The available
systems e.g., animals and fish larvae, are relatively not easy and do not provide
a quick response. The usage of these animal systems is also ethically
objectionable. The lack of consistency in the results using these systems raises
the need for developing alternative measures to determine environment
pollutants. Moreover dependence of a single method of detection and
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measurement of toxicity is an insufficient evaluation of adverse biological Biosensors
impact of a pollutant in a generally diverse ecosystem.

Learner should understand that there are several environmental pollutants and
their diverse nature of toxicity on different life forms may be altogether
different. Therefore, several different robust techniques are needed to detect
and analyze the presence of environment pollutants. The detection and analysis
of differentenvironmental (biological and chemical) pollutants has entered in
a new phase during the last decade. Up gradation and improvements in
instrumentation of available techniques have become essential to keep-up with
the requirements to detect the pollutants at low levels (ppb or ppt) range, as
well as to achieve quick and fast results.

12.3 BIOSENSORS
What is a Biosensor?

You will be eager to know about the biosensor. Biosensor is a portable analytical
device composed of the sensing element of biological origin and a physio-
chemical transducer. The International Union of Pure and Applied Chemistry
(IUPAC) has defined biosensor as a self-reliant integrated device capable of
makingdefinite quantitative or semi-quantitative analytical information using
a biological recognition element, i.e.biochemical receptor. The biological
receptor makes contact through spatial with a transduction element. Biosensors
are nowadays used widely in environment pollution control programs. Please
see Figure-1. It shows different components of the Biosensor.

Fig. 1: Components of a Biosensors

Let us discuss different components of biosensor as shown in the above figure:

A) Bioreceptor:As the name suggests, bioreceptor has biological origin, i.e.

whole cells/ microorganisms, tissue or organelles. It may be enzyme,
antibody or nucleic acids or any other biologically derived material (Figure-
2).The bioreceptor chosen should be sensitive to detect the changes and
the level of changes in the environment pollutants.
B) Transducer or the detector element: The principle of working of this
elementis aphysico-chemical way that converts the signal being generated
from the interaction of the analytes, i.e. sample, with the biological element
into another signal that can be more easily measured and quantified.
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Block 3 C) Signal processor: The processor generally displaysthe results in a user-
friendly way.

Fig. 2: Bioreceptor for Biosensors

After learning about the biosensor, learner may be interested in determining

how biosensorworks.

12.3.1 Working of biosensor

A biosensor makes use of inherent biological characteristics of the bioreceptor
or biomatrix with their chemical or physical attributes or properties. The
bioreceptor binds to the analyte and then biochemical interactions are converted
to an electric or an optic signal whose amplitude depends on the concentration
of distinct analytes in the solution. The biomatrix is generated and fixed by
traditional methods, i.e. physical or membrane entrapment, non-covalent or
covalent binding. Biosensor has an interface architecture where biological event
takes place. Furthermore, contact is made between the fixed biological material
and the transducer element. The transducer signal (from the in-coupling angle
of a laser beam to the current produced at an electrode) is transformed to an
electronic signal and amplified by a detector circuit which is then sent for
processing by computer software. The software converts the signal to a
meaningful physical parameter and the resulting quantity is presented through
an interface to the human operator.Sometimes the analyte is changed to a
product which is generally associated with the release of energy gas (oxygen),
electrons or hydrogen ions. In such scenario, transducer converts the product
related changes into electrical signals that can be amplified measured and
displayed using the electronic system. Several types of biosensors have been
designed and developed using the different combinations of bioreceptor and
transducer.
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A successful biosensor should be: Biosensors

1. Highly specific for functional analysis.

2. Stable under standard storage conditions and show a lowerdisparity

between assays.

3. The reaction should be independent and manageable of such physical

parameters as stirring,pH and temperature.

4. Efficient, precise, reproducible and linear over the concentration range

without dilution or concentration.

5. Free from electrical or other transducerinduced noise.

6. Probe must be tiny biocompatible, having no toxic or antigenic effects.

Biosensor should not beprone to inactivation or proteolysis.

7. Economical, portable and operational by semi-skilledpersonnel.

SAQ 1
Do as directed

a. Which one is the physic-chemical component [enzymes/Transducer] (Pick

the correct option?)

b. Give two examples of biologically derived material

c. What is the full form of IUPAC?

d. A successful biosensor should beexpensive and non-linear (True/False)

e. Transducer, b-Nucleic acid, enzymes, antobody, c- International Union

of Pure and Applied Chemistry, d- False.

12.4 CLASSIFICATION OF BIOSENSORS:

Biosensors are typically classified according to the bioreceptor element used
in the biological recognition process (e.g., enzymes, immunoaffinity recognition
elements, whole-cells of micro-organisms, plants or animals, or DNA
fragments), or according to the physicochemical transducer used (e.g.,
electrochemical, optical, piezoelectrical or thermal). The main classes of
bioreceptor elements that are applied in environmental analysis are whole cells
of microorganisms, enzymes, antibodies and DNA. The biosensors are based
on the basis of bio-electrochemistry, the reaction occurring at bioreceptor level
that would either produce a measurablecurrent (amperometric), a measurable
potential or charge accumulation (potentiometric) or visiblyalter the conductive
properties of a medium (conductometric) between electrodes. Other types of
detection techniques includeimpedimetric, which measures impedance (both
resistance and reactance), and field-effect, which uses transistor technologyto
measure current as a result of a potentiometric effect at a gate electrode.

In most of the biosensors used for environmental applications electrochemical

transducers are extensively used.
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Block 3 12.4.1 Electrochemical Biosensors:
The development of electrochemical biosensors is probably one of the most
promising ways to solve problems concerning sensitive, fast and economical
analytical techniques for environment monitoring. Learners are well aware of
the fact that enzymes are well known biocatalysts in the biological systems.
Secondly the enzymes are very specific in nature. So, electrochemical biosensors
are based on enzymatic biocatalysis of a reaction that generates or use electrons.
The electrochemical process involves mainly redox enzymes. Antibodies, nucleic
acids and cells/microorganisms are also some of other bioreceptors.

The sensor substrate typically contains three electrodes; a reference electrode

a working electrode and a counter electrode. An Ag/AgCl made reference
electrode is kept back at a distance from the reaction site to maintain a
recognized and constant potential.The working electrode is also called as
sensing or redox electrode as it serves as the transduction element in the
biochemical reaction and the third electrode is counter electrode that set up a
connection to the electrolytic solution so that a current can be applied to the
working electrode.On the surface of active electrode reaction takes place in
the presence of target analyte that may cause either electron transfer across
the double layer (producing a current) or can contribute to the double layer
potential (producing a voltage). The measurement of current (rate of flow of
electrons is proportional to the analyte concentration) at a fixed potential or
the potential can be measured at zero current.The electrodes used in
electrochemical biosensors are both conductive and chemically stable. Silicon,
gold, platinum or graphite electrodes are generally being used.The chosen
function of a specific electrode, the electrode material, its surface modifications
or its dimensions influences the detection ability of the biosensor.

Nanostructures are the new and important components in recently developed

electrochemical biosensors. Nanowires, carbon nanotubes, nanoparticles
and nanorods are some of the well-known objects thathave shown promise
to becomecritical elements of upcoming bioelectronic devices and
biosensors.

In electrochemical biosensors, you should know that generally in amperometry,

the current is measured at a constant potential and in voltammetry current is
measured during controlled variations of the potential.

12.4.2 Amperometric devices are a kind of electrochemical

sensor.
These electrodes can continuously determinethe current ensuing from the
oxidation or reduction generated in the biochemical reaction involving
electroactive species.Classic example of amperometric devices are Clark
oxygen electrodes.In these electrodes, current generated is in proportion
to the oxygen concentration. The reduction of oxygen is determined at a
platinum working electrode in reference to a reference electrode (Ag/AgCl)
at a given potential. The amperometric devices are believed to be more
sensitive than potentiometric devices.
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12.4.3 Cyclic Voltammetry (CV) Biosensors

Cyclic voltammetry is one of the widely used electrochemical sensors to

get information regarding the redox potential generated by the rate of
electrochemical reaction of analyte. Voltammetry method is based on
analyzing the analyte depending on the measurement of current by varying
the potential. This elctro-analytical method is therefore, an amperometric
technique. There are several different ways to vary a potential resulting in
different forms of voltammetry, such as: polarography (DC Voltage), linear
sweep, differential staircase, normal pulse, reverse pulse, differential pulse
and more.

In cyclic voltammetry, voltage is brushed between two values (V2 and V1)
at a fixed rate. The voltage is determined between the reference electrode
and the working electrode and current is analyzed between the working
electrode and the counter electrode. As you know, when results
(measurements) obtained are plotted as current vs. voltage we obtain
voltammogram. A higher voltage toward the electrochemical reduction
potential of the analyte is followed by an increase in the current. If the
voltage surpasses the reduction potential (V2), then the current will decrease
leading to the formation of peak since the concentration of analyte near
the surface of electrode gets reduced along with the high rise in oxidation
potential.So when the voltage is reversed to V1, the electrochemical reaction
will beginreoxidising the product from the initial reaction. This generates
sharprise in the current of opposite polarity as compared to the forward
scan whichfor a second timefalls, leading to the formation of a second
peak as the voltage scan continues toward V1. The scan rate V2-V1 and the
duration of scan determine the shape of voltammogram for a given analyte.

SAQ 2
Answer the following:

a. _______________ Biosensors uses the movement of electrons produced

during redox reactions. (Fill in the Blank)

b. Voltammetry method is based on analyzing the analyte depending on the

measurement of ____________by varying the __________.

c. Name the three electrodes used in electro-chemical biosensor.

d. Name two metals used in making electrodes.

a- Amperometric biosensor, b-current, potential c- reference electrode,

working electrode and counter electrode, d- Silicon, gold, platinum or
graphite electrodes.

12.4.4 Conductometry Biosensors

Conductometric biosensors are based on the measurement of the capability of
an analyte or a medium(e.g.nanowires) to conduct an electrical current between
electrodes or reference nodes. These biosensors are helpful to determine large
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Block 3 spectrum of compounds on account of several reactions and mechanisms.
Conductometric devices are associated with enzyme catalyzedreactionsthat
leads to changes in the ionic strength or conductivity of a solution between
two electrodes.Therefore, in conductometric biosensorsthe usage or production
of charged species ultimately leads to change in the ionic composition of the
sample to be tested. These biosensors offer several other advantages. Some of
these are:

1) Miniaturisationusing thin-film electrodes and large scale production using

inexpensive technology,
2) No need for reference electrode,
3) Transducers are not light sensitive,
4) Driving voltage isadequately low to reduce the power consumption
significantly,

Generally the liquids analyzedhave low background conductivity, which is

easily customized by different factors.The low selectivity of this method makes
its potential usagedifficult for wider applications.The erratic ionic background
of samples and the requisite to determine small conductivity changes in media
of high ionic strength limit the applicability of conductometric
biosensors.However, with the development of integrated microbiosensor,
severallimitations can be overcome using a differential measuring scheme that
compensates for changes in background conductivity, temperature variations
and other factors.

Conductometric measurement is basicallymeasuring the conductivity of a

solution between two parallel electrodes. The conductivity of liquids used in
biosensors came from the dissociation of the dissolved substance, an electrolyte,
into ions. In an electric field, these ions will migrate to the oppositely charged

Figure 3. Ion migration and electrolyte conductivity in conductometric biosensors.

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electrodes (learners should know that cations will move to cathode and anions Biosensors
will move towards anode). When a potential difference is applied to the
electrode, current generated in the electrolyte is caused by the movement of
ionstowards the electrodes where oppositely charged ions loses their charge
and collected as neutral atoms. Transducer used in conductometric devices is
usually a tiny two-electrode devicedesigned to determine the conductivity of
the thin electrolyte layer adjoining to the electrode surface. These transducers
measure the changes in the electrolyte conductivity.Electrodes made of metals
such as platinum, gold, aluminium, nickel copper, titanium, chromium, silver
and carbon are used as interdigital transducers.

Electrodes from metals i.e titanium, chromium and aluminium electrodes

have low sensitivity to changes in the ion strength of solution and get
saturatedwithin a short span.Microelectronics techniques –
photolithography and vacuum spraying are used to make conductometric
transducers.

Nearly 30% of the total organic carbon, proteins is used as markers of

urban pollution. Proteins are hydrolysed by enzyme proteinase K into
constituent ionic amino-acids that leads to changes in the local conductivity.
Conductometric biosensors using bovine serum albumin (BSA) as standard
protein are being used. Similarly, conductometric tyrosinase biosensor has
been developed for the detection of toxic compounds containing diuron,
atrazine, and copper ions. The presence of formaldehyde in aqueous
solutions can be detected using interdigitated thin-films planar electrodes
immobilized with enzyme alcohol oxidase. A highly sensitive, fast and
stable conductometric enzyme biosensor for determination of nitrate
inwaters has also been developed.

Table enlists several conductometric biosensors developed for monitoring

environmental pollution. Local conductivity changes caused by algae
enzymes such as alkaline phosphatase and acetylcholinesterase enzyme
activities can be determined and measured. Distinct family of toxic
compounds: heavy metals for alkaline phosphatase, carbamatesand
organophosphorous pesticides for acetylcholinesterase are recognized as
potent inhibitors of alkaline phosphatase and acetylcholinesterase.

12.4.5 Potentiometric biosensors

Potentiometric biosensors in an electrochemical cell are based on thebuildup
of a charge potential at the working electrode compared to the reference
electrode when zero or no significant current flows between them (Figure 4).
The ion activity (potential/pH variation) in an electrochemical reaction is
recorded and these determinations are applicable in clinical or environmental
monitoring. The analytical signal is due to changes in the concentration of an
ionic species. In these biosensors biorecognition element is connected withthe
transducer that senses the variation in the quantity of protons (or other ions)
and the recorded analytical signal is logarithmically correlated with the analyte
concentration.
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Block 3

Figure 4: A simple potentiometric biosensor. A semi-permeable membrane (a) surrounds

the biocatalyst (b) entrapped next to the active glass membrane (c) of a pH probe (d). The
electrical potential (e) is generated between the internal Ag/AgCl electrode (f) bathed in
dilute HCl (g) and an external reference electrode (h).

Do you recall Nernest equation from Physics/Chemistry textbooks, it is as

given below:

Ecell = Eocell–(RT/nF) –RQ

Nernst equation shows the relationship between concentration and potential.

EMF or Electromotive force or Ecell = Observedcell potential at zero current.
Eocell = Constant potential contribution to the cell,
R =Universal gas constant,
T = Absolute temperaturein degrees Kelvin,
n =Charge number of the electrode reaction,
F = The Faraday constant
Q = Ratio of ion concentration at the anode to ion concentration at the cathode

Direct measurement of the analyte concentration using Nernst equation is called

as direct potentiometry.The wide analytical range and the low accuracy and
precision of these sensors is due to the logarithmic relationship of the potential
with the ionic concentration. Their normal range of detection is 10-4 - 10-2 M.
Some of them may be ten-fold more sensitive. The response times vary from
one and five minutes allowing up to 30 analyses every hour.Ion-selective
electrodes in potentiometric devices gave the advantage of detecting analytes
at lower levels. However, such electrodes are quiet limited.

The simplest transducer in the development of potentiometric biosensors is

the glass pH electrode. It consists of an immobilized enzyme surrounding the
probe from a pH-meter. The enzyme catalyzing the reaction helps in generating
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or absorbing hydrogen ions. This leads to change in pH which can be read Biosensors
directly. These electrodes help to measure the electrical potential at very high
impedancethat permits zero current flow.

There are three types of ion-selective electrodes which are of use in biosensors:

1. Normal pH Electrodes: These electrodes are also known as glass electrodes

for cations. A concentration-dependent competition between the cations
for specific binding sites produces a transverse electrical potential that is
detected by sensing element composed of a very thin hydrated glass
membrane. The selectivity of this membrane is determined by the
composition of the glass. The sensitivity to H+ is higher than that of NH4+.

2. Glass pH electrodes for carbon dioxide (CO2), NH3 or hydrogen sulfide

(H2S): These electrodes are coated with a gas-permeable membrane
selectivefor CO2, NH3 or H2S.Diffusion of gas through the permeable
membrane causes a change in the pH of a sensing solution which is then
measured.

3. Solid-state electrodes: In these electrodes has a thin membrane of a specific

ion conductor made from a mixture of silver sulphide and a silver halide
in place of the glass membrane. The iodide electrode is helpful for
measuring I- ions in the peroxidase reaction as well as cyanide ions.

Table 1: Reactions involving the release or absorption of ions that may be

utilised by potentiometric biosensors.

(a) H+ cation,

Enzyme: Glucose Oxidase

Enzyme: Penicillinase

Enzyme: Urease (pH 6.0)

Enzyme: Lipase

(b) NH4+ cation,

Enzyme: L-amino acid oxidase

Enzyme: Asparaginase ‘

Enzyme: Urease (pH 7.5)

H2NCONH2 + 2H2O + H+ 2NH4+ + HCO3
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Block 3 (c) I- anion,

Enzyme: Peroxidase

(d) CN-anion,

Enzyme: Glucosidase

12.4.6 Optical Biosensors

One of the important classes of biosensors is optical biosensors (Figure-5).
These biosensors are much more advantageousas they permit direct, real-time
and label free detection of numerous biological and chemical substances. Other
benefits include high specificity, sensitivity andeconomy.The latest
developments in optical biosensors include several advanced concepts with
multidisciplinary approaches such as microelectronics, microelectromechanical
systems (MEMSs), micro/nano-technologies, molecular biology, biotechnology
and chemistry.

The basis of optical detection in these biosensors is the nature of interaction of

the optical field with a bioreceptor/biorecognition element. Generally there
are two modes of optical biosensors: label-free and label-based. In the first
model (label-free), detected signal is produced directly by the interaction of
the analysed material with the transducer. However, in the latter mode (label-
based), label sensing is used and the optical signal is then produced by
spectroscopic methods such as colorimetric, fluorescent or luminescent.

Several biological materials, such as enzymes, antibodies, antigens, receptors

and nucleic acids are used as biorecognition elementsin the optical
biosensors.The biorecognition sensing element is integrated with an optical
transducer system in these biosensors. These biosensorsare compact in nature.
The vitalpurpose of an optical biosensor is to generate a signal proportional to
the concentration of analyte.

Figure-6 : Schematic representation of an optical biosensor

Several optical biosensors are available in the market by multiple

manufacturers. They vary in their nature of construction (biorecognition
element or transducer).
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Surface Plasmon resonance (SPR) based biosensors are currently predominantly Biosensors
used. Pharmacia Biosensor AB, which was later renamed Biacore launched
first commercial SPR based biosensor instrument. The SPR phenomenon takes
place on the metal surface when a polarized light at a certain angle illuminates
at the interface of two media (usually glass and liquid). The event produces
surface plasmons is followed by the reduction of the intensity of reflected
light at a specific angle (resonance angle). The induced effect is proportional
to the mass on the surface. The measurements in the shift of reflectivity, angle
or wavelengths against timehelps to generate a sensorgram.The SPR
phenomenonin all of its configurationsfacilitatenonstop, label-free and real-
time changes of refractive index at the surface of sensor proportional to the
concentration of analyte. SPR instrument has several components such as an
optical detector thatdetects intensity shift, a metal surface (gold) sensor chip
and a layer enabling ligand immobilization, which is incorporated with a fluidics
structure enabling a flow-through operation.

SAQ 3
Answer the following:

a. Optical biosensors have fast response time but the sensitivity is reduced.
(True/False)

b. Voltammetry method is based on analyzing the analyte depending on the

measurement of ____________by varying the __________.

c. Name the three electrodes used in electro-chemical biosensor.

d. Name two metals used in making electrodes.

a- False, b- current, potential c- reference electrode, working electrode and

counter electrode, d- Silicon, gold, platinum or graphite electrodes.

12.5 APPLICATIONS OF BIOSENSORS:

Environmental pollution constitutes five types of pollution i.e water, soil, air,
noise and light. For the safety of environment, technologies that are sensitive,
specific, economical, user and ecofriendly as well as portable with minimal
power consumption are urgently required. Nowadays, biosensorsare extensively

Figure-6: Applications of Biosensor in Environmental Pollution

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Block 3 used as environmental quality monitoring tools for detection and quantification
of pollutants (Figure-6). They are very helpful in the assessment of biological/
ecological quality as well as for the chemical monitoring of both inorganic
and organic priority pollutants.The major applications of biosensors are
discussed below for different types of environmental pollutants such as heavy
metals, organic and inorganic pollutants, toxins, antibiotics and contaminating
microorganisms.

Heavy metals

As you know, accumulation of some of the well known heavy metals such as
copper (Cu), cadmium (Cd), mercury (Hg), Lead (Pb), zinc (Zn), etc., lead
tograve environmental pollution problems. These heavy metalsused in different
fields of industry and/or agriculture have high toxicity index and their
accumulation in the ecological food chain pose serious risk. These metals are
transported by runoff water and contaminate water sources downstream from
the industrial site. Since heavy metals can bind to the surface of microorganisms
and then may get transported inside the cell. Inside cell during the course of
metabolic reactions, these metals can be changed chemically and may disrupt
its functioning.The method to determine traces of heavy metal using biosensors
has a vibrant trend and is mainly applied for improving the “quality of life”,
due to biosensor’s sensitivity, selectivity, and simplicity. Bacterial biosensors
using biocatalysts such as enzyme or DNA as bioreceptors and electrochemical
transducer are being used for detection and analysis of heavy metals. Optical
biosensor based on the inhibition of alkaline phosphate (AP) present on the
outer membrane of microalgae (Chlorella vulgaris)isbeing used to detect heavy
metals such as lead (Pb) and cadmium (Cd).

Biochemical oxygen demand (BOD)

Biochemical oxygen demand (BOD) is one of the prominent parameters used

for characterizing the organic pollution of water and wastewater. BOD is
measured by determining the amount of oxygen required by aerobic
microorganisms for degrading organic matters in wastewater. In routine,
conventional BOD method is the well-known BOD5 which needs 5-day
incubation at 20°C in the dark.

A quick and efficient determination of BOD is possible using biosensors.

Several biosensors have been developed for BOD using the different type of
biological components and multiple strategies of techniques for immobilization.
In one of the biosensor, immobilized Pseudomonas putida bacterium membrane
was placed on the top of an optode that was linked to a photo diode to detect
fluorescence signal. The response time of biosensor is 15min for chloride up
to 1000mg/L.BOD sensor based on immobilizing multispecies BOD seed for
wastewater monitoring has also been developed in the flow system. Some
online systems based on biofilm-reactor-based approach are used for detecting
organic pollutants. A BOD biosensor based on the microbial fuel cell principle
is being used for online and in situ monitoring of biodegradable organic content
of domestic wastewater.
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Nitrogen compounds Biosensors

Nitrogen compounds and its derivatives are commonly used by food industries
as preservatives and as fertilizer in agriculture sector to increase the fertility
of the soil.These compounds also contaminate the surface and groundwater
which can be toxic for aquatic environment. An excessive use of nitrogenous
compounds causes severe adverse effects on human health. Nitrogen
compounds react irreversibly with hemoglobin thereby decreasing its oxygen
carrying capacity. Several researchers have developed different biosensors to
detect and analyze presence of nitrogenous compounds in the environment. A
few of them are listed below:

1- Amperometric biosensor using enzyme (cytochrome c nitrate reductase)

as bioreceptor is used for the determination of nitrate.

2- A veryspeedy, stable and sensitive conductimetric enzymatic based

biosensor has also been reported for the determining presence of nitrate in
water samples.

Polychlorinated biphenyls (PCBs)

As you know, PCBs are well known toxic compounds, and are called as
universal environmental pollutants. The production and usage of these
compounds have been prohibited in several countries. Several biosensors have
been developed to detect PCBs in the environment such as:

i. Chronopotentiometric biosensors with DNA bioreceptor.

ii. Fluorescent Immunosensors.

iii. Electrochemical sensors.

Phenolic compounds

Phenols and phenolic derivatives are widely distributed commonly in the

environment. They are mainly used in the production of dyes, drugs, plastics,
pesticides, detergents, etc. As these compounds are highly toxic, their
accumulation in the environment is hazardous and therefore, their effective
detection and monitoring is essential to save the environment. Some of the
commonly used biosensors for detection and monitoring of phenolics are:

i. Amperometric biosensor with enzyme (tyrosinase) as bioreceptor for

selective detection of phenol in effluent.

ii. A flow-injection chemiluminescence fiber-optic biosensor for the detection

of chlorophenols.

Organophosphorus (OP) compounds

Pesticides are most extensively distributed in water, soil and food. Increase in
the usage of pesticides containing organophosphorus in order to achieve high
productivity in agriculture for controlling pests, weeds and vectors has led to
serious health issues. Organophosphoruses (OP) are toxic compounds that
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Block 3 interfere with the proper functioning of enzyme acetylcholinesterase (AChE)
and finally affect the central nervous system (CNS). Organophosphates are
considered to be highly toxic to bees, wildlife, and humans. So, there is an
urgent need for regular and continuous assessment of these compounds. AChE
based enzymatic biosensors are being reported as the most promising tool for
detection and analysis of pesticides to control toxicity and for environment
conservation. Immunological based amperometric biosensors have also been
developed for determining the pesticides in water. Amperometric and optical
transducers are employed to detect herbicides such as phenylureas and triazines.
OP compounds such as dioxins are polychlorinated compounds are discharged
as byproducts of chemical processes. These are well known potentially toxic
and carcinogenic substances. The surface Plasmon resonance (SPR) biosensors
are used for the detection of dioxins.

SAQ 4

Answer the following:

a. BOD is the amount of oxygen required by aerobic microorganisms for

degrading organic matters in wastewater(True/False)

b. Give full form of PCB.

c. Dyes, drugs, plastics, pesticides, detergents contain __________

compound which are toxic in nature.

d. Organophosphoruses (OP) are toxic compounds that interfere with the

proper functioning of enzyme __________________.

a- True, b- Polychlorinated biphenyls (PCBs), c- Phenol, d-

acetylcholinesterase.

12.6 LET US SUM UP

Biosensor is a biological sensor. Itoffers many rewards over conventional
detection and monitoring techniques for broad spectrum of environmental
contaminants. The function of biosensor depends on the specificity of the
biologically active bioreceptor. The sensitivity and selectivity of the bioreceptor
presents a chance for the development of exceedingly specific devices for
real-time analysis in complex mixtures, without the requirement for extensive
sample pre-treatment or large sample volumes. The components of biosensor
includesbio-recognition element, a biotransducer and an electronic system
composed of a display, processor and amplifier.Biosensors are further classified
on the basis of sensor devices and biological material. They have been applied
in many fields such as food industry, medical sector and monitoring of
environmental pollution. Application of biosensors in environment pollution
involves detection and measurement of various pollutants including heavy
metals, phenolics, organic and inorganic pollutants, toxins and contaminating
microorganisms. There are several challenges in the advancement of cost
effective, efficient, specific and reliable biosensor. However, using the advances
306
in bio- and electrochemistry, solid-state and surface physics, bioengineering, Biosensors
integrated circuit silicon technology and data processing the possibility of a
new generation of widely applicable biosensors is soon emerging.

TERMINAL QUESTIONS

1. Illustrate working of a biosensor.

2. What are the advantages ofconductometric biosensors?

3. Enlist three reactions involving the release or absorption of ions that may
be utilised by potentiometric biosensors.

4. Discuss several applications of biosensors.

ANSWERS
Self Assessment Questions (SAQ):

1. a- Transducer, b-Nucleic acid, enzymes, antibody, c- International Union

of Pure and Applied Chemistry, d- False.

2. a- Amperometric biosensor, b- current, potential c- reference electrode,

working electrode and counter electrode, d- Silicon, gold, platinum or
graphite electrodes.

3. a- False, b- current, potential c- reference electrode, working electrode and

counter electrode, d- Silicon, gold, platinum or graphite electrodes.

4. a- True, b- Polychlorinated biphenyls (PCBs), c- Phenol, d-

acetylcholinesterase.

Terminal Answers:

1. Refer to section: Working of biosensor

2. Refer to section:Conductometric biosensors

3. Refer to Table 6.2

4. Refer to section: Applications of Biosensors.

12.7 REFERENCES:
1. Pavel Damborsky, Jurajd vitel and Jaroslav Katrlik. Optical Biosensors.
Essays in Biochemistry, 2016, 60 91-100.

2. Jitendra Kumar and S. F. D’Souza. Biosensors for Environmental and

Clinical Monitoring, 2012,324, 344-38.

3. Tejpal Dhewa. Biosensors for Environmental Monitoring: An Update. Octa

Journal of Environmental Research. 3(3): 212-218

4. Lívia Maria da Costa Silva. Biosensors for Environmental Applications

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Block 4
UNIT 13 MICROARRAYS
Structure of Unit

13.0 Introduction

13.1 Objectives
13.2 History of DNA Microarray

13.3 Substrates used for Microarray Fabrication

13.4 Preparation of DNA Arrays

13.4.1 Sample preparation and labelling

13.4.2 Array hybridisation

13.4.3 Image acquisition

13.5 Types of DNA microarrays

13.5.1 Glass cDNA microarrays

13.5.2 Oligonucleotide microarrays/ In situ oligonucleotide array

13.6 Advantages of Microarray

13.7 Oligonucleotide Array

13.7.1 Short oligonucleotide arrays: in situ synthesis

13.7.2 Long oligonucleotide arrays: in situ synthesis

13.8 Advantages of Oligonucleotide Arrays

13.9 Disadvantages of Oligonucleotide Arrays

13.10 Applications of Microarrays in Environmental Studies

13.11 Applications of Microarry

13.12 Let Us Sum Up

13.13 Glossary

13.14 Terminal Questions

13.0 INTRODUCTION
A DNA microarray is commonly known as DNA chip or a biochip. It is one of
the most promising methods in functional genomics.It is a collection of
microscopic DNA spots deposited or synthesized in a two dimensional or
three dimensional array on a solid surfacelike glass, silicon chips or nylon
membrane by covalent or non-covalent interactions.It is thus an extension of
Southern and Northern hybridization blots, which have been to detect and
characterize nucleic acids in diverse biological samples. They are used to
measure expression of multiple genes simultaneously or to genotype multiple
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regions of a genome. DNA spotted on the solid surface contains a specific Microarrays
DNA sequence or a short section of a gene known as probes or reporters or
oligos. The sample spot sizes are typically less than 200 microns in diameter.
These probes are further used to hybridize a c-DNA or antisense RNA target
under high-stringency conditions. The probe - target hybridization is then
detected and quantified by using fluorophore (Fluorscent chemical compound
that can re-emit light upon light excitation) or chemiluminescence (emission
of light as the result of a chemical reaction) labeled targets to determine relative
abundance of nucleic acid sequences in the target.

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

z understand various Substrates used for Microarray Fabrication

z describe various methods of preparation of microarrays

z classify DNA Arrays

z explain advantages and disadvantages of microarrays

13.2 HISTORY OF DNA MICROARRAY

Microarray has been thought to be evolved from the southern blotting method.
In 1975, Grunstein and Hognesswas reported to use colony hybridization
method to screen bacterial clones.DNA of interest was arbitrarily cloned into
E. coli plasmids plated agar plates covered with nitrocellulose filters. The
cells were lysed and the denatured DNA was fixed to nitrocellulose filter
producing a random and unordered collection of DNA spots representing the
cloned fragments. A radioactively labeled probe was then added which binds
to the complimentary DNA within the sample. Here a labeled probe is being
utilized to identify complimentary base pair binding and thus can be considered
as one of the first examples of a DNA array. Gerganet al. in 1979 used a
mechanical pin device for placing samplesin well microplates which permitted
the production of arrays for over a thousand different bacterial colonies.

Multiple hybridization targets were automated in the late 1980s and early 1990s
using robotic technology to array clones from microtiter plates onto filters
thereby allowing parallel hybridizations. The development of complimentary
DNA (cDNA) cloning led to the creation of reference sets of cDNA and
corresponding filter arrays for whole genomes. Radioactively labelled cDNA
sequences on nitrocellulose membrane were used byAugenlicht and his
colleaguestostudy the variation in gene expression pattern in various types of
colon tumors in different stages of malignancy. Awide range of oligonucleotides
was used to determine DNA sequence by hybridization methods.The
inconsistent specificity of hybridization limited the use of hybridization
techniques for routine sequence determination. In the mid 1990s the reverse
dot-blot scheme for monitoring genomic expression was developed. In 1995
high-speed robotic printing of cDNA on glass was constructedthrough which
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Block 4 the expression of many genes could be monitored in parallel.Various substrates
like nylon membranes, plastic and glass were used to prepare arrays of DNA
fragments and synthetic oligonucleotides.

13.3 SUBSTRATES USED FOR MICROARRAY

FABRICATION
The quality of data obtained from microarray depends on the substrate used
for printing microarrays. A non-uniform surface will cause variations in the
amount of the attached DNA while low surfacetreatment may result in less
attachment of the DNA probes to the slide.High background fluorescence is
created due to the residual substances deposited on the slide surface during
amicroarray experiment. Thus, the selection of appropriate substrates is the
most critical part in designing a microarray experiment.Two types of substrates
are commonly employed for fabricating microarrays: porous and non-porous
substrates.

1. Non-porous substrates:

The most common type of substrateused for printing arrays is non-porous

solid surface. Non-porous materials like glass andpolypropylene are
suitable for microarray fabrication. The most commonly used substrates
are glass slides because of their inexpensive and physicalcharacteristics
beneficial to hybridization. Apart from this glass slides can be easily
modified for nucleicacid attachment and synthesis.

Advantages of non porous substrates

1. Small amounts of molecules may be deposited at precise, predefined

positions on the substrate surface thus enabling high density capacity of
microarrays.
2. Non-porous solid surfaces molecules do not have to diffuse in and out
ofthe pores hybridization between target and probe occurs at a much faster
rate.
3. High probe concentration, rapid hybridization kinetics and high sensitivity
can be achieved using small sample volumesapplied to a nonporous surface
under a coverslip.
4. Unbound labeled materials can be easily removed as non-porous
substratesprevent the absorption of reagents and samples into pores. This
reduces background and improves reproducibility.
5. Non-porous substrate allows the use of fluorescence detection, the most
critical requirements in large-scale genomic analysis

Porous substrates:

Nitrocellulose and nylon membranes have been used as porous materials for
microarray fabrication. Membranebasedmicroarrays can be reused several times
and the pores in these membrane provide a large surface area for binding which
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enables to immobilize large volumes and concentrations of samples.Capillary Microarrays
flow helps in the immediate distribution of deposited samples into the
membrane thereby obtaining homogeneous spots. Thus, it offers high sensitivity
and betterrange for quantitative comparison of samples.

SAQ 1

1. What types of substrates are used for microarray fabrication?

................................................................................................................
................................................................................................................
................................................................................................................
2. Which type of substrate is best for large-scale genomic analysis?
................................................................................................................
................................................................................................................
................................................................................................................

13.4 PREPARATIONOF DNA ARRAYS

A microarray experiment involves the comparison of expression pattern of
genes in a specific set of conditions and the genes that are expressed in the
cells/tissue to be analysed. The principle of DNA microarray technology is
based on the fact that complementary sequences of DNA can be used to
hybridiseimmobilised DNA molecules (Fig 1). This involves three major steps

1. Microarrays manufacturing: DNA is spotted on a chip or a glass slide

using robotics to produce microarrays

2. Sample preparation and array hybridisation: Fluorescent labelling of cDNA

probes obtained from isolated mRNA or DNAand hybridisation of the
sample to the immobilised target DNA.

3. Image acquisition and data analysis: It uses sophisticated software programs

to scan microarrays and image analysis to quantify and interpret the data.

13.4.1 Sample preparation and labelling

There are various methods to prepare and label samples for DNA microarray.
The term “sample” refers the immobilised DNA is known as reporter element
and the free, fluorescently labelled cDNA.There are a number of different
ways in which a DNA microarray sample is prepared and labelled. The term
“sample” is used to refer the free, fluorescently labelled cDNA and the
immobilised DNA is known as reporter element. The first step in sample
preparation involves isolating total RNA containing mRNA which represents
the quantitative copy of genes expressed at the time of sample collection. Thus
the success of microarray experiment depends on the quality of the RNA. The
sample mRNA extracted from the biological sample of interest and the reference
are separately converted into cDNA using a short primer and reverse-
transcriptase enzymeandlabelled with fluorescent cyanine dyes like Cy3 and
Cy5 for tracking.

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Block 4 13.4.2 Array hybridisation
The process of joining two complementary strands of DNA to form a double-
stranded molecule is called hybridization.The labelled cDNA from sample
and control are mixed together and contaminants such as primers,
unincorporated nucleotides, cellular proteins, lipids and carbohydrates are
removed using filter spin columns. To reduce non specific bindingbefore
hybridisation the microarray slides are incubated at high temperature with
solutions of saline-sodium buffer (SSC), sodium dodecyl sulfate (SDS) and
bovine serum albumin (BSA). The mixed labelled cDNA is then competitively
hybridised against denatured PCR product or spotted on a glass slide. On the
immobilised array the labelled cDNA will bind to its appropriate
complementary target sequence. To increase stringency of the experiment and
to reduce cross hybridisation the slides are washed after hybridisation, to
remove the labelled cDNA that did not hybridise on the array.

13.4.3 Image acquisition

Image acquisition and data analysis is the final step of microarray experiments.
This step produce an image of the surface of the hybridised array. To determine
the quantity of bound labelled cDNA to each target spot the microarray slide
is dried and placed into a laser scanner. A confocal laser microscope measures
the laser excitation spectra of the incorporated targets. To represent genes
upregulated compared to control microarray the software uses green spots
while red spots are used to represent those genes that are downregulated in the
experimental sample and yellow spots represent genes of equal abundance in
both experimental and control samples.

Fig 1. DNA Microarray Synthesis

312
 SAQ 2 Microarrays

1. Mention the steps involved in the preparation of microarrays

................................................................................................................
................................................................................................................
................................................................................................................
2. Define reporter element
................................................................................................................
................................................................................................................
................................................................................................................

13.5 TYPES OF DNA MICROARRAYS

On the type of immobilized probes microarrays can be divided into two major
types. Both types differ from each other in themethods of printing DNA spots
on the slide/chip, size of printed DNA fragments and the images generated
from microarray experiments.

1. DNA microarrays/ Glass cDNA microarraysare constructed by micro

spotting of pre-fabricated cDNA fragments or with DNA fragments
generated using the polymerase chain reaction on a glass slide.

2. Oligonucleotide microarrays/ In situ oligonucleotide arrayformat is

designed with shorter or longer oligonucleotide sequences complementary
to specific coding regions of interest. These are often referred as a chip.

13.5.1 Glass cDNA microarrays

It was the first type of DNA microarray technology. It was pioneered by Patrick
Brown and his colleagues at Stanford University. A robotic device is used to
spot nanoliters of cDNA (50-150 µm in diameter) to a glass slide surface. The
spots are at a distance of approximately 200-250 µm from each other and each
spot represents one gene. On an area of 3.6 cm2these glass cDNA microarrays
bear about more than 10,000 spots. Glass cDNA microarrays use specially
manufactured glass slides with desired physico-chemical characteristics like
chemical resistance against solvents, good mechanical stability and low intrinsic
fluorescence properties (Fig 2).

Manufacturing a glass cDNA microarray involves selecting genes from public

databases/repositories or institutional sources to spot onto the microscope glass
surface. This procedure is followed by the preparation and purification of DNA
sequences representing the gene of interest. Universal primers or gene specific
primers are used to amplify the DNA by polymerase chain reaction (PCR)
from library of interest. The purity of PCR generated DNA fragments are
checked by sequencing or using an agarose gel electrophoresis. To achieve
similar reaction kinetics for all hybridizations the PCR generated DNA
fragments representing genes of interest should be of similar concentration/
molarity.

To spot DNA, the glass slides are chemically modified with poly L-lysine or
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Block 4 other cross-linking chemical coating material like polyethyleneimine polymer,
p-aminophenyltrimethoxysilane. Spotting of DNA sample is done by precisely
controlled robotic pins or other delivering technology like inkjet printing. The
negatively charged phosphate groups in the DNA molecule form ionic bonds
with the positively charged amine-derivatisedglass surface.Spotting is followed
by the post-print processing step. It involves drying of DNA on the slide
overnight at room temperature and the use of UV cross-linking to prevent
subsequent binding of DNA and to decrease the background signal upon
hybridization of a labelled target.

Fig 2. Glass cDNA Microarray

13.5.2 Oligonucleotide microarrays/ In situ oligonucleotide

array
It is a sophisticated microarray technologydeveloped by Stephen Fodor et al.
(1991). It usesin situ chemical synthesis for manufacturing microarrays.
Affymetrix is the industry leader in the field of in situ oligonucleotide
microarrays has pioneered its technology to manufacture high density
oligonucleotide based DNA arrays called GeneChips (Fig 3).

Short single strands of DNA are constructed onto 5-inch square quartz wafers
using photolithography and combinatorial chemistry.The genes on the chip
are designed based on sequence information. An industry chip synthesizer is
used tosynthesize the sequences directly onto the surface of the 5-inch square
quartz wafer at pre-selected positions.

The production of Affymetrix’s GeneChips using DNA photolithography

process starts by the derivatization of the solid support (quartz) with a covalent
linker molecule terminated with a photolabile protecting group. The first step
involved in derivatization is washing quartz to ensure uniform hydroxylation
314
across its surface. The hydroxylated quartz is then immersed in silane where Nanobioanalytical
Techniques
the hydroxyl groups of the quartz react with silane to form a matrix of covalently
linked molecules.

Along with derivatizaton, in situ synthesis of oligonucleotides is carried out

which consecutively adds A, C, G and T nucleotides to the appropriate gene
sequences on the array.At each step in synthesis the oligonucleotide chains
are deprotected by light at the appropriate positions by a mask. The derivatized
quartz (chip) is further flooded with a solution containing activated adenine
nucleotides with a removable protection group, which are coupled to the
deprotected positions. Uncoupled adenine residues are washed away and
another mask is applied to carry out deprotection of the next nucleotide. The
process is repeated approximately 70 times with 70 different masks to
synthesize array of thousands of 25-mer oligonucleotides in parallel.

Fig 3.DNA GeneChip (Affymetrix)

SAQ 3

1. Mention the types of DNA microarrays

................................................................................................................
................................................................................................................
................................................................................................................
2. Describe the type of microarray technology manufactured using in situ
chemical synthesis
................................................................................................................
................................................................................................................
................................................................................................................

13.6 ADVANTAGES OF MICROARRAY

Microarrays offer the following advantages over conventional nucleic acid
based approaches.

1. High-throughput and Parallel Analysis

It allows uniform deposition of thousands ofarray elements or probesin a

very small areaon the surface of a non-porous substrate. It permits the
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Block 4 expression ofthe entire gene content of a genome of interestto build a
complete integrated view of a complexbiological system. It can be also
used to analyzeconstituents of a microbial community

2. High Sensitivity

Microarray hybridization uses a very small volume of probe and the target
nucleic acid in a small area which enables high sample concentrations and
rapid hybridization kinetics.

3. Differential Display

Multifluorescence detection schemes allow differential display of different

biological samples. In a single assay it allows the simultaneous analysis
of two or more biological samples. Different target samples can be labeled
with different fluorescent tags and then hybridized in parallel to the same
microarray. Multicolor hybridization detection minimizes variations
resulting from inconsistent experimental conditions and allows direct and
quantitative comparison of target sequence abundance among different
biological samples.

4. Low background signal noise

The amount of non-specific hybridization can be reduced by using non-

porous surfaces. The organic and fluorescent compounds that attach to
microarrays during fabrication and hybridization procedures can be rapidly
removed by post-hybridization washing, resulting in considerably less
background signal noise than is typically encountered with porous
membranes.

5. Real time data analysis

Easy real-time data analysis is possible as hybridization and detection are

relatively simple and rapid.

6. Automation

Microarray technology is cost-effective compared to traditional

hybridization methods as it is acquiescent to automation.

13.7 OLIGONUCLEOTIDE ARRAY

It is a sophisticated microarray technologydeveloped by Stephen Fodor et al.
(1991). It uses in situ chemical synthesis for manufacturing microarrays.
Affymetrix is the industry leader in the field of in situ oligonucleotide
microarrays has pioneered its technology to manufacture high density
oligonucleotide based DNA arrays called Gene Chips. The commercial versions
of Affymetrix Gene Chips hold up to 500,000 probes in a 1.28 cm2 chip area.
These chips contain a large information of genes and hence widely used in
hybridization based detection and analysis of mutations and polymorphisms
such as single nucleotide polymorphisms, disease-relevant mutations analysis
etc.
316
13.7.1 Short oligonucleotide arrays: in situ synthesis Microarrays

The production of Affymetrix’s GeneChips using DNA photolithography

process starts by the derivatization of the solid support (quartz) with a covalent
linker molecule terminated with a photolabile protecting group. The first step
involved in derivatization is washing quartz to ensure uniform hydroxylation
across its surface. The hydroxylated quartz is then immersed in silane where
the hydroxyl groups of the quartz react with silane to form a matrix of covalently
linked molecules.

Along with derivatizaton, in situ synthesis of oligonucleotides is carried out

which consecutively adds A, C, G and T nucleotides to the appropriate gene
sequences on the array.To remove the exposed groups and to direct light to
predetermined areas on the substrate a mask is used. The de-protected groups
react with bi-functional deoxynucleosides, resulting in chemical coupling. A
new mask is used to direct coupling at other sites, and the step is repeated until
the desired sequence and length of oligonucleotide is synthesized.

13.7.2 Long oligonucleotide arrays: in situ synthesis

The photolithographic method is inconvenient and expensive for the creation
of newarrays with added or different gene content but it is very efficient to
produce thousands or millions of identical arrays. The new method adopted
for the production of new arraysvia synthesisof long 60-mer oligonucleotides
uses ink-jet printingprocess.Modified ink-jet pumps areused to dispense
DNAmonomers onto a hydrophobic surface containing chemically active
hydroxyl groups. The DNAmonomers react and bind to the matrix by
covalentbonds. This step is followed by washing and deprotection. The process
is repeated until the desired oligonucleotide length is reached. This method
offers the advantage that no masks are required and synthesis is faster because
each cycle attaches onebase whiefour cycles per base are required with
photolithography. New arrays can be prodced by programmingthe computer
with directions on how to synthesizethe new set of oligonucleotide sequences.
This versatile system can routinely produce arrays with more than 25,000
elements.

SAQ 4
1. Differentiate between short oligonucleotide and long oligonucleotide
array
................................................................................................................
................................................................................................................
................................................................................................................
................................................................................................................
2. Mention the applications of oligonucleotide array
................................................................................................................
................................................................................................................
................................................................................................................
................................................................................................................
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Block 4
13.8 ADVANTAGES OF OLIGONUCLEOTIDE
ARRAYS
i) In situ oligonucleotide array offers speed, specificity and reproducibility.

ii) Prior knowledge of the genome sequence is required to design the

oligonucleotide arrays. This reduces the time spent in the preparation and
precise determination of handling bacterial clones, PCR products or
cDNAs.

iii) In situ oligonucleotide array format uses multiple, short sequences for
representing the unique sequence of genes which render high specificity
and reproducibility.

iv) Oligonucleotides are synthesized as perfect match and mismatch pairs.

Oligonucleotide sequences designed to be perfectly complementary to a
target gene sequence are called perfect match while the mismatch
oligonucleotide has a one-base mismatch in the center position. This
sequence mismatch strategy increases specificity and helps to identify and
minimise the effects of non-specific hybridisation and background signal.

13.9 DISADVANTAGES OF OLIGONUCLEOTIDE

ARRAYS
i) In situ oligonucleotide array formats need expensive specialisedequipments
to carry out the hybridisation, staining of label, washing, and quantitation
process.

ii) The readymadein situ oligonucleotide array format(GeneChips) are

expensive.

iii) Short-sequences used on the array confer high specificity, they may have
decreased sensitivity/binding compared with glass cDNA microarrays
which can be compensated by using multiple probes.

iv) In situ oligonucleotide array format also offers reduced flexibility.

v) The cost and time needed to manufacture the in situ oligonucleotide is

very high.

13.10 APPLICATIONS OF MICROARRAYS IN

ENVIRONMENTAL STUDIES
Microarray-based technology is well suited for detecting microorganisms in
natural environments. The functional genes involved in biogeochemical cycling
are highly diverse. It is difficult to identify conserved regions for designing
PCR primers or oligonucleotide probes. Microarray based approach does not
require sequence conservation, because the diverse gene sequences from
different populations of the samefunctional group can be fabricated on arrays
and used as probes to monitor theircorresponding populations.Three different
318
types of microarray formats have been developed for use in environmental Microarrays
studies

1. Functional gene arrays (FGAs)

2. Phylogenetic oligonucleotide arrays (POAs)
3. Communitygenome arrays (CGAs).
4. Metagenomic arrays

1. Functional gene arrays

For assessing the physiological status and functionalactivities of microbial

populations and communities in natural environments genes encoding
functional enzymes involved in various biogeochemicalcycling processes
like carbon, nitrogen, sulfate etc. are very useful and can be used
asmolecular signatures.Microarrays containing functional gene sequence
information are called as functional gene arrays, because they are used for
the functional analysis of microbialcommunity activities in environments.
Depending on the part of gene used as probe, functional gene arrays may
detect the same gene in a wide variety of organisms. The information
produced is not of taxonomic nature unless species specific parts of genes
are targeted. The most popular version of functional gene arrays is
‘Geochip’introduced by He et al. (2007). Oligonucleotides and PCR-
amplifiedDNA fragments corresponding to functional genes can also be
used for fabricatingfunctional gene arrays.The probes for the construction
of FGAs can be generated in three ways.

1. Extraction of genomic DNA from pure bacterialcultures followed by

amplification of desiredgene fragments using specific primers or by
using vector-specific primers for plasmid clones containing the desired
gene insert.

2. By using PCR-based cloning methods the desired gene fragments can

be recovered from natural environments.

3. Designing oligonucleotides (usually 50–70-mers) based on the

functional sequences available in public databases and those
synthesized for microarray fabrication.

Since environmental samples contain a mixture of target and non-target

templates, the presence of non-target templates affect microarray based
quantification. Microarray hybridization signal intensities and its quantification
are dependent on the target genes present in environmental samples which
may havedifferent degrees of sequence divergence. Since, very limited studies
have been carried out to evaluate specificity, sensitivity, sequence divergence
and quantitation of DNA microarrays for environmental applications FGAs
for microbial detection are still in the developmental stages.

2. Phylogenetic oligonucleotide arrays (POAs)

Ribosomal RNA genes can be used as a powerful molecule for analyzing

microbial communitystructure in natural environments and to study
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Block 4 phylogeneticrelationships among different organisms. rRNA genes are very
usefultargets for developing microarray-based detection approaches
because of the following reasons.

i. These genes exist in all organisms and contain highly conserved and
variable regions which can be used in differentiating microorganisms
at different taxonomic levels.

ii. A very large database of ribosomalRNA genes is available making

them ideal molecules fordeveloping microarray-based detection tools.

iii. Cells have multiple copies of rRNA genes.

iv. rRNA accounts for the majority of total RNA isolated from a any
samples making their detection sensitivity higher.

Phylogenetic oligonucleotide microarrays (POAs) are oligonucleotide

microarrays containing information from rRNA genes.These microarrays
were primarily used for phylogenetic analysis of microbial communities.
They can be constructed for different phylogenetic taxa and can be used
inmicrobial community analysis studies. The oligonucleotide probes can
bedesigned in varying level of sequence conservation ranging from highly
conserved sequences (for broad taxonomic groupings) to hypervariable
sequences (for genus and species levelgroupings). Since highly conserved
universal primers for amplifying rRNAgenes are available, POA-based
hybridization can be easily coupled with PCRamplification for the
implementation of highly sensitive assays.

rRNAgene-based oligonucleotide arrays are in the early stages of

development and only a few studies have applied POAs in the analysis of
microbial communities from environmental samples.

3. Community genome arrays

It is based on the principle of reverse sample genome probing which does

not require prior knowledge of genome sequences. It uses a non-porous
glass surfaceto spot whole bacterial genome for microarray fabrication
and fluorescence-based detection. A quickand reliable identification of
unknown strains is possibleby hybridizing genomic DNA from unknown
strains with this type of microarray. The main disadvantage of the CGAis
that only the cultured components of a community can be monitored,
becausethe construction requires the availability of pure isolates.With the
recent advances in environmental genomics, high-molecular-weightDNA
from uncultivated microorganisms could be accessed through
bacterialartificial chromosomes (BACs).

4. Metagenomic array

It relies on sequence information from environmental DNA. Probes are

generated using bioinformatic analysis of large dataset obtained by means
of high throughput sequencing of environmental samples. Probes are
designed without any prior knowledge of species. This technique has gained
320
 popularity because sequencing environmental DNA has become much Microarrays
powerful and cheaper using next generation sequencing technology.

SAQ 5

1. Why rRNA genes are usefultargets for developing microarray-based

detection?
....................................................................................................................
....................................................................................................................
....................................................................................................................

2. Define phylogenetic oligonucleotide microarray

....................................................................................................................
....................................................................................................................
....................................................................................................................

1. Multiple and singe channel microarrays

(i) Multiple channel microarrays (Two colour microarray) Genes from

different sample is analyzed in a single test. The target gene is labelled
with fluorochrome having different fluorescence emission. This sample
is hybridized with a single microarray probe in a single test. The ratio
of different gene can be quantified by scanning with microarray scanner
after excitement with different corresponding wavelengths. It makes
use of only one test to give multiple results and is used to detect
different genes present in one mixed sample.

(ii) Single channel microarrays (One colour array) A probe is hybridized

with target DNA labelled with one colour fluorochrome. Itis employed
to estimate the amount of gene expression of same sample or between
samples. They provide more accurate results but different testsare
required to quantify different gene expression.

13.11 APPLICATIONS OF MICROARRAY

i) Diagnostic applications: They can be used to detect the presence of genetic
diseases, mutations, polymorphism, cancer etc.

ii) Gene expression profiling: Monitoring the gene expression is known as

gene expression profiling. Microarrays can be used to detect the number,
quantitative and qualitative expression rate of genes.

iii) Genomics: Single nucleotide polymorphisms(SNPs) refers to different

nucleotides present at the same base position in different alleles. Micro
arrays can be used to identify minute differences between different
individuals or different alleles of same genes

iv) Sequencing: A microarray is prepared with oligonucleotides of specific

length. When hybridized with target sequences of unknown lengths, the
results obtained indicate the length of the target sequence.

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Block 4
13.12 LET US SUM UP
DNA microarrays have provided a potential and powerful tool to perform
molecular biology and clinical diagnostic assays. DNA microarray technology
immobilizes micrometer-sized known DNAsequences called probes on a solid
surface and specifically hybridizes a complementary sequenceof the analyte
DNA or a target. A fluorescently labeled reporter facilitates fluorescent
detectionof the presence or absence of a particular target or gene in the sample.
By usinglaser-scanning and fluorescence detection devices different
targethybridization patterns can be read on the microarray and the results
quantitatively analyzed.DNA microarray technology is a versatile tool for
genomic research and diagnostics, biomedical researchof cancer and genetic
diseases, pharmacogenomics, food safety and quality etc.

13.13 GLOSSARY
1. Probe and Target:

A probe refers to a small nucleotide sequences with known bases. This is

fixed onto a solid substrate to form a neat arrangement called an array.
Purified mRNA, isolated DNA, cDNA produced from mRNA; all can form
probes. Target refers to test a DNA sequence which has to be studied for its
position, composition, gene expression, mutations etc; these are labelled
using fluorochrome or other labels which aid in quantifying the results
after hybridization.

2. c DNA and Oligonucleotide arrays

cDNA arrays:

The probes consisting of complementary DNA is spotted onto the glass

substrate with the help of fine needles and a robotic arm.

Oligonucleotide arrays:

The probes here are oligonucleotides formulated in situ or produced

externally and then immobilized on the arrays by different techniques like
photolithography. Earlier the oligonucleotide array was termed as DNA
chips. But recently the term DNA or gene chips are applicable to both the
kind of array.

13.14 TERMINAL QUESTIONS

1. What are microarrays? What are their applications?

2. What are the main technologies used in microarrays?

3. Describe the advantages and disadvantages of microarray technology?

4. Discuss briefly the microarray process for cDNA microarrays?

5. Discuss briefly the microarray process for oligonucleotides microarrays?

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6. What is the general flow of the microarray process? What are the essential Microarrays
pieces of informationthat need to be stored in each phase of this process?

7. Compare the cDNA and oligonucleotide technologies from a data analysis

perspective.

8. Describe the Affymetrix microarray technology.

9. Define the term oligonucleotides

10. Differentiate between multichannel and single channel microarrays

ANSWERS
All the answers to the given questions are mentioned as such in the chapter
under their respective headings. Few answers are mentioned for reference.

1. A microarray is a collection of microscopic DNA spots deposited or

synthesized in a two dimensional or three dimensional array on a solid
surfacelike glass, silicon chips or nylon membrane by covalent or non-
covalent interactions.It is thus an extension of Southern and Northern
hybridization blots, which have been to detect and characterize nucleic
acids in diverse biological samples. They are used to measure expression
of multiple genes simultaneously or to genotype multiple regions of a
genome.

2. The main technologies used in microarray are as follows:

a) Manufacturing of microarrays: This step involves the use of robotics to

produce microarrays by spotting DNA on a chip or a glass slide.

b) Sample preparation and array hybridisation step: This step involves mRNA
or DNA isolation followed by fluorescent labelling of cDNA probes and
hybridisation of the sample to the immobilised target DNA.

c) Image acquisition and data analysis: It involves microarray scanning and

image analysis by using sophisticated software programs that allows us to
quantify and interpret the data.

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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
334
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

335
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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