Introduction and Review •

Introduction

Pesticides are being increasingly used for enhancing yield and quality in agricultural

produce. The impact of pesticides on non-target organisms depends on their toxicity,

metabolites and transpmt pathways through the hydrological cycle, soil and food chains

(.Hinsch et a!., 2006). Indiscriminate use without following proper guidelines causes the

contamination of soil and water and apart from being an operational hazard, posing a serious

threat to human health. The risk of acute exposure to these compounds is a constant threat

and found to be responsible for numerous cases of poisoning annually in nontarget wildlife

and for acute mammalian toxicity and neurotoxicity (Gilden et a!., 201 0). European

Economic Communities (EEC) directive, 1980 fixed the maximum residue levels (MRLs) for

drinking water of each individual pesticide at 0.1 11g/l and the total amount of pesticides at

0.5 llg/1.

In order to comply with the strict EEC norms, pesticide residue analysis are

performed using chromatographic techniques such as gas chromatography (GC), liquid

chromatography (LC) and supercritical fluid chromatography (SFC), together with specific

detection, in combination with mass spectrometry (MS) (Schroder, 1993; Hoff and Zoonen,

1999). In general, the above procedures are expensive, time consuming, labour intensive

(Kramer, 1996) and also required some toxic solvants (Tones et a!., 1996). To avoid the

general drawbacks of the above methods, application of immunoanalytical techniques has

increased significantly (Suri et a!., 2002; Singh et a!., 2004). Immunoanalytical techniques

are characte1ized by the high affinity and specificities of the antibody-antigen interaction,

offering detection limits at the low parts per billion (ppb) to parts per tiillion (ppt) levels.

Consequently, these techniques are rapid, simple and cost effective. In addition these can be

used by untrained personnel and performed on-site monitoring without requiring sample

transfer to an analytical laboratory.

Pesticides are small molecules therefore do not elicite immune response. These

molecules are first derivatized (called hapten) containing an appropriate group for attachment

to a catTier protein. The size of the spacer ann bridging the protein and hapten in very

impmtant for good antigen presentation and 4-6 atoms are often quoted as an optimal range

(Szurdoki et a!., 1995). Different labels used are radioactive tracers (Fatori and Hunter,

1980), enzymes (Matuszczyk eta!., 1996) and fluorescent probes (Niessner eta!., 1995).

Recently, antibody technology has evolved as a major tool to detect pesticides

whereby monoclonal antibodies with desired specificity can be selected (Kohler and

Introduction and Review •

Milestein, 1975). Antibody genes can be cloned and expressed in various host systems, thus

further improving the sensitivity and the reproducibility of immunoassay (Nord et al, 1997;

Hoogenboom and Chames, 2000). Exploitation of antibody enzyme conjugate play an

important role in recombination technology where the scFvs can be conjugated to other

moieties like enzyme by gene fusion technique (Yang et al. 2004; Dhillon et al. 2003) and a

luminescent enzyme aquorin have been employed as labels (Casadei et al. 1990).

Fluorobodies/luminobodies, the fusion protein product of antibody fragment and fluorescent

protein can be produced in culture of bacterial cells (Griep et al., 1999; Schwalbach et al.,

2000). Unlike FITC/ RITC/ HRP conjugated antibodies, scFv fusion proteins do not show

intensity degradation and have a longer life span. The utilization of fusion proteins is likely to

improve detection signal and reduce the time of analysis. Green Fluorescent Protein (GFP)

from Aequorea Victoria i.e., which emits green light when illuminated with long wave UV­

light showed the promise of being used as a very convenient portable marker for gene

expressiOn.

1.1 Pesticides

A pesticide may be a chemical substance, biological agent (such as a virus, fungus or

bacteria), antimicrobial, disinfectant or device used against pests including insects, plant

pathogens, weeds, mollusks, birds, mammals, fish, nematodes (roundworms) and microbes

that compete with humans for food, destroy property, spread or are a vector for disease or are

a nmsance.

1.1.1 History

The first known pesticide was elemental sulfur dusting used in Sumeria about 4,500

years ago. By the 15th century, toxic chemicals such as arsenic, mercury and lead were being

applied to crops to kill pests. In the 17th century, nicotine sulfate was extracted from tobacco

leaves for use as an insecticide. In 1939, Paul Miiller discovered that DDT was a very

effective insecticide. It quickly became the most widely-used pesticide in the world.

However, in the 1960s, it was discovered that DDT was preventing many fish-eating birds

from reproducing which was a huge threat to biodiversity. DDT is now banned in most parts

of the globe, but it is still used in some developing nations to prevent malaria and other

tropical diseases by killing mosquitoes and other disease-carrying insects.

1.1.2 Pesticides: Types and Classification

Today pesticides have become indispensable tools in the efficient control of animals,

insects, plants and fungi which otherwise have a detrimental effect on the crop production.

There are over 800 pesticides and 20,000 pesticide products, which are currently in use.

2

Introduction and Review •

Apart from being classified according to the chemical class the pesticide belongs to, they can

also be classified broadly into different groups (Sanborn eta!., 2002) as

Insecticides: are usually organophosphorous (e.g. malathion, chlorpyriphos etc),

organochlorine (e.g. DDT) or carbamates (e.g. carbofuran, carbmyl etc). Other insecticide

chemicals include Pthalates and hydrazines, Pyrezoles and Pyrethroids etc.

Herbicides: Organic compounds such as Ureas and sulfonylureas, Chlorophenoxy

acids, Triazines, Carbamates, benzoic acid, dinitrophenols and Naphthalene acetic acid

derivatives (e.g. atrazine, 2,4-D, metachlor etc.) or inorganic compounds (e.g. Arsenicals,

Sodium Chlorate).

Fungicides: Sulphur and copper salts (copper sulfate etc) or organic compounds such

as Azoles, Benzimidazoles, Carboxyimides, Dithiocarbamates and substituted Benzenes (e.g.

Captan, Mancozeb etc.)

Antimicrobials: Pesticides commonly used as sterilizers, disinfectants, sanitizers,

antiseptics and ge1micides intended to kill or inhibit growth of microorganisms such as

bacteria (Bactericides/bacteriostat) include organic chemicals such as Phenols and

chlorinated phenols, Hyadantoins, Isothiazolones and Quarternary ammonium salts.

Rodenticides: Used to control rodents and related species. Organic chemicals such

as Coumarins and Indandiones are used as rodenticides world over.

According to chemical group they can be classified as

Organophosphate Pesticides:- These pesticides affect the nervous system by

disrupting the enzyme that regulates acetylcholine, a neurotransmitter. Most

organophosphates are insecticides. They were developed during the early 19th centu1y, but

their effects on insects, which are similar to their effects on humans, were discovered in 1932.

Carbamate Pesticides:- Affect the nervous system by disupting an enzyme that

regulates acetylcholine, a neurotransmitter. The enzyme effects are usually reversible. There

are several subgroups within the carbamates.

Organochlorine Insecticides: Commonly used in the past, but many have been

removed from the market due to their health and environm~ntal effects and their persistence

(e.g. DDT and chlordane).

Pyrethroid Pesticides: were developed as a synthetic vers10n of the naturally

occurring pesticide pyrethrin, which is found in ch1ysanthemums. They have been modified

to increase their stability in the enviromnent. Some synthetic pyrethroids are toxic to the

nervous system.

3

Introduction and Review •

1.1.3 Pesticides Production and use: Global and Indian Scenario

1.1.3.1 Global Scenario:

In many countries population growth has raced ahead of food production in recent

years (United Nations Development Programme, 1997). The world grain harvest increased

about 1 per cent annually between 1990 and 1997, less than the average population growth

rate of 1.6 per cent in the developing world. Between 1985 and 1995 food production lagged

behind population growth in 64 of 105 developing countries studied by F AO. The average

amount of grain land per person dropped by almost half between 1950 and 1996 from 0.23

hectares to 0.12 hectares. By 2030, when world population is projected to be at least 8 billion,

there would be just 0.08 hectares of grain land per person (Leach and Fairhead, 2000). As for

developing countries, in 1992, there were about 0.2 hectares of arable land per person. By

2050, this figure could fall to about 0.1 hectare per capita (Rosenzweig, 2000).

Agriculture is the main source of income for more than 2.5 billion people and 96% of

the fatmers are living in developing countries. This would require a 40-45% increase in food

production. To meet the demand of increase in food production, pesticides are a major fmm

input with many commercial cash crop operations spending 5-l 0% of cash operating

expenses on these products. The world pesticide market is valued at about $ U.S. 3 7 billion

and India accounts for about 2.5% of this amount. India is normally ranked 121h in the world

pesticide market while the Europe is ranked 151 with close to 33% followed by US with nearly

20% of the global market share. World Pesticide consumption is about 2.5 million tones and

Europe is the largest consumer with over 32% followed by the US with around 20%, Asia

accounts for about 12% pesticide usage and Canada and Africa are the lowest consumers with

about 4% share each, the rest of the world makes for the remaining 20% pesticide

consumption (Pimentel, 1995).

The world pesticide, agrochemicals and agriculture trade industry is dominated by a

relatively small number of corporations; manufacturers (approximately 15) supplying a large

number of active ingredients. According to UN food and Agriculture organization studies

(http://apps.fao.org/faostat/default.jsp) it is estimated that 10 of these companies produce

90% of the world's active ingredients (Table 1.1 ). Just four companies, based in the US and

linked in two alliances (Cargill/Monsanto and Novm1is/ ADM) control over 80% of the world

seed market and 75% of the world agrochemical market.

4

Introduction and Review •

Table 1.1: Major Pesticide Producers of the world along with their Flagship products

Serial Company Prime Product

Number

1. AgrEvo Liberty

2. Cyanamid Pursuit

3. Monsanto RoundUp

4. Dow Blanco 2,4-D

5. Novartis Dual

6. BASF Banvel

7. Zeneca Achieve

8. Bayer, Ontario Furadan

9. Rhone-Poulenc Select

10. Bayer Admire

1.1.3.2 Indian Scenario

Agriculture is the lynchpin of the Indian economy. Ensuring food security for more

than 1 bn Indian population with diminishing cultivable land resource is a herculean task.

This necessitates use of high yielding variety of seeds, balance use of fertilisers, judicious use

of quality pesticides along with education to farmers and the use of modem farming

techniques. Use of pesticides in India began in the year 1948 when DDT was imported for

Malaria control and the agricultural use started in 1949 with introduction of Benzene

hexachloride (BHC) for Locust control (Gupta PK, 2004). The production of pesticides

started in the year 1952 with production of BHC followed by DDT production plants.

Pesticide Industry in India is second largest in Asia (behind China) and twelved largest in

World. In value terms, the size of the Indian pesticide industry was estimated at Rs.74 bn for

2007, including exports of Rs.29 bn. Group wise consumption of pesticides during 1995-

2005 in India is show in table 1.2. Among the states, Punjab uses the highest anount of

pesticide followed by Haryana (Fig. 1.1 ). Comparision of pesticide use in India and the world

is shown in fig. 1.2.

5

Introduction and Review •

Table 1.2: GroupWise consumption ofpesticides (MT) during 1995-2005 in India

Pesticide 1999-2000 2000-01 2001-02 2002-03 2003-04 2004-05

Group

Insecticide 28,926 26,756 29839 28197 25627 25929

Fungicide 8,435 8,307 9222 10712 9087 6397.4

Weedicide 7,369 7,299 6979 7857 5610 7364

Others 1,465 1,222 1308 1398 438 1660

Total 46,195 43 ,584 47348 48146 40762 41350.4

Jtt.:v Pt<Jdesh ••••••

Tarnltl.'IOU ••••• PunJab ················· M<!hara!>l•ll'3 •••

~ j3 Madhya Pradesh -li'J

'<arn<~Jaka ••••

Haryana ••••••••••••••••

GuJarat ••••••

t..ndilra Pradesh ••••••

0 200 .100 £00 800

PestrcidesconSllm ptron (gmslhecta re } m 1999.01

Figure 1.1: State wise pesticide consumption in India.

6

1000

Introduction and Review •

8U 76

70

O lrda 13J

;50 44 l':l -c: ;~,...,

~

"' ... 30 ~ 30 11.

21 20

10 5

0

nse~'JO? Oten;

Figure 1.2: Comparison of pesticide consumption in India and world.

1.1.4 Pesticide Pollution and public health

1.1.4.1 Pesticide Pollution: A Global Concern

Pesticide use has gained popularity due to their ability to control the pest problem at a

very reasonable cost thereby increasing food supplies at lower prices and also in preventing

transmission of diseases such as malaria through insects. Pesticides, depending upon their

water solubility can either remain in the soil and are broken down by action of

microorganisms, or washed off, eventually into surface and ground waters (Rehana et al.

1996; Agarwal, 1999; Suri et al. 2009). The persistency of pesticides and their degradation

products in the geosphere causes environmental problems. The transfer of pesticides from

treated soil to surface and ground water leads to contamination of drinking water resources

and to subsequent intake of pesticides by human populations. They may also present hazards

to human health directly as in spray drift of pesticides or indirectly as accumulated pesticide

in edible plant or animal tissue.

Rachel Carson was the first to predict a massive destruction of planet ' s fragile

ecosystem through her book 'Silent Spring' (1962). The title of the book was based on the

fact that birds died more in the area where pesticides were being used through aerial spraying.

She was the first to point out the damage being done to non-target species by uncontrolled

use of pesticides through direct and indirect toxicity. Working on chlorinated pesticide she

showed that DDT was harmful to fish and crabs and not only to the insects for which it was

being used. Indirect toxicity on the other hand was due to the persistency of the pesticides.

7

Introduction and Review •

Bioaccumulation, is the tendency of the persistent pesticides to accumulate and get

concentrated in an organisms tissue and Biomagnification, is the increase in the

concentration of pesticides higher up in the food chain, which leads to indirect toxicity.

Carson's work disclosed the fact that even when the pesticide DDT had a very low

concentration in water 3 ppt the concentration in zooplanktons was 0.04 ppm, was found to

be 0.5 ppm in minnows and further increased to 2 ppm in fish and the birds feeding on fish

were found to have a concentration of 25 ppm. In most countries DDT was banned in 1972

but it can still be found in various ecosystems even 3 decades after the ban on it's use in

seals, fishes, invertebrates, amphibians, birds and human (Ramakant, 2004; Muir et al., 2003;

Minh et al., 2002; Wong et al., 2002). It is now evident that even in small amounts these

pesticides can cause death, irritate eyes and skin, damage nervous system, disrupt hormonal

balance and immune system, effect ability to reproduce and can cause cancer (Hooghe et al.,

2000; Garry et al., 1996). Insecticides and herbicides are the two main classes of pesticides,

which are most commonly used and are found in environment as pollutants. Due to a large

number of chemicals which are being used and deposited in the environment, WHO

recommended the classification of pesticides based on the threat it poses to human race,

calculated on the basis of Oral and Dermal LD50 values. Table 1.3 shows the classification

categories in which harmful chemicals are divided according to the WHO recommendations.

Insecticides are the major cause of concern because the effect produced by their

mode of action such as enzyme inhibition, hormonal imbalance etc on the pest animals and

insects is also applicable on the humans (Jintana et al., 2009). Acute and long-term exposure

to organophosphates leads to elevated neurological, psychiatric symptoms and poor

neuropsychological response (Jokanovic and Kosanovic, 201 0). Organochlorine pesticides

were used extensively till 1970's but the health hazards associated with their use implied

severe restrictions on their use. Organochlorines are known to disrupt hormonal balance,

retard growth in children and also adversely affect the immune system (Vine et al., 2000;

Karamus et al., 2002).

Herbicides owe their indiscriminate use to their effectiveness in controlling weeds

and also to their apparent tolerability by animal species including farm animals and humans.

Recent studies on long-term exposure to different types of organic herbicides in uses such as

heterocyclic aromatic compounds (e.g. Triazines etc.) or phenoxy acids (e.g. 2,4-D etc.) have

been reported to cause great concern for human health (Kniewald et al., 2000; Cox, 2001;

Narotsky et al., 2001). Phenoxy and triazine herbicides are also known to disrupt hormonal

balances (Hayes TB, 2006). Herbicides are linked to increased incidence of cancer and

8

Introduction and Review •

chromosomal breakage in various studies (Van-Leeuwen, 1999). In an in vitro study triazines

were found to change the production of two chemicals, Dopamine and Norepinephrine, both

of which act as neurotransmitters in the central nervous system and are thus potential

neurotoxic agents (Das et al. 2000, 2001).

Table 1.3: WHO recommended classification of pesticides by hazard

Class Hazard level LDso for the rat, oral (mg kg-1

body mass)

Solids Liquids

Ia Extremely hazardous ::;;s ::;;20

lb Highly hazardous 5-50 20-200

II Moderately hazardous 50-500 200-2000

III Slightly hazardous >500 >2000

III+ Unlikely to present hazard >2000 >3000

in normal use

1.1.4.2 Pesticide Pollution: Indian perspective

Despite the fact that the consumption of pesticides in India is very low, about 0.5

kg/ha of pesticides against 6.60 and 12.0 kg/ha in Korea and Japan, respectively, there has

been a widespread contamination of food commodities with pesticide residues, basically due

to non-judicious use of pesticides. In India, 51% of food commodities are contaminated with

pesticide residues and out of these, 20% have pesticides residues above the maximum residue

level values on a worldwide basis. It has been observed that their long-term, low-dose

exposure are increasingly linked to human health effects such as immune-suppression,

hormone disruption, diminished intelligence, reproductive abnormalities, and cancer (Gupta,

2004).

The first report of poisoning due to pesticides in India came from Kerala in 1958

where, over 100 people died after consuming wheat flour contaminated with parathion

(Karunakaran, 1958). Since then several cases of mass poisoning have been reported but

minor case still go unreported. Pesticides have been found in food· stuffs, human blood, milk

and fat (Bhatnagar, 2001). A study on 356 workers in four different manufacturing units of

Hexachlorohexane (HCH) showed enhanced neurological symptoms (21 %) along with

significant increase in detoxifying enzyme levels related to the degree of exposure (Nigam et

al., 1993). Rajendran and co workers (1999) reported the presence of Chlorinated pollutants

9

Introduction and Review •

in air from a tropical coastal environment (Parangipettai - southeast coast of India). DDT and

HCH ranged in concentrations from 0.16 to 5.93 ng m-3 and 1.45 to 35.6 ng m-3 respectively.

The ban on DDT in agriculture is reflected from the low residue levels recorded,

predominantly by metabolites other than the parent compounds. Excessive use of

agrochemicals has definitive environmental consequences that have reached alarming levels

in Panjab and Haryana, which are also the highest user of agrochemicals per hectare in India

(Singh, 2000).

The world's worst industrial disaster occurred in India on the night of December 2-

3, 1984, at the Union Carbide plant in Bhopal (population: 900,000) a pesticide

manufacturing company. More than 2,500 people died almost instantly, and over 16,000

people have died as a result of health problems related to their exposures to MIC an

intermediate product in the manufacture of carbaryl (carbamate pesticide). More than 50,000

people are still suffering significant long term health impacts such as ocular, respiratory,

reproductive, immunological, genetic, and psychological (Dhara et al, 2002).

1.1.5 Methods for Pesticide Detection

The conventional methods for detection and analysis of pesticide residues include

physico-chemical techniques like Gas Chromatography (GC), High-pressure liquid

chromatography (HPLC), Capillary electrophoresis (CE), Mass spectrometry (MS) and their

combinations thereof such as GC-MS, LC-MS, GC-MS-MS etc (Osselton and Snelling,

1986).

1.1.5.1 Chromatographic Techniques

a) Thin Layer Chromatography

TLC and HPTLC complement gas chromatography (GC) play a very important role in

the determination of pesticide residues, their metabolites and transformation products in

environmental waters. They have the following unique advantages over column

chromatography: single use of the layer simplifies sample preparation procedures; simplicity

of development by dipping the plate into a mobile phase in a chamber; high sample through­

put with low operating cost because multiple samples can be run simultaneously with

standards on a single plate using a very low volume of solvent. Thin layer

radiochromatography (TLRC) is used routinely for metabolism, degradation, and other

studies of pesticides in plants, animals, and the environment, and these applications will be

covered as well as studies of lipophilicity and pesticide migrations through soils. Screening of

10

Introduction and Review •

265 pesticides in water by TLC with automated multiple development was published

(Hamada and Wintersteiger, 2002).

b) Gas Chromatography:

Gas Chromatography (GC) is most commonly used for separation of thermostable

pesticides. Various kinds of fused-silica capillary columns with bonded phases of different

polarities are commercially available. The popularity of GC is based on a favorable

combination of very high selectivity and resolution, good accuracy and precision, wide

dynamic concentration range and high sensitivity (Santos and Galceran, 2003).

Combination of GC with detection methods such as flame ionization detection (FID)

and nitrogen-phosphorus detection (NPD) are most popular; FID gives universal response to

organic compounds, while NPD is selective for compounds containing nitrogen or

phosphorus and gives much lower detection limits than FID. GC-NPD thus is very suitable

for detection of amino group containing pesticides. Electron-capture detection (ECD) is used

for sensitive determination of the compound containing halogen or nitro group(s) in a

molecule (Stalikas and Konidari, 2001).

c) Liquid Chromatography:

Liquid Chromatography (LC) is the method of choice for highly polar, thermolabile

and/ or high-molecular mass compounds, which are not amenable to GC. The compatibility

of the water samples with the reversed-phase chromatographic separation systems and the

possibility of performing derivatization in aqueous solution made LC the preferred technique.

Liquid chromatography approach for analysis of pesticides uses various detectors such as

ultraviolet (UV), fluorescent Detector (FD) and Refractive index detector (RID) (Kumazawa

and Suzuki, 2000). These techniques though highly sensitive, however suffer from several

drawbacks

1. Complex sample preparation: Derivatization of the sample to be analyzed is a

prerequisite for most of the pesticides, before or after using any particular technique

for their efficient detection for example some pesticides such as Glyphosate, bialaphos

which contain phosphonic and amino acid groups needs to be derivatized to be less

polar and more volatile before they can be detected using GC. The derivatization

process may be very lengthy and include the use of highly toxic chemicals (Stalikas

and Konidari, 200 I). A post chromatographic derivatization to get a fluorophore or

chromophore attached to such pesticides for detection has also been reported for

detection by HPLC (Oppenhuizen et al., 1991).

11

Introduction and Review •

2. Large time for sample analysis: The chromatographic methods used for the final

determination require extraction of the residues from the matrix and subsequent clean­

up procedure before they become suitable for analysis (Bruzzoniti et al., 2000). Over

60% of the analysis time is used for sample preparation (Smith, 2002; Krutz et al.,

2003; Eskilsson and Bjorklund, 2000).

3. Sophisticated instrumentation and trained personnel to operate: Mass

selective detection (MS) is now used more often with GC and LC after the mass

detectors such as ion trap detectors and bench top quadrupole instruments were

improved in their detector design and operation and acquisition software (Kumazawa

and Suzuki, 2000). Thus the personnel performing the analysis should be highly

trained so as to correctly determine which detector to use for which compound

(Stalikas and Konidari, 2001).

4. Unsuitable for field studies and in situ monitoring of samples: These

techniques due to their bulky and sophisticated instrumentation are limited to the lab

and are unsuitable for filed applications. Furthermore, collection, transport and

storage can affect sample characteristics, and subsequently, the validity and hence

usefulness of analysis. Field analytical methods in combination with an appropriate

number of laboratory analyses for field data validation could provide a good

monitoring strategy (Mallat et al., 2001; McMohan, 1993; Giese, 2000; Suri et al.,

2002).

5. Large cost of analysis per sample: The main driving force in the emergence of

alternate technology is the increasing cost and number of samples associated with

environmental compliance. Therefore, more cost effective tools for water monitoring

are urgently needed (Mallat et al., 2001, Nister and Emneus, 1999).

1.1.5.2 Electrophoretic Techniques

Recent advances in technology have found application of electrophoresis in the field

of environmental pollution monitoring. Small amounts of pollutants can be detected using

Capillary electrophoresis, where separation is performed in fused-silica capillaries with

internal diameters of 25-1 00 Jlm and they provide very high theoretical plate numbers and

along with sensitive detection methods such as UV -absorbance and fluorescence make it a

highly promising technique. This technique is fast gaining popularity because of its various

modes, which can be used for separation of polar as well as non-polar compounds (Martinez

et al., 2000).

12

Introduction and Review •

In capillary zone electrophoresis (CZE) separation is based on differences in the

electrophoretic mobility (determined by size and charge) of charged analytes in an electric

field. However, since many environmental pollutants are uncharged or have very similar

chemical structures, they often cannot be separated from interfering components by CZE and

Micellar electrokinetic capillary chromatography (MEKC) has to be used (Altria, 2000).

1.1.5.3 Mass spectrometry

The analysis of organic compounds by mass spectrometry (MS) first involves

producing gas phase charged molecular ions and fragment ions of the parent molecules in the

ion source of the instrument, and subsequently separating these ions according to their mass­

to-charge ratio (m/z), and finally measuring the intensity of each of these ions. A mass

spectrometer accomplishes this through a sequence of events within its three main

subsystems namely: (i) the ion source in which the ionization of the organic molecules takes

place, and from which the ions are then accelerated (or extracted) into (ii) the mass analyzer

which separates the ions according to their m/z values, and finally (iii) the detector where the

relative intensities (abundances) of the separated ions are determined. The instrument is

maintained at low pressure (high vacuum) by a system of turbo molecular or oil diffusion

pumps. A mass spectrum displays the values of the mlz ratio for molecular ions and fragment

ions along the X-axis, increasing in value from the origin. The relative intensities of the

detected ions are displayed on the Y -axis, generally as the % relative abundance compared to

the most intense ion in the spectrum. MS has gained popularity more as a detector for specific

compounds separated using one of the separation techniques i.e., GC, LC and CE (Careri et

al., 1996; Pico et al., 2004).

1.1.5.4 Immunochemical Methods

Immunosensors are based on the binding interactions between immobilized

biomolecules (Ab or Ag) on the transducer surface with the analyte of interest (Ag or Ab ),

resulting in a detectable signal. The sensor system takes advantage of the high selectivity

provided by the molecular recognition characteristics of an Ab, which binds reversibly with a

specific Ag. In solution phase, Ab molecules interact specifically and reversibly with an Ag

to form an immune complex (Ab-Ag) according to the following equilibrium equation:

Ka Ab+Ag Ab-Ag

Kd

where Ka and Kd are the rate constants for association and dissociation, respectively. The

equilibrium constant (or the affinity constant) of the reaction is expressed as follows:

K = Ka/Kd = [Ab-Ag]/ [Ab] [Ag]

13

Introduction and Review •

The equilibrium kinetics of Ab binding with Ag in solution suggests that both association and

dissociation are relatively rapid. The direction of equilibrium depends on the overall affinity,

which is basically the summation of both attractive and repulsive non-covalent forces. An

immune complex usually shows low Kd values (in the range 10-6-10-12) and also displays

higher affinity (i.e. K value typically 1 04). Immunosensors, which usually incorporate Abs

immobilized on solid matrix do not show the same kinetics of reaction established for those

reactants in solution. Immobilization of biomolecules on a solid surface can alter the

properties of the reactants (measured in reaction-rate constants) or the surface characteristics

(e.g., surface charge and hydrophobicity) of the support itself. Until the mid-1980s,

immunosensors were mainly used for clinical diagnostics, mostly for the detection of

macromolecules (Karube and Suzuki, 1986). Not much work had been reported m

environmental monitoring using immunosensors. Development of immunosensors for

environmental pollutants, mainly for pesticide analysis was delayed for several reasons. First,

getting Abs against pesticide molecules has been a tedious task, primarily because of the low

molecular weight of pesticide molecules (usually less than I kD), as is evident from the fact

that not many Abs against pesticides are available. Second, repeated measurements are less

efficient, especially with high affmity Abs (and unlike enzymes). However, development of

Ab technologies and advancement in the transducer system have provided further impetus

(Kaur et al., 2008; Kim et al., 2003; Singh et al., 2004).

Pesticides, organic compounds of molecular mass, are usually non-immunogenic, so

they will not elicit an immune response unless coupled with some macromolecules (e.g.,

proteins). It is therefore necessary to modify these small substances (haptens) to couple them

with macromolecules (carriers) so as to make a stable carrier-hapten complex. The carrier­

hapten conjugate developed can be used to generate Ab against pesticides. This section

highlights Ab generation and optimization of assay techniques for the detection of pesticide

molecules. Abs are specialized proteins capable of recognizing Ags with high specificity. The

quality of the Ab employed greatly influences the specificity and the sensitivity of an

immunosensor. Polyclonal Abs (pAbs), conveniently prepared from sera, comprise a wide

variety of Ab molecules of different specificities and affinity (Hill et al., 1993). Hybridoma

technology provides an excellent alternative, whereby monoclonal Abs (mAbs) can be

produced in unlimited quantities with constant characteristics. Besides, mAbs of desired

affinities can also be selected. Recombinant DNA technology has been exploited to engineer

recombinant Abs (rAbs), whereby Ab genes can be cloned and expressed in various host

systems, thus further improving the sensitivity and the reproducibility of immunoassay

14

Introduction and Review •

(Scheller et al., 2001). The choice between pAbs, rnAbs or rAbs for development of an

immunosensor depends upon the various aspects of the assay (e.g., cost of development,

relative specificity and sensitivity of the Abs, ease of screening and selection of Abs) (Suri et

al., 2008).

1.1.5.4 Classification of immunosensors

In general, immunosensors can be distinguished from imrnnunoassays where the

transducer is not an integral part of the analytical system. If a transduction is achieved using

labeled species, the principles are similar to immunoassays. Depending on if labels are used

or not, immunosensors are divided into two categories: labeled type and label-free type.

1.1.5.4.a Labeled formats

This procedure involves a label to quantifY the amount of Ab or analyte bound during

an incubation step. Widely used labels involve enzymes (e.g. glucose oxidase, horseradish

peroxidase (HRP), p-galactosidase and Alkaline phosphatase (AP), nanoparticles, and

fluorescent or electrochemiluminescent probes. (Wilson et al., 1997; Keay and McNeil, 1998;

Danielsson et al., 2001; Seydack, 2005; Wilson, 2005). Fig. 1.3 shows the schematic of

labeled immunosensors. Commonly, two different formats for labeled immunosensors are

available: sandwich assays and competitive assays. A sandwich assay consists of two

recognition steps. In the first step, the Ab is immobilized on a transducer surface, allowing it

to capture the analyte of interest. In the second step, labeled secondary Ab is added to bind

with the previously captured analyte. The immunocomplexes (immobilized Ab-analyte­

labeled Ab) are formed and the signals from labels increase in proportion to the analyte

concentration (Sadik and Van Emon, 1996). In competitive assays, the analyte competes with

labeled analyte for a limited number of antibody binding sites. As the analyte concentration

increases, more labeled analyte are displaced, giving a decrease in signal if antibody-bound

labeled analyte is detected (Bange et al., 2005).

Use of fluorescence for detection provides a very sensitive method of detection. If

these substances are excited by polarized light, the molecules change their orientation and the

emitted light is also depolarized. Fluorescence polarization (FP) is determined by exciting

these compounds with vertically polarized light and measuring the intensity of both the

vertically (lv) and horizontally (lh) polarized components of the fluorescence light emitted.

The polarization (P) value is determined as

15

Introduction and Review •

A B 0 ~ Product 0 Substrate ~ Product SubStrate

...).{~ ...J..<,. ..).{~ Enzyme-labeled hapten

fir~· ~ .~ • • ' ' • Free anaJyte Free analytee

.. ! , • ,$ ·,k~ • • 'If~ •

• AnU-pestiCide antibody

•

~Enzyme-labeled ~secondal)' antibod

AnUbody analyte complex

Figure 1.3: Principle of labeled immunosensor. Concentration of analyte to be monitored is corelated with the amount of labeled antigen (A) or labeled antibody (B) to the corresponding ligand coated on the transducer surface.

the ratio of the difference and the sum of Iv and lh components:

P = (Iv - lh)/(Iv + Ih)

Theoretically, the value of P is 0 or 0.5 for full depolarization or fixed molecules without

movement, respectively. The use of FP-based immunoassay (FPIA) for the detection of

pesticides has been described extensively (Eremin, 1998). FPIA measures the increase in FP

when a tracer (fluorophore-labeled Ag) is bound to a specific Ab; the FP signal is decreased

when free analyte competes with the tracer for binding. FPIA for small pesticide molecules is

a competitive method based on detection ofthe difference ofFP between a small fluorescent­

labeled Ag and its immune complex with a specific Ab. When there is no analyte present in

the sample, the tracer will be completely bound to the specific Ab and the FP value will

increase. However, if the analyte concentration in sample is greater than the concentration of

the tracer, the Ab-binding sites will be occupied by the analyte so there will be free tracer.

This will decrease the FP value of the reaction mixture.

Sensitive method based on an optical immunosensor was developed to determine

different types of organic pollutants in water samples (Rodriguez-Mozaz et al., 2005). The

system, called river analyzer (RIANA), is based on a rapid, solid-phase, indirect, inhibition

immunoassay that takes place at an optical transducer chip chemically modified with an

analyte derivative. Fluorescence produced by labeled Abs bound to the transducer is detected

16

Introduction and Review •

by photodiodes and can be correlated with analyte concentration. Similarly, a parallel

affinity-sensor array (P ASA) system, based on chemiluminescence labels (peroxidase/

luminal) and charge-coupled device (CCD) detection was developed for monitoring pesticide

contaminants in water (Weller et a!., 1999). An LOD of this system of 20 ng/1 was achieved

for terbutylazine. The assay could be regenerated more than 100 times.

After the chemiluminescence (CL) phenomenon of luminal reported by Albrecht in

1928, investigation of effective catalysts for such CL reactions has increasingly been carried

out, including metal ions, metal complex, and enzymes. The catalyzed luminal CL has been

successfully applied in bioanalysis and immunoassay or as sensitive detectors for high­

performance liquid chromatography (HPLC) or capillary electrophoresis (CE).

Recently, gold nanomaterials as biological labels for immunoassays have attracted

keen interest for their unique size dependent optical and electronics properties and great

analytical potential (Rosi and Mirkin, 2005; Seydack, 2005; Daniel and Astruc, 2004). These

nanoparticles have been widely used for their catalysis of gas I liquid phase redox reaction

(Wallace and Whetten, 2002) and also as catalyst for some chemiluminescence (CL)

reactions (Zhang et al., 2005; Cui et al., 2005) in liquid phase systems. In a CL immunoassay

format based on gold nanoparticles, Fan et al., (2005), developed a stripping CL

immunoassay which was based on the dissolving of gold nanoparticles to Au (III) after

immunoreactions and reacting with luminol to produce CL signals. However, these methods

employed stripping procedure, i.e., dissolution of gold nanoparticles under extremely strict

conditions, which could result in high CL background. In a non-stripping CL immunoassay

based on gold nanoparticles, Li and co-workers (2007) demonstrated that the gold

nanoparticles with irregular geometry could greatly enhance the CL intensity of the luminol­

H202 system.

A luminal-peroxide chemiluminescent system has attracted particular interests in the

field of bioanalytical chemistry. For generating the light emission, the oxidation of luminol

proceeds for the first step. Metal ions and organometallic compounds are known to catalyze

the oxidation of luminal with peroxide in strong alkaline solution (pH I 0-13) (Huang et al.,

1996; Obata eta/., 1993). Table 1.4 shows some examples of immunosensors using labels for

the determination of residual pesticides

17

Introduction and Review -

Table 1.4: Some examples of immunosensors using labels for the determination of residual

pesticides

Pesticide Detector Label Detection limit

Atrazine Fluorescent Nano-particle ELISA

Chlorsulfuron Amperometric Glucose- oxidase 0.01-1 ng/ml

Glyphosate Fluorescent Glyphosate-peroxidase 0.021 ng/ml

Isoproturon Fluorescent Fluorescein 0.1 ng/ml

Simazine Potentiometric HRP 3 ng/ml

Mesotrione Fluorescent Fluorescein 0.04 ng/ml

1.1.5.4.b Label-free formats

This procedure detects the binding of pesticide and the Ab on a transducer surface

without any labels. There are also two basic types in this format: direct and indirect. In the

first type, the response is directly proportional to the amount of pesticides present. The vital

advantage of these direct immunosensors is the simple, single-stage reagentless operation.

However, such direct immunosensors are often inadequate to generate a highly sensitive

signal resulting from Ab-Ag binding interactions and it is still difficult to meet the demand of

sensitive detection. The second type, also based on competitive formats, is carried out as a

binding inhibition test. The antigen (pesticide-protein conjugate) is first immobilized onto the

surface of a transducer, and then pesticide-antibody mixtures are preincubated in solution.

After being injected on the sensor surface, the antibody binding to the immobilized conjugate

is inhibited by the presence of target pesticide. It is advanced transducer technology that

enables the label free detection and quantification of the immune complex (Fig. 1.4). It has

been proven to be an effective method for the determination of pesticide. Some examples of

label-free immunosensors for the determination of residual pesticides are presented in Table

l.5.

18

Introduction and Review •

Y Y Y Y Membrane potential changes

.. ".? ~X~ XX c:::::===>

Solid Matrix YYYY A

._X-YY_'(_ '

Electrode potential changes

Optical properties change

Micromechanical approach

Figure 1.4: Principle of labeled free immunosensor. Antigen binding to antibody-coated surface changes the physical properties of the transducer that can be converted into detectable electrical signal.

Table 1.5: Some examples of label-free immunosensors for the determination of residual pesticides SI.No. Pesticide Detector Detection limit Reference

1. 2,4-D Impedimetric 45 nmol/1 Navr ' atilov ' a and

Skl 'adal (2004)

2. 2,4-D SPR 0.1 ng/ml Gobi et al. (2007)

3. Atrazine Impedimetric 20 ng/ml Hleli et al. (2006)

4. Atrazine Impedimetric 8.34± 1.3 7 ng/ml Valera et al. (2007)

5. Atrazine Piezoelectric 1.5 ng/ml (direct) Pvribyl et al. (2003)

6. Chlorpyrifos SPR 45-64 ng/1 Mauriz eta!. (2006a)

7. DDT N anomechanical Below nM range Alvarez eta!. (2003)

8. Trifluralin Optical waveguide I 00 ng/ml (direct) Sz'ek'acs et al. (2003)

i) Piezoelectric crystal immunosensor

The PZ crystal immunosensor works on the principle of measuring a small change in

resonant frequency of anoscillating PZ crystal due to a change in mass on the sensor surface,

as a result of the formation of the A b-Ag complex. The resonant frequency of a PZ crystal is

mass dependent, giving LODs for analyte concentration at the sub-ng level (Suri, 2006). Fig.

1.5 (A-C) shows the PZ crystal vibrating in thickness shear mode and the formation of

immunocomplex (Ab- Ag) on electrodes. Fig. 1.5 D shows a prototype of a quartz-crystal

19

Introduction and Review •

microbalance (QCM) system that our group developed with an auto-flow attachment (patent

filed) for pesticides monitoring. The basis of this mass-frequency dependency [i.e. the surface

mass change (Om) and resonant frequency shift (OF) of a PZ crystal vibrating at fundamental

frequency (F)] is attributed to the Sauerbrey equation (Sauerbrey, 1959):

~F=-k F2 run; A = -2:26 x 1 o-6F2 run; A

where ~F is the change of frequency (Hz) due to coating, k is the proportional constant

depending upon density and shear modulus of quartz crystal (for AT -cut quartz, the density is

2.648 g/cm3 and shear modulus is 2.947 X 10 11 dynes/cm2), F is the fundamental frequency

(MHz) of the quartz crystal, run is mass (g) of coating deposited, and A is the coated area of

the crystal ( cm2). The oscillating frequency of the quartz crystal immersed in a aqueous

medium is also influenced by the density (q) and the viscosity (TJ) of the medium, as

described by the following equation (Kanazawa and Gordon, 1985):

~F= -2:26 xw-6F312 (q .11) 112

This equation gives a shift in fundamental frequency of the order of 7 kHz for a 1 0-MHz

quartz crystal with only one face in contact with aqueous medium. Characterization of mAbs

to 2,4-D using a PZ crystal microbalance in solution has also been reported. Because oftheir

low cost and high quality factors (Q), these miniaturized sensors have fast response time and

high sensitivity, and they can be mass-produced using standard fabrication techniques.

(Steegbom and Skla 'dal, 1997)

A / Metal electrode(s) B

~I___., '---=====''=:j Quartz wafer

c D

• C ty'ital Substrnte

Figure 1.5: (A) Piezoelectric crystal construction, (B) crystal vibrating in thickness shear mode, (C) piezoelectric crystal bound with receptor molecules for antigen detection, and (D) qua~1z-crystal set-up that our group designed and developed for pesticide analysis. The quartz-crystal microbalance (QCM) unit is coupled to a flow-through system for on-line monitoring applications.

20

0 0

.:J aJ -

[

f

Introduction and Review •

ii) Electrochemical immunosensors

Formation of Ab-Ag complex in electrochemical transducers alters the change in ion

concentration or electron density on the electrode surface, which, in turn, is measured by

electrodes. Electrochemical transducers, classified as amperometric, potentiometric,

conductimetric and capacitative, measure changes in current, potential (voltage), conductance

and capacitance, respectively. Applications of electrochemical transducers for detection of

different pesticide molecules have been reported. A rapid assay based on an immunoenzyme

electrode and peroxide conjugates was developed for 2,4-D and 2,4,5-trichlorophenoxy acetic

acid (2,4,5-T) (Dzantiev et a/., 1996). The assay monitors the competitive binding of free

pesticide and pesticide-peroxide conjugate with anti-pesticide Ab immobilized on a graphite

electrode by measuring peroxide activity in the immune complex on the electrode surface.

LODs for 2,4-D and 2,4,5-T were about 50 ng/rnl showing no interference with serum protein

present in solution. A simple electrochemical immunosensor-based assay for the field-based

quantification of 2,4-D in soil extracts has been demonstrated (Kroger et a!., 1998). The

sensor utilized a competitive immunoassay format, incorporating an immobilized Ag

complex at the surface of a disposable, screen-printed, working electrode element. The extent

of glucose oxidase-labeled Ab binding to the Ag electrode was determined amperometrically

and was related to the sample-analyte concentration. A disposable immunosensor with

liposome enhancement and amperometric detection technique for monitoring herbicide

triazine in real samples has also been presented (Baumner and Schmid, 1998).

In a different approach, an acetylcholine (Ach) receptor-based electrochemical

biosensor was developed (Eldefrawi et a!., 1998). The Ach receptor was fixed to the gate of

an ion selective field-effect transistor (ISFET) and binding of Ach with the receptor resulted

in a potential change that was detected by the ISFET.

iii) Micromechanical immunosensors

The merging of silicon-microfabrication techniques with surface-functionalization

chemistry offers an exciting new opportunity in developing ultra-sensitive microscopic

immunoanalysis devices. Micromechanical transducers make use of cantilevers as force

sensors in an atomic force microscope (AFM), which can transduce a signal caused by the

formation of immune complex onto the cantilever surface into a mechanical motion with high

sensitivity (Browning-Kelley et a!., 1997). The origin of this nanomechanical bending of

such a hybrid structure has been shown to be by a change in the surface stress due to ligand­

receptor binding (Wu et a!., 2001 ), so detection of nanomechanical bending offers a

quantitative measure of both the occurrence and the kinetics of specific molecular binding

21

Introduction and Review •

events on the microcantilever beam. The selectivity of molecular interactions is dictated by

the molecular probe (receptor) layer on the microcantilever surface, while the sensitivity is

governed by the extent of microcantilever deflection resulting from the molecular binding

event (Raiteri eta!., 2000; Thaysen eta/., 2000). Binding of 2,4-D to its rnAb was monitored

on a thin gold layer (30 nm) deposited on a cantilever. Binding characteristics of anti-2,4-D

Ab to the protein-2,4-D conjugate were analyzed by measuring the bend of cantilever due to

Ab-Ag interaction on its surface. Recently, we demonstrated label-free detection of highly­

specific atrazine Ab-Ag interactions at the nm scale on microcantilevers up to ppt sensitivity

(Suri et a/., 2008).

Fig. 1.6 shows the preparation of the thiolated anti atrazine Ab and the corresponding

site-directed immobilization scheme on the gold-coated cantilever. The absolute deflection of

the cantilever, Dz, was measured using an optical detection system in liquid with constant

flow of Ag (Sentric System, Veeco Instruments). The deflection signal resulted in a

differential surface stress, ~cr, so, using Stoney's equation:

~cr =~ (t!Li E/(1-u) ~z;

where Lis the effective length of the cantilever, tis the thickness, and E/(1- u) is the ratio

between the Young 's modulus, E (130 GPa), and Poisson ratio m of Si (0.28). Because the

cantilever deflection also strongly depended on the geometry, all the cantilevers used in these

measurements were exactly similar in their geometry. As a result, the surface-stress change

due to chemical interactions was directly related to quantitative measurements for the

bindings.

22

Introduction and Review •

Figure 1.6: Nanomechanical detection of atrazine antibody-antigen interactions on a cantilever. Atrazine in solution binds with the antibody, causing the cantilever to bend downward due to compressive surface stress. Antibody molecules are already immobilized on the cantilever in a site-directed orientation pattern to expose antigen-binding sites free for binding.

iv) Surface-plasmon resonance

In a different evanescent format, usmg SPR, the excitation is provided by the

evanescent field, which is produced at the site of total internal reflection from a glass-metal

interface. In this format, laser light first enters the optical substrate at certain angle and then

strikes the glass-metal boundary by first passing through the layer coated with biomolecules

on the metal film before contacting the glass. The intensity of the reflected light is a function

of the refractive index at the glass-metal interface. Any change in the refractive index, caused

by the f01mation of the immune complex on the metal surface, signifies immunological

recognition (Fig. I. 7). The SPR phenomenon was first utilized in an immunosensing

23

Introduction and Review •

application by Liedberg and his team (Chegel et al., 1998). Immunosensor systems based on

SPR detection have been developed for various pesticides in aqueous solutions (Liedberg et

a!. , 1998; Mouvet et a!. , 1997). An inhibition immunoassay using the BIAcore system

achieved an LOD of 0.005 ppb for atrazine in water with the analysis time of approximately

15 min. A competitive immunoassay based on SPR for detection of pesticide 2,4-D was

reported recently (Svitel eta!. , 2000). The interaction between anti-2,4-D Ab and the surface­

bound concanavalin A-2,4-D conjugate was monitored by SPR and the response was used to

quantify 2,4-D. The dynamic range ofthe curve was 3-100 ng/rnl. SPR-based immunoassays

for small molecules have been reviewed by Mullett and his colleagues (Mullet et al., 2000).

v) Evanescent wave

An optical immunosensor based on EW combines the potential of molecular

recognition by the Ab with the good signal-transduction capability of a waveguide or fiber­

optic probes. Signal generation can be obtained directly or enhanced by coupling a reactant

with a label. When light is propagated through a waveguide (gl) by multiple total internal

reflections in the x direction, an electromagnetic (EM) wave (called an EW) is generated in

the optically less dense external medium (g2) with g 1 > g2. For visible light, the evanescent

field penetrates 50-500 run in they direction, so only surface-bound molecules interact with

the incident light. This allows the absorption of energy by the molecules located in the

evanescent field, leading to attenuation of the light reflected in the waveguide. For

immunosensing applications, planar-waveguide systems exploit the same optical

phenomenon as fiber optics with some minor variations (Marazuela, 2002). The waveguide

can be mass-produced at very low cost by injection molding. A suitable detector monitors the

formation of Ab-Ag complex. A major advantage of this system is minimal interference from

the bulk media. There are two possible methods for measuring EW: attenuated total reflection

(ATR) and total internal reflection fluorescence (TIRF). In ATR mode, the energy absorbed

by the surface bound molecules is monitored as an attenuation of the internally reflected

light. Since the absorbed light is usually a small percentage of the total incident light, the

sensitivity of the assay is very poor. In TIRF mode, the evanescent photons captured by

surface-bound molecules are re-emitted at a longer wavelength as fluorescence. Fig. 1. 7 A

shows the basic principle of an EW biosensor where light is directed into the waveguide and

generates an EM field , allowing excitation of fluorophores used as labels with Abs or Ags.

EW biosensors gave limits of detection (LODs) for 2,4-D and simazine were 0.035 ng/rnl and

0.026 ng/ml, respectively (Klotz eta!., 1982).

24

Introduction and Review ·

• · # B A

Figure 1.7. Evanescent wave (EW)-based optical immunosensor. (A) Optical waveguide, and (B) surface-plasmon resonance (SPR) configuration. The monochromatic laser light is directed into the prism/waveguide, which generates an electromagnetic (EM) field allowing excitation of fluorophores used as tracers in optical waveguide and resonance shift in SPR, respectively.

1.2 Hapten Synthesis

Hapten, a derivative of the target molecule, contains an appropriate group for

attachment to carrier protein. Direct coupling of haptens with carrier proteins is possible if

the target compound contains functional groups (e.g., -NH2, -COOH, -CHO, or -SH).

However, it is necessary to derivatize the target compounds such as pesticide with such

functionality before conjugation with carrier protein. Design of haptens for linking with

carrier proteins is the most important aspect of specific Ab generation against pesticide

molecules. Hapten molecules are synthesized so that they:

• Mimic the structure of the compound; and,

• Contain a reactive group that can form a covalent linkage with the carrier proteins.

Minimal structure alteration to the hapten ensures that the Ab recognizes the

unmodified hapten in the assay system. The hapten should also preserve to a great extent the

chemical structure and the spatial conformation of the target compound. Careful hapten

selection might be important for generating class-specific or compound-specific Abs for the

immunosensor system. Class specificity of the Abs developed could be achieved by

preserving Ag determinants common among related compounds. Similarly, masking these

common determinants and targeting a distinct molecular entity could obtain compound

specificity (Kim eta/., 2003).

25

Introduction and Review •

For the bioconjugation of hapten with carrier-protein molecules, the functional group

of the hapten governs the selection of the conjugation method to be employed. However, the

stability of the hapten while conjugating with the carrier protein is an important factor. The

problem of hapten stability during conjugation has been highlighted by various groups (Hill

et al., 1993; Harrison et al., 1991 ). It is also important that the hapten-protein conjugate is

designed so as to preserve and enhance the epitopic regions on the hapten for getting an Ab

specific against a target molecule. The site of linkage of hapten and protein should usually

occur away from any suspected Ag determinants. Using a spacer arm (bridging groups)

between hapten and protein structure also helps in the production of hapten-specific Abs. The

spacer arm can be envisioned as a pedestal for the hapten to accommodate recognition and

binding by the large Ab molecule. In general, increasing the length or the hydrophobicity of

the spacer arm helps generating Abs of higher specificity and good titer. The same hapten

employing a shorter spacer arm may not elicit the Ab response. Design of a suitable hapten

and selection of a spacer arm and a suitable carrier protein are therefore critical to ensure

successful generation of Abs having the desired specificity (Kim et al., 2003).

Immunogens contain a B-cell epitope and a T -cell epitope, both of which are

necessary for the production of antibodies against a particular antigen of interest. When

hapten-labeled proteins are introduced into a suitable host animal, they generate antibodies •

against hapten or protein or hapten protein conjugate (Brinkley, 1992). In addition to these

intrinsic groups, specific reactive groups may be introduced into the protein by chemical

modification. Some of the reactive groups found on proteins are amines, thiols, phenols, and

carboxylic acids.

1.2.1 Bioconjugate Preperation

1.2.1.1 Protein Modification Reagents

There are different reagents that are available for the purpose of protein modification.

These reagents are designed to have specific reactivity with functional groups contained in

each reactant (Annunziato et al., 1993) and may be grouped according to the reactive groups

that they target on the protein to be modified (Brinkley, 1992). These may be grouped as

follows:

a) Amine-Reactive Reagents are those that react primarily with lysines and a-amino groups

of proteins to give amide products. N-hydroxy-succinimide (NHS) ester of S-acetylthioacetic

acid (SATA) is one of the commercially available amine-reactive reagents. These reagents

have intermediate reactivity toward amines, with a high selectivity toward aliphatic amines.

26

Introduction and Review •

Other reagents that have been used to modify ammes of proteins are acid anhydrides

(Brinkley, 1992).

b) Thiol-Reactive Reagents are those that will couple to thiol groups on proteins to give

thioether-coupled products (Brinkley, 1992). Maleimides react with thiols resulting

information of a thioether bond.

c) Carboxylic Acid-Reactive Reagents. Amines can be coupled to carboxylic acids of

proteins via activation of the carboxyl group by a water-soluble carbodiimide followed by

reaction with the amine (Brinkley, 1992) and resulting in formation of stable amide bonds.

d) Bi-functional reagents or cross-linking reagents are specialized reagents that will f01m a

bond between two different groups, either on the same molecule or two different molecules

(Brinkley, 1992). Those with the same reactive group at each end of the molecule are called

homobifunctional while those with different reactive groups at each end of the molecule are

called heterobifuntional. Succinimidyl (acetylthio) acetate (SA TA) contains both an amine

reactive and a protected thiol group. After de-protection, the thio-containing protein is reacted

with a thiol-reactive group on the other protein. p-Maleimidophenyl isocyanate (PMPI) is

another heterobifunctional cross-linking agent containing a thiol reactive (maleimide) and a

hydroxyl reactive group (Annunziato eta!., 1993). The isocyanate group reacts with amines

to fom1 ureas and with alcohols to f01m carbamates. The heterobifunctional reagents allow

coupling to be can·ied out in a stepwise manner and result in better control of the conjugation

chemistry (Annunziato eta!., 1993).

1.2.1.2 Carrier Proteins

A canier protein is a large molecule capable of stimulating its own immune response

(Harlow and Lane, 1988) and it must come from a species different from the animal to be

immunized. The carrier molecule provides the T -cell epitope for the production of a

successful immune response (De Silva eta!., 1999). The proteins to be used as caniers must

possess functional groups that can easily be substituted (Brinkley, 1992). Many different

canier proteins have been used for coupling with peptides to create immunogens. The choice

of the carrier to use is based on immunogenicity, solubility, and whether adequate

conjugation with the hapten can be achieved. Keyhole limpet hemocyanin (KLH) is one of

the commonly used caiTiers, however due to its large molecular weight(~ 8,000,000 Da) the

serum antibody response to the canier often obscures that of the antigen of interest (De Silva

et al., 1999) resulting in a reduced antigen-specific antibody concentration in the lgG

fraction.

27

Introduction and Review •

1.3 Antibody generation and characterization

Antibodies have become important research tools for many applications including the

detection and purification of desired targets. Antibodies are host proteins produced in

response to the presence of foreign molecules in the body (Harlow and Lane, 1988). They are

synthesized primarily by plasma cells, a te1minally differentiated cell of the B-lymphocyte

lineage, and circulate throughout the blood and lymph where they bind to foreign antigens.

An antibody response however is the culmination of a series of interactions between

macrophages, T lymphocytes and B-lymphocytes all reacting to the presence of a foreign

material (antigen). The extreme specificity at the molecular level of each immunoglobulin for

its antigen has made antibodies valuable therapeutic and diagnostic tools. Monoclonal and

polyclonal antibodies are useful in a variety of circumstances for their different and unique

prope1iies.

1.3.1 Polyclonal Antibodies

A general protocol of immunization, for the generation of either pAbs or mAbs,

involves injecting the immunogen mixed with the adjuvant (first using complete Freund's

adjuvant followed by boosters with Freund's incomplete adjuvant) at multiple sites. Sera are

then collected for the isolation of pAbs. For mAb generation, spleen cells of an immunized

animal are fused with an appropriate myeloma cell line, and extensive screening is can·ied out

to select a clone secreting the mAb desired. In our earlier work (Kaur et a/., 2008), we

extensively described production and characterization of Abs against low molecular-weight

pesticides (e.g., atrazine) with major emphasis on immunization protocols. Cross-reactivity of

an Ab to unexpected metabolites is a major problem in any immunodetection technique for

pesticide applications. Depending upon the conjugate used for immunization and the class of

chemicals under investigation, cross-reactivity of the Abs with metabolites similar to the

analyte of interest are frequently observed (Hanison et a!., 1991 ). Cross-reactivity, in

general, could be minimized by using compound-specific (monospecific) pAbs or mAbs,

because there are then fewer binding epitope-recognizing analogues and metabolites other

than the target compound.

The principal animals that have been used as polyclonal antibody sources are rabbits,

sheep, goat and donkey; however, there has been a surge of interest in the utilization of

chicken egg-yolk immunoglobulin in recent years (Losso et a/., 1998). Chicken antibody, or

7S immunoglobulin, is refened to as chicken IgG or lgY (Losso et al., 1997). The antibody in

egg yolk differs in molecular weight and isoelectric point from mammalian IgG (Hansen et

28

Introductiou and Review •

a!., 1998). IgY originates from the semm and is transferred preferentially by the follicular

epithelium of the ovary to the developing ova via specific receptors 4-5 days before

ovulation; it ranges from 9 to 25 mg/ml of egg yolk (Losso et a!., 1997). lg Y is transported

from the hen to the egg to protect the offspring against infections until the chick's immune

system has matured so that it can provide the young animal with sufficient amounts of

antibodies. The active transp011 of Ig Y from the semm to the egg results in a higher

concentration in the yolk than in the semm and therefore more antibodies can be produced

per month in a laying hen than in a rabbit (Larsson and Sjoquist, 1990). Immunoglobulins

(IgY) from egg yolk can be a valuable source of antibodies when obtained from hens

immunized against the target antigen. The advantages that IgY offers over conventional

sources of antibody include (I) the potential to produce gram quantities (2) low cost of

production, the cost of feeding and handling is considerably lower for a hen than for a rabbit,

(3) purification of lgY is relatively simple, and (4) the convenience of collecting antibodies

from eggs rather than bleeding animals, which facilitates compliance with animal welfare

considerations (Losso et a!., 1998 and Losso et a!., 1997). Chicken antibodies also have

biochemical advantages over mammalian antibodies (e.g., rabbit antibodies) that can be used

to improve immunoassays where antibodies are used. Due to great evolutionary distance

between birds and mammals, a chicken is able to produce antibodies against more epitopes

on a human antigen than a rabbit, which will give a stronger signal in immunological assays

(Carlander eta!., 2003). Antibodies are purified from the yolk usually by separating the lipid

fraction from the water-soluble faction. Several methods have been used for the purification

oflgY. Polson eta!., (1985) used polyethylene glycol precipitation, Jensenius eta!., (1981)

used dextran sulfate precipitation, Hassi and Aspock ( 1988) used hydrophobic interaction

chromatography and gel filtration to obtain pure egg yolk immunoglobulins. Hatta et a!.,

(1990) used xanthan gum precipiation. Akita and Nakai (1992) used a water dilution method

in which water-soluble plasma proteins were separated from the granular proteins as the egg

yolk granules aggregated with dilution. Factors that affect this purification method are extent

of dilution, incubation time, pH of diluted egg yolk and addition of sodium chloride. This

method was employed in this study. Akita and Nakai (1993) compared the water dilution

method with the polyethyleneglycol, dextran sulfate and xanthan gum methods in tetms of

yield, purity, ease of use, potential scaling up and immunoactivity oflgY. They rep011ed that

the water dilution method gave the highest yield, followed by dextran sulfate, xanthan gum

and polyethylene glycol method in that order. The underlying principle for the purification of

29

Introduction and Review •

lgY by the four methods compared is the precipitation of granules leaving the lgY in the

water-soluble fraction or supernatant.

1.3.2 Monoclonal Antibodies

Presence of an immunogenic molecule in an animal stimulates B-lymphocytes (B

cells), which undergo proliferation, differentiation, and maturation such that numerous B

cells produce antibodies to combat the invasion. Each B-cell produces a single type of

antibody molecule (monoclonal) such that the overall response involves different antibody

molecules (polyclonal) from different B-cells (Liddell and Cryer, 1991). Monoclonal

antibody production aims at selecting a B-cell producing a single type of antibody that is

specific to the antigen and proliferating it in vitro to produce the desired amount of antibody.

The cells that are isolated from the immunized animal, however, do not continue to grow in

tissue culture on their own. Kohler and Milstein (1975) showed that the prope1iy of

pe1manent proliferation could be added to the B-lymphocyte by fusing it with a tumorigenic

plasma cell (myeloma) fl"om the same animal species. The myeloma cells provide the conect

genes for continued cell division and the antibody secreting cells provide the functional

antibody genes. The hybrid cell or hybridoma can then be maintained in vitro and will

continue to secrete antibodies with a defined specificity. Myeloma can be induced in a few

strains of mice by injecting mineral oil into the peritoneum (Harlow and Lane 1988). Potter

(1972) isolated myelomas from BALB/C mice and they are the most commonly used partners

for fusion. Polyethylene glycol (PEG) is the most common fusogen for hybridoma

production. It fuses the plasma membranes of adjacent myeloma and/or antibody-secreting

cells, forming a single cell with two or more nuclei (Harlow and Lane, 1988). Usually,

myeloma has a mutation in one of the enzymes of the salvage pathway of purine nucleotide

biosynthesis (Liddell and Cryer, 1991 ). Selection with 8-azaguanine yields a cell line

harboring a mutated hypoxanthine-guanine phosphoribosyl transferase gene (HGPRT). The

addition of any compounds (azaserine, aminopte1in, etc) that block the de novo nucleotide

synthesis pathway will force the cells to use the salvage pathway. Cells containing

nonfunctional HGPRT protein will die in these conditions, while the hybrids between

myelomas with a nonfunctional HGPRT and antibody secreting cells (B-cells) with a

functional HGPRT will be able to grow.

1.3.3 Phage Display Library

Animal immunisation followed by hybridoma technology has been used to generate

monoclonal antibodies against a variety of antigens. Over the past ten years, advances in

molecular biology have allowed for the use of Escherichia coli to produce recombinant

30

Introduction and Review •

antibodies. By restricting the size to either a Fab, a Fv or a linker-stabilised single chain Fv

(scFv) such antibody fragments cannot only be expressed in bacterial cells but also displayed

by fusion to phage coat proteins (Griffiths and Duncan, 1998). The phage display concept

was first introduced for shm1 peptide fragments in 1985 (Smith, 1985). Fragments of the

EcoRI endonuclease, displayed as a polypeptide fusion (phenotype) to the gene 3 protein

(g3p), were encoded on the DNA molecule (genotype) encapsulated within the phage

particle. The linkage of genotype to phenotype is the fundamental aspect of phage display.

Subsequently, phage display of functional antibody fragments was shown (McCafferty et a!.,

1990) when the VH and VL fragments of the anti-lysozyme antibody were introduced, with a

linker, into a phage vector at theN tetminus of g3p. Since then, large scFv, Fab and peptide

repe11oires have been generated using a variety of phage display formats. One of the major

advantages of phage display technology of antibody fragments compared with standard

hybridoma technology is that the generation of specific scFv/Fab fragments to a particular

antigen can be performed within a couple of weeks (Arndt eta!., 2001 ).

1.4 Problem Definition: Overall Aims and Objectives

This study was aimed at the development and characterization of a highly specific,

sensitive, reliable and cost effective method for detection and monitming of pesticides in

environmental samples. Biosensors have of late become a method of choice for

environmental monitoring. Consisting of a biological detection element in close association

with a physical transducer gives a fast method of monitoring biomolecular interactions. The

specific interaction of biomolecules with target analyte generates a physical response and the

transducer detects this physical change and produces a corresponding electronic signal. We

chose antibodies for use as biological recognition element because the vertebrate immune

system is practically capable of producing antibodies specific for any target analyte provided

it is able to initiate an immune response. Thus the overall aim of the cmTent study was to

develop and characterize immunobiosensors for detection of pesticides in environmental

samples.

1.4.1 2,4-Dichlorophenoxyacetic acid

2,4-D has a molecular weight of 221.0 and a molecular formula of C8H60 3Ch. It is

soluble in organic solvents. The reported solubility of the free acid in water varies

considerably and is given as 0.09 % or 900 mg/1 at 25°C. The phenoxy herbicides, and 2,4-D

in pa11icular, ushered in the chemical weed control revolution in the mid 1940's. Even 51

years after its introduction, 2,4-D continues to be the most commonly and widely used

31

Introduction and Review •

herbicide worldwide. Monochloroacetic acid and 2,4-dichlorophenol were used to make 2,4-

D. Subsequently, salts were made by adding the approp1iate amine or inorganic hydroxide to

the acid. Esters were synthesized by reacting 2,4-D with the appropriate alcohols. A systemic

pesticide moves inside a plant following absorption by the plant. This movement is usually

upward (through the xylem) and outward. Increased efficiency may be a result. Systemic

insecticides which poison pollen and nectar in the flowers may kill needed pollinaters.

Physical Properties:

Table 1.6: Chemical characteristics of 2,4-D

Molecular weight 221.04

Melting point 135-142uC

Boiling point (at .4 rnmHg) 160uC

Water solubility (average of two values near 3.39xl0" ppm

25°C at pH7)

Vapor pressure( at 25°C) 1.4x10-1mmHg

Hydrolysis half-life (average of two values at 39 days

25°C, pH7)

Aqueous photolysis half-life (at 25uC) 13.0 days

Anaerobic aquatic half-life 312 days

Aqueous aerobic half -life 15.0 days

Aerobic half-life 66.0 days

Soil photolysis half-life 393 days

Field dissipation half-life 59.3 days

1.4.2 Discovery and development of 2,4-D

Botanists have long been intrigued with plant shoot and root growth and the

mechanisms causing plants to respond to stimuli. Kogl and Haagen-Smit, 1934 reported the

isolation of indole acetic acid (IAA) from plants and human urine and identified it as the

principal naturally occun·ing hormone (later called an auxin) in plants. Because IAA was

unstable outside of plants, researchers began synthesizing and investigating the effect of IAA

derivatives and homologs on plant growth activity. Pokorny in 1940 synthesized 2,4-D and

2,4,5-T while looking in vain for a fungicide. In June 1941, Pokorny described the chemical

synthesis of2,4-D.

Kraus at the University of Chicago had observed smce 1936 that ce11ain growth

regulators were phytotoxic (Peterson, 1967) and in 1941 he was first to propose that growth

32

Introduction and Review •

regulators might work as herbicides, because they often killed test plants. In November 1942,

the United States Army began developing Camp Detrick in Frede1ick, Maryland, as the

center for research and testing of chemicals for biological warfare with special emphasis on

crop destroying chemicals.

In November 21, 1962 United States and South Vietnam military personnel sprayed

2,4,5-T plus 2,4-D (Agent Orange) in Vietnam to defoliate trees along military routes to

reduce sniper activity. All biocidal research activities were terminated in 1972, following a

declaration by President R. M. Nixon that the United States would no longer conduct

biological warfare research or develop biological weapons.

In 1942, Zimmennan and Hitchcock reported that the phenoxy acetic acids could

induce seedless tomatoes and that they were potent synthetic plant hmmones. Franklin D.

Jones with the American Chemical Paint Company (ACPC) filed on March 20, 1944 and

received use patent 2,390,941 in December 1945 for 2,4-D as a herbicide. In June 1945,

ACPC marketed 2,4-D under the brand name Weed one, which was the first selective,

systemic herbicide produced and sold on a commercial scale.

1.4.3 Mode of action

2,4-D is thought to increase cell-wall plasticity, biosynthesis of proteins and the

production of ethylene. The abnmmal increase in these processes is thought to result in

uncontrolled cell division and growth, which damages vascular tissue. 2,4-D is generally

applied to the foliage of broadleaf plants or directly to the soil as both a liquid and a granular

product. It is also applied as an aqueous solution to sapwood cuts in the bark known as a hack

and squi11 application. Plants absorb 2,4-D through their roots and leaves within 4-6 hours

after application (Munro et al., 1992). Following foliar absorption, 2,4-D progresses through

the plant in the phloem, most likely moving with the food material. If absorbed by the roots,

2,4-D moves upward in the transpiration stream (EPA, 1988). Accumulation of the herbicide

occurs in the meristematic regions of the shoots and roots. 2,4-D mimics the effect of auxins,

or other plant growth regulating hmmones, and thus stimulates growth, rejuvenates old cells,

and over stimulates young cells leading to abnormal growth patterns and death in some plants

(Mullison, 1987). Plants treated with 2,4-D often exhibit malfmmed leaves, stems, and roots.

2,4-D affects plant metabolism by stimulating nucleic and protein synthesis, which affects the

activity of enzymes, respiration, and cell division (EPA, 1988). Often, cells in the phloem of

treated plants are crushed or plugged, interfering with normal food transport (Mullison,

1987), which can leave pmts of the plant malnourished or possibly lead to death.

33

Introduction and Review •

1.4.3 Impacts of 2, 4-Dichlorophenoxyacetic acid

In mammals, 2,4-D disrupts energy production (Zychlinkski and Zolnierowicz, 1990),

depleting the body of its primary energy molecule, ATP (adenosine triphosphate) (Palmiera

et a!., 1994). 2,4-D has been shown to cause cellular mutations, which can lead to cancer.

This mutagen contains dioxins, a group of chemicals known to be hazardous to human health

and to the environment (Littorin, 1994). Numerous epidemiological studies have linked 2,4-D

to non-Hodgkin's lymphoma (NHL) among farmers (Zahm, 1997; Fontana et al., 1998, Zahm

and Blair, 1992, Morrison et a!., 1992). Multi-center studies in Canada and in Sweden of

members of the general public found 30-50% higher odds of 2,4-D exposure among people

with NHL (McDuffie et a!., 2001, Bardell and Eriksson, 1999, Sterling and Arundel, 1986).

The teratogenic, neurotoxic, immunosuppressive, cytotoxic and hepatoxic effects of 2,4-D

have been well documented (Blakley et al., 1989; Sulik eta!., 1998; Bamekow eta!., 2000;

Rosso et al., 2000; Venkov eta!., 2000; Charles et al., 2001; Madrigal- Bujadar eta!., 2001;

Osaki et a!., 2001 ;Tuschl and Schwab, 2003). Other researchers publishing in the open

scientific literature have reported oxidant effects of 2,4-D, indicating the potential for

cytotoxicity or genotoxicity. For example, Bukowska (2003) repmted that treatment of

human erythrocytes in vitro with 2,4-D at 250 and 500 ppm resulted in decreased levels of

reduced glutathione, decreased activity of superoxide dismutase, and increased levels of

glutathione peroxidase. These significant changes in antioxidant enzyme activities and

evidence of oxidative stress indicate that 2,4-D should be taken seriously as a cytotoxic and

potentially genotoxic agent. 2,4-D causes significant suppression of thyroid hmmone levels

(Rawlings et a!., 1998). Similar findings have been repmted in rodents, with suppression of

thyroid hormone levels, increases in thyroid gland weight, and decreases in weight of the

ovaries and testes (Charles eta!., 1996). The increases in thyroid gland weight are consistent

with the suppression of thyroid hmmones, since the gland generally hypertrophies in an

attempt to compensate for insufficient circulating levels of thyroid hom1ones. Thyroid

hom1one is known to play a critical role in the development of the brain. Slight thyroid

suppression has been shown to adversely affect neurological development in the fetus,

resulting in lasting effects on child leaming and behavior (Haddow et al., 1999). 2,4-D causes

slight decrease in testosterone release and significant increase in estrogen release from

testicular cells (Liu et a!., 1996). In rodents, this chemical also increases levels of the

hmmones progesterone and prolactin, and causes abnmmalities in the estrus cycle (Duffard et

a!., 1995). In Minnesota, higher rates of bitth defects have been observed in areas of the state

with the highestuse of 2,4-D and other herbicides of the same class. This increase in bitth

34

Introduction and Review •

defects was most pronounced among infants who were conceived in the spring, the time of

greatest herbicide use (Garry et al., 1996). 2,4-D also interferes with the neurotransmitters

serotonin and dopamine. Females are more severely affected than males. Rodent studies have

revealed a region-specific neurotoxic effect on the basal ganglia of the brain, resulting in an

array of effects on critical neurotransmitters and adverse effects on behavior (Bortolozzi et

al., 2001; Rosso et al., 2000). This herbicide specifically appears to impair normal deposition

of myelin in the developing brain (Duffard et al., 1996). The neurotoxic and anti thyroid

effects of 2,4-D make it highly likely that fetuses, infants, and children will be more

susceptible to longterm adverse health effects from exposure to this chemical although they

may appear normal at birth. Young animals can also be exposed to 2,4-D through maternal

milk (Sturtz et al., 2000).

2,4-D is a moderately persistent chemical with a half- life between 20 and 200 days.

Unfortunately, the herbicide does not affect target weeds alone. It can cause low growth rates,

reproductive problems, changes in appearance or behavior, or death in non-target species.

Due to the widespread use of 2,4-D on agricultural land, the environmental effects of this use

are emerging in scientific studies. Donald et al., (1999) found agricultural pesticides in

wetlands, and 2,4-D was the most commonly detected pesticide.

Thus keeping above described observations in mind, our objective is aimed at

developing a sensitive system for detection of 2, 4-D in soil and water. The brief outlines of

the objectives are

OBJECTIVES

1. Hapten synthesis, bioconjugate preparation and characterization.

2. Antibody generation in mice/rabbit/chicken their characterization and affinity

purification.

3. Labeling of antibody with fluorophore/nanoparticles for immunoassay development.

4. Development ofimmunochemical methods for biomonitoring of2, 4-D in water and soil.

5. Optimization and validation of developed immunochemical technique.

35

Introduction and Review •

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